InteractiveFly: GeneBrief

Inositol 1,4,5,-tris-phosphate receptor: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Maintenance of homeostatic plasticity at the Drosophila neuromuscular synapse requires continuous IP3-directed signaling

Synapses and circuits rely on neuroplasticity to adjust output and meet physiological needs. Forms of homeostatic synaptic plasticity impart stability at synapses by countering destabilizing perturbations. The Drosophila melanogaster larval neuromuscular junction (NMJ) is a model synapse with robust expression of homeostatic plasticity. At the NMJ, a homeostatic system detects impaired postsynaptic sensitivity to neurotransmitter and activates a retrograde signal that restores synaptic function by adjusting neurotransmitter release. This process has been separated into temporally distinct phases, induction and maintenance. One prevailing hypothesis is that a shared mechanism governs both phases. This study shows the two phases are separable. Combining genetics, pharmacology, and electrophysiology, a signaling system consisting of PLCbeta, inositol triphosphate (IP3), IP3 receptors, and Ryanodine receptors was shown to be required only for the maintenance of homeostatic plasticity. It was also found that the NMJ is capable of inducing homeostatic signaling even when its sustained maintenance process is absent (James, 2019).

The Drosophila melanogaster NMJ is an ideal model synapse for studying the basic question of how synapses work to counter destabilizing perturbations. At this NMJ, reduced sensitivity to single vesicles of glutamate initiates a retrograde, muscle-to-nerve signaling cascade that induces increased neurotransmitter vesicle release, or quantal content (QC). As a result, the NMJ maintains a normal postsynaptic response level. Mechanistically, this increase in QC depends upon the successful execution of discrete presynaptic events, such as increases in neuronal Ca2+ influx and an increase in the size of the readily releasable pool (RRP) of synaptic vesicles. The field has termed this compensatory signaling process as presynaptic homeostatic potentiation (PHP) (Delvendahl, 2019). Two factors that govern the expression of PHP are the nature of the NMJ synaptic challenge and the amount of time elapsed after presentation of the challenge. Acute pharmacological inhibition of postsynaptic glutamate receptors initiates a rapid induction of PHP that restores synaptic output in minutes. By contrast, genetic lesions and other long-term reductions of NMJ sensitivity to neurotransmitter induce PHP in a way that is sustained throughout life (James, 2019).

Previously work has identified the Plc21C gene as a factor needed for PHP (Brusich, 2015). Plc21C encodes a Drosophila Phospholipase Cβ (PLCβ) homolog known to be neuronally expressed, but recent ribosomal profiling data also indicates possible muscle expression of Plc21C (Chen, 2017). In canonical signaling pathways, once PLCβ is activated by Gαq, it cleaves the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3). DAG can affect synaptic function by activating Protein Kinase C (PKC), while IP3 binds its receptor (IP3R) to trigger release of calcium from intracellular stores. It is not understood which aspects of this signaling machinery are mobilized during PHP. Potential downstream consequences of PLCβ activity at the NMJ include phosphorylation of neuronal proteins, modulation of ion channel activity, and changes in localization of neurotransmission machinery (James, 2019).

This study scrutinized PLCβ-directed signaling further. Tests were performed to see whether PLCβ-directed signaling was required solely for the maintenance of PHP or if it could also be required for induction. In addition to PLCβ, the IP3 Receptor (Drosophila Itpr, herein IP3R) and Ryanodine receptor (Drosophila RyR) were identified as being part of the same signaling process. Neither PLCβ, nor IP3R, nor RyR are required for the rapid induction of PHP. Additionally, it was found that the rapid induction of PHP is still possible in synapses already sustaining PHP. Surprisingly, it was found that NMJs are capable of rapidly inducing PHP -- even when the sustained expression of PHP is already blocked by impairments in PLCβ, IP3R, or RyR signaling. Taken together, these data show that the induction and maintenance of PHP are separable. Even though there is compelling evidence that parts of the induction and maintenance signaling mechanisms overlap (Goel, 2017), it is also true that acute PHP is possible in scenarios where long-term PHP is not (James, 2019).

This study divides the acute induction and chronic maintenance stages of presynaptic homeostatic potentiation. The data support two core findings. The first is that the short-term induction and long-term maintenance of PHP are separable by genetic and pharmacological manipulations. The second is that an IP3-mediated signaling system is specifically required for the maintenance of PHP (see Model depicting PLCβ/IP3R/RyR signaling underling the maintenance of PHP in both muscle and neuron.) (James, 2019).

For several years, one assumption has been that both the acute and chronic forms of PHP are executed in a similar way, and possibly by shared mechanisms. The issue has been clouded by the fact that both PhTox and a GluRIIA deletion mutant -- the primary reagents utilized to induce PHP -- have the same molecular target, that is GluRIIA-containing glutamate receptors. The process of combining these acute and chronic forms of plasticity within a single genotypic background was cumbersome due to a lack of reagents available to conduct temporally separate targeting experiments (James, 2019).

Several groups ascertained insights into temporal requirements by targeting potential homeostatic signaling genes. The main finding has been that the majority of molecules identified are essential to both the acute and chronic forms of PHP. Neurons tightly control neurotransmitter release probability, and the core presynaptic machinery directly responsible for increasing quantal content is shared. These shared components include the CaV2-type voltage-gated calcium channel or factors gating influx through the channel. They also include factors that regulate the size of the readily releasable pool (RRP) of presynaptic vesicles or factors that control the baseline excitability or plasticity of the presynaptic motor neuron, and neurotransmitter fusion events themselves. As a result, both the acute and chronic forms of PHP signaling cause increases in readily releasable pool (RRP) size and CaV2-mediated calcium influx; and in turn, these presynaptic mechanisms underlie the increases in QC which constitute PHP (James, 2019).

This study shows that although the acute and chronic processes might overlap, they are functionally separable. The fact that they are separable is not necessarily surprising. This finding mirrors data for discrete molecules required for long-term PHP maintenance, such as Target of Rapamycin (Tor), the Rho-type guanine exchange factor Ephexin, the transcription factor Gooseberry, C-terminal Src Kinase, innate immune signals molecules IMD, IKKβ, Relish and the kinesin adaptor Arl8. Importantly, this list contains molecules implicated both in neuron and muscle. This study has added PLCβ (Brusich, 2015) and its effectors IP3R and RyR to this list (James, 2019).

Recent studies have augmented the idea of overlapping signaling pathways and added a degree of specificity. Both acute and chronic forms of PHP begin as instructive retrograde signals after perturbations are detected in the muscle. These forms of PHP involve a decrease in phosphorylation of muscle CaMKII levels, and converge upon the same signaling components in the presynaptic neuron. These studies suggest that Tor signaling converges on the same molecular targets as acute forms of PHP (Goel, 2017). However, the precise roles for Tor and CaMKII in either form of PHP are as yet unknown (James, 2019).

These data appear to contradict the idea of PHP pathway convergence (Goel, 2017). Yet, the current findings are not incompatible with this idea. Multiple lines of evidence indicate discrete signaling requirements for acute forms of PHP on both sides of the synapse. A convergence point is undefined. Accounting for the separation of acute and chronic forms of PHP -- as well as their discrete signaling requirements -- long-term maintenance of PHP might integrate multiple signals between the muscle and neuron over time. For future studies, it will be important to clearly define roles of signaling systems underlying PHP and how distinct signaling systems might be linked (James, 2019).

This work presents unexpected findings. The first is that even in the face of a chronic impairment or block of homeostatic potentiation, the NMJ is nevertheless capable of a full rapid induction of PHP. Given that most molecules required for PHP identified to date are needed for both phases, significant functional separation between them was not expected. A priori it was expected that a failure of the chronic maintenance of PHP would make core machinery unavailable for its acute induction. The second unexpected finding is how quickly the chronic maintenance of PHP can be nullified by pharmacology (10 min), resulting in a return to baseline neurotransmitter release probability after only minutes of drug exposure. This study showed that homeostatic potentiation in GluRIIA mutant larvae or GluRIIC knock-down larvae was abrogated by four different reagents previously known to block IP3R or RyR. Those findings are reminiscent of prior work showing that acute blockade of DAG/ENaC channels with the drug benzamil abolishes PHP in both a GluRIIA mutant background, as well as in the presence of PhTox (Younger, 2013). A difference between benzamil application and the pharmacological agents used in the current study is that the drugs employed in this study only abolished PHP in a chronically challenged background (James, 2019)?

It is unclear how signaling systems that drive homeostatic plasticity transition from a state of induction to a state of maintenance. It is also not understood how interdependent short-term and long-term HSP implementation mechanisms are. A more complete understanding of the timing and perdurance of these properties could have important implications for neurological conditions where synapse stability is episodically lost (James, 2019).

The current findings parallel recent data examining active zone protein intensities in the contexts of induction of PHP and maintenance of PHP at the NMJ. There are multiple results informative to this study. First, the expression of any form of PHP (acute or chronic) appears to correlate with an increased intensity of active zone protein levels, such as CaV2/Cacophony, UNC-13, and the Drosophila CAST/ELKS homolog Bruchpilot. Unexpectedly, however, two of these studies also reported that the rapid induction of PHP does not require this protein increase in order to be functionally executed. These are conundrums for future work. How does rapid active zone remodeling happen in minutes on a mechanistic level? In the absence of such remodeling, how is PHP able to be induced rapidly? Moreover, is the observed short-term active zone remodeling the kernel for the longer-term changes to the active zone and release probability - or is some other compensatory system triggered over long periods of developmental time (James, 2019)?

The current findings add a new dimension to those puzzles with the data that IP3 signaling is continuously required to maintain PHP. If active zone remodeling truly is instructive for PHP maintenance, then it will be interesting to test what roles IP3 signaling and intracellular calcium release play in that process. The screen did include a UAS-RNAi line against unc-13 and an upstream GPCR-encoding gene methuselah. Moreover, a previously study of PHP used a UAS-cac[RNAi] line (Brusich, 2015). Chronic PHP maintenance was intact for all of those manipulations. Those findings are not necessarily contradictory to the recent work from other groups. For instance, knockdown of an active zone protein by RNAi is not a null condition. As such, RNAi-mediated knockdown should leave residual wild-type protein around. In theory, that residual protein could be scaled with homeostatic need (James, 2019).

The data strongly suggest that intracellular calcium channel activation and store release fine tune neurotransmitter release that is implemented by PHP. The exact mechanism by which IP3R and RyR function to maintain PHP at the NMJ is unclear. It appears to be a shared process with IP3. If these store-release channels are acting downstream of IP3 activity, then the data suggest that this would be a coordinated activity involving both the muscle and the neuron, with loss of IP3 signaling in the neuron being more detrimental to evoked release (James, 2019).

It remains unclear what signals are acting upstream. PLCβ is canonically activated by Gαq signaling. From prior work, evidence was garnered that a Drosophila Gq protein plays a role in the long-term maintenance of HSP (Brusich, 2015). Logically, there may exist a G-protein-coupled receptor (GPCR) that functions upstream of PLCβ/IP3 signaling. A screen did not positively identify such a GPCR. Several genes encoding GPCRs were examined, including TkR86C, mAChR-A, GABA-B-R1, PK2-R2, methuselah, AdoR, and mGluR. Genes encoding Gβ subunits or putative scaffolding molecules were also examined, including CG7611 (a WD40-repeat-encoding gene), Gβ13F, and Gβ76C, again with no positive screen hits (James, 2019).

The data are consistent with dual pre- and post-synaptic functions of IP3. This could mean dual pre- and post-synaptic roles for calcium store release, through an undetermined combination of RyR and IP3R activities, again either pre- or post-synaptic. Both RyR and IP3R have been shown to be critical for specific aspects of neuroplasticity and neurotransmission. Activities of both RyR and IP3R can activate molecules that drive plasticity, such as Calcineurin and CaMKII. At rodent hippocampal synapses, electrophysiological measures like paired-pulse facilitation and frequency of spontaneous neurotransmitter release are modulated by RyR and/or IP3R function, as is facilitation of evoked neurotransmitter at the rat neocortex. In addition to vesicle fusion apparatus, activity of presynaptic voltage-gated calcium channels is modulated by intracellular calcium. Work from this lab at the NMJ has shown that impairing factors needed for store-operated calcium release can mollify hyperexcitability phenotypes caused by gain-of-function CaV2 amino-acid substitutions (Brusich, 2018; James, 2019 and references therein).

Within the pre-synaptic neuron, IP3R and RyR could activate any number of calcium-dependent molecules to propagate homeostatic signaling. Some candidates were tested in a screen, but none of those tests blocked PHP. One possibility is that the reagents utilized did not sufficiently diminish the function of target molecules enough to impact PHP in this directed screen. Detection of downstream effectors specific to muscle or neuron might also be ham>pered by the fact that attenuation of IP3 signaling in a single tissue is insufficient to abrogate PHP. Another possibility is that presynaptic store calcium efflux via IP3R and RyR may directly potentiate neurotransmitter release, either by potentiating basal calcium levels or synchronously with CaV2-type voltage-gated calcium channels (James, 2019).

Both pre- and post-synaptic voltage-gated calcium channels are critical for the expression of several forms of homeostatic synaptic plasticity. Much evidence supports the hypothesis that store-operated channels and voltage gated calcium channels interact to facilitate PHP. In various neuronal populations, both RyR and IP3R interact with L-type calcium channels physically and functionally to reciprocally impact the opening of the other channel. In presynaptic boutons, RyR calcium release follows action potential firing. Calcium imaging experiments show that both the acute expression and sustained maintenance of PHP requires an increase in presynaptic calcium following an action potential. Because IP3Rs are activated by both free calcium and IP3, elevated IP3 levels in the case of chronically expressed PHP could allow IP3Rs and RyRs to open in a way that is time-locked with CaV2-mediated calcium influx or in a way to facilitate the results of later CaV2-mediated influx (James, 2019).

Associative learning drives longitudinally graded presynaptic plasticity of neurotransmitter release along axonal compartments

Anatomical and physiological compartmentalization of neurons is a mechanism to increase the computational capacity of a circuit, and a major question is what role axonal compartmentalization plays. Axonal compartmentalization may enable localized, presynaptic plasticity to alter neuronal output in a flexible, experience-dependent manner. This study shows that olfactory learning generates compartmentalized, bidirectional plasticity of acetylcholine release that varies across the longitudinal compartments of Drosophila mushroom body (MB) axons. The directionality of the learning-induced plasticity depends on the valence of the learning event (aversive vs. appetitive), varies linearly across proximal to distal compartments following appetitive conditioning, and correlates with learning-induced changes in downstream mushroom body output neurons (MBONs) that modulate behavioral action selection. Potentiation of acetylcholine release was dependent on the Ca(V)2.1 calcium channel subunit Cacophony. In addition, contrast between the positive conditioned stimulus and other odors required the inositol triphosphate receptor, which maintained responsivity to odors upon repeated presentations, preventing adaptation. Downstream from the MB, a set of MBONs that receive their input from the γ3 MB compartment were required for normal appetitive learning, suggesting that they represent a key node through which reward learning influences decision-making. These data demonstrate that learning drives valence-correlated, compartmentalized, bidirectional potentiation, and depression of synaptic neurotransmitter release, which rely on distinct mechanisms and are distributed across axonal compartments in a learning circuit (Stahl, 2022).

Compartmentalized plasticity in neurotransmitter release expands the potential computational capacity of learning circuits. It allows a set of odor-coding MB neurons to bifurcate their output to different downstream approach- and avoidance-driving downstream output neurons, independently modulating the synaptic connections to alter action selection based on the conditioned value of olfactory stimuli. The KCs modify the encoded value of olfactory stimuli through bidirectional plasticity in odor responses, which vary in a compartment-specific manner along the length of the axons. These changes were observed following pairing an olfactory CS with gustatory/somatosensory US (sucrose feeding or electric shock) in vivo. The CS+ and CS- drive unique patterns of plasticity in each compartment, demonstrating that olfactory stimuli are reweighted differently across compartments following learning, depending on the temporal associations of the stimuli. Different molecular mechanisms govern the potentiation of trained odor responses (CaV2/Cac) and maintenance of responsivity over time (IP3R). Finally, one set of γ output neurons, γ3/γ3β'1, is important for appetitive short-term memory (Stahl, 2022).

Learning-induced plasticity of ACh release in the MB was bidirectional within the compartment, depending on the valence of the US, and was coherent with the valence of the MBON downstream of the compartment. Notably, the γ2 and γ3 MB compartments, which relay information to approach-promoting MBONs, exhibited plasticity that was coherent with promoting behavioral approach following appetitive conditioning and avoidance after aversive conditioning. There was an increase in the relative CS+:CS- ACh responses after appetitive conditioning, and conversely reduced CS+:CS- ACh responses following aversive conditioning. Fhis study focused on the time point 5 min following conditioning, which is consistent with behavioral short-term memory. Aversive conditioning was previously reported to decrease neurotransmitter release from KCs. Indirect evidence, via Ca2+ imaging in presynaptic KCs, suggested that increases in presynaptic neurotransmission could also be associated with learning. Pairing odor with stimulation of appetitive PAM dopaminergic neurons potentiates odor-evoked cytosolic Ca2+ transients across the KC compartments. Appetitive conditioning increases odor-evoked Ca2+ transients across KC compartments. Stimulation of dopaminergic circuits associated with reward learning potentiate MB γ4 connections with the respective γ4 MBON. Statistically significant effect was not observed in γ4 with appetitive or aversive classical conditioning, though the CS+ and CS- trended in the same direction as the adjacent γ5 compartment following conditioning. Overall, the present data demonstrate that there are bidirectional changes in neurotransmitter (ACh) release from MB compartments following appetitive vs aversive learning and provide a window into the spatial patterns of plasticity across compartments following associative learning (Stahl, 2022).

Behavioral alterations following conditioning involve changes in responses among the MBONs. As the KCs provide presynaptic olfactory input to the MBONs, it was a logical a priori assumption that presynaptic plasticity in the KCs could be altered in a compartmental manner and contribute to the changes in MBON responses after conditioning. Yet data from previous Ca2+ imaging experiments have not completely supported this model. Compartmentalized effects have been observed in KCs with non-associative learning protocols and within the γ4 compartment following associative learning. In contrast, classical conditioning produces no compartmentalized differences in odor-evoked Ca2+ responses. Appetitive conditioning with odor + sucrose pairing increases odor-evoked cytosolic Ca2+ transients in KCs across the γ lobe compartments. Aversive conditioning produces no net change across the compartments, but alters synapse-specific Ca2+ responses at the individual bouton level. If the compartmental effects of conditioning (observed with Ca2+ imaging) in KCs drove a proportional change in neurotransmitter release, both the approach- and avoidance-promoting MBONs would be simultaneously potentiated. Extracellular influx of Ca2+ through voltage-gated calcium channels is a primary driver of neurotransmitter release; however, there are multiple sources of Ca2+ in the cytosol that could contribute to the GCaMP signals. A major conclusion of the present study is that learning drives compartmentalized plasticity in neurotransmitter release that is coherent with the behavioral valence of the corresponding MBON (Stahl, 2022).

At least two major molecular mechanisms govern the spatial patterns of plasticity across the MB compartments: a Cac-dependent CS+ potentiation and an IP3R-dependent maintenance of sensory responses over trials/time. This suggests that different sources of Ca2+ play different roles in regulating KC synaptic responses. Cac is the pore-forming subunit of the voltage-sensitive, presynaptic CaV2 Ca2+ channel in Drosophila. CaV2 channels regulate several forms of synaptic plasticity, including paired-pulse facilitation, homeostatic plasticity, and long-term potentiation. The current data suggest that these channels regulate the spatial patterns of learning-induced plasticity in the MB unidirectionally (from baseline), with Cac underlying potentiation but not depression. CaV2 channel activity is modulated by presynaptic calcium and G protein-coupled receptor activity, and channel localization in the active zone dynamically regulates synaptic strength. Thus, Cac insertion into, or increased clustering within, the active zones may underlie learning-induced potentiation (e.g. in the γ1-γ2 compartments following appetitive conditioning). Conditional knockdown of Cac, which reduced Cac levels by ~29%, impaired this potentiation, likely by decreasing the number of available channels for modulation. Baseline stimulus-evoked neurotransmitter release was maintained during Cac knockdown, mediated either by the significant residual Cac expression or compensation by other intracellular Ca2+ channels/sources. In contrast to the Cac effect on potentiation, IP3R was necessary to maintain normal odor responsivity when odors were presented repeatedly across multiple trials (whether those were pre/post trials in the conditioning protocol or 10x odor presentations in the adaptation protocol). This is broadly consistent with the temporal role of IP3R in maintenance of presynaptic homeostatic potentiation at the neuromuscular junction. In addition, dopaminergic circuits associated with reward learning drive release of Ca2+ from the endoplasmic reticulum when activated with KCs in a backward pairing paradigm ex vivo, potentiating MB γ4 connections with the respective γ4 MBON. This is consistent with a role for ER calcium in positively regulating synaptic strength (Stahl, 2022).

Potentiation and depression of ACh release was observed across multiple MB compartments following conditioning, providing a presynaptic mechanism that potentially contributes to shaping conditioned MBON responses. Importantly, by comparing the CS+ and CS- responses to those of untrained odors, plasticity was ascribed to potentiation or depression (accounting for any non-associative olfactory adaptation) within each compartment. This is relevant for modeling efforts, where it has been unclear whether to include potentiation (along with depression) in the learning rule(s) at KC-MBON synapses. In addition, the experiments revealed an additional layer of spatial regulation in the γ1-γ3 compartments: a gradient of CS+ potentiation to CS- depression following appetitive conditioning. Specifically, the CS+/CS- relationship changed in a linear gradient down the γ1-γ3 compartments following appetitive conditioning. Appetitive conditioning increased CS+ responses in the γ1 compartment, while decreasing the CS- responses in the γ3 compartment. The γ2 compartment yielded a mix of these responses. These patterns of plasticity have the net effect of increasing the relative response to the CS+ odor (↑CS+:CS-). Since the MBONs postsynaptic to these compartments drive behavioral approach, this would bias the animal to approach the CS+ if it encountered both odors simultaneously. Such a situation occurs at the choice point of a T-maze during retrieval in a classical conditioning assay. The CS+ and CS- produce different patterns of plasticity at different loci (e.g. γ1 vs γ3), which presumably coordinate to regulate behavior via temporal integration of the odor and US cues. The CS+ is temporally contiguous with the US, while the CS- is nonoverlapping. Therefore, the timing of CS/US pairing drives plasticity differently in each compartment. These patterns of plasticity presumably coordinate to regulate memory formation and action selection during retrieval. For instance, while the γ1-γ3 compartments exhibited ↑CS+:CS- following appetitive conditioning, the γ5 compartment exhibited plasticity in the opposite direction: decreasing the relative response to the CS+ odor (↓CS+:CS-). As the γ5 compartment is presynaptic to an avoidance-promoting MBON, this plasticity pattern would coherently contribute to biasing the animal toward CS+ approach (reducing CS+ avoidance). Thus, it would work in concert with the plasticity in γ1-γ3 to bias the animal toward behavioral approach. Overall, plasticity is regulated in each MB compartment individually by the timing of events and the valence of the US, with the changes coordinated across multiple compartments to coherently drive behavior (Stahl, 2022).

Behaviorally, MBONs innervating the γ lobe variably drive behavioral approach or avoidance when stimulated. Despite the approach-promoting valence of the γ2α'one and γ3/γ3β'1 MBONs, among them, only the γ3/γ3β'1 MBONs produced a loss-of-function phenotype in behavioral appetitive conditioning. This suggests that redundancy and/or different weighting across approach promoting MBONs renders the system resilient to silencing some of them. A previous study found effects of blocking the γ2α'1 MBONs, though not γ3/γ3β'1 MBONs, when blocking individual steps of memory processing (acquisition, retention, and/or retrieval) with a 1 hr appetitive memory protocol. This suggests that the different MBONs have differing roles across time, with some redundancy in appetitive processing. Blocking synaptic output of γ3/γ3β'1 MBONs reduced appetitive conditioning performance in these experiments immediately following conditioning, suggesting that these neurons play a specific role in appetitive short-term memory (Stahl, 2022).

The present and previous studies suggest that alterations of MBON activity following learning are the product of both presynaptic and postsynaptic plasticity at the KC-MBON synapses, as well as feedforward inhibition. Blocking synaptic output from KCs impairs the acquisition of appetitive memories (30-60 min after conditioning), suggesting a role for postsynaptic plasticity. However, this does not rule out presynaptic plasticity, as blocking KC output (with R13F02) leaves signaling from reinforcing dopaminergic neurons partially intact, which likely shapes the presynaptic KC responses via heterosynaptic plasticity. At the circuit level, polysynaptic inhibition can convert depression from select MB compartments into potentiation in MBONs following learning; in one established example, reduction of odor-evoked responses in the GABAergic γ1pedc MBON following aversive conditioning disinhibits the downstream γ5β'2 a MBON (Stahl, 2022).

KC-MBON synapses represent one node of learning-related plasticity, which is distributed across multiple sites during learning. Short-term memory-related plasticity has been observed in multiple olfactory neurons, such as the antennal lobes. In addition, connectomics studies have revealed complex connectivity within and beyond the MB, which is a multi-layered network including circuit motifs that influence the propagation of information and generation of plasticity during learning. Such connections include recurrent feedback. Some of these recurrent connections are from cholinergic MBONs that synapse within the MB, which could have contributed to the ACh signals observed in this study. For instance, the γ2α'1 MBON is a cholinergic MBON that sends ~6% of its output back to the γ lobe. Some of the recurrent connections are formed by dopaminergic neurons, such as the PAM γ4<γ1/y2. In addition, reciprocal connections between KCs and dopaminergic neurons in the vertical lobes are necessary for memory retrieval. This adds another layer of recurrent circuitry that may participate in reinforcement during associative learning. Across these circuits, some neurons corelease several neurotransmitters and act on an array of postsynaptic receptors, which contribute to plasticity distributed across multiple sites (Stahl, 2022).

Overall, plasticity between KCs and MBONs may guide behavior through biasing network activation to alter action selection in a probabilistic manner. Appetitive conditioning drives compartmentalized, presynaptic plasticity in KCs that correlates with postsynaptic changes in MBONs that guide learned behaviors. Prior studies documented only depression at these synapses at short time points following conditioning. This study observed both potentiation and depression in ACh release in the MB, suggesting that bidirectional presynaptic plasticity modulates learned behaviors. These bidirectional changes likely integrate with plasticity at downstream circuit nodes that also undergo learning-induced plasticity to produce network-level alterations in odor responses across the olfactory pathway following salient events. Thus, plasticity in ACh release from KCs functions to modulate responsivity to olfactory stimuli features across graded plasticity maps down the MB axons (Stahl, 2022).

Circadian pacemaker neurons display cophasic rhythms in basal calcium level and in fast calcium fluctuations

Circadian pacemaker neurons in the Drosophila brain display daily rhythms in the levels of intracellular calcium. These calcium rhythms are driven by molecular clocks and are required for normal circadian behavior. To study their biological basis, this study employed genetic manipulations in conjunction with improved methods of in vivo light-sheet microscopy to measure calcium dynamics in individual pacemaker neurons over complete 24-h durations at sampling frequencies as high as 5 Hz. This technological advance unexpectedly revealed cophasic daily rhythms in basal calcium levels and in high-frequency calcium fluctuations. Further, the rhythms of basal calcium levels and of fast calcium fluctuations were found to reflect the activities of two proteins that mediate distinct forms of calcium fluxes. One is the inositol trisphosphate receptor (ITPR), a channel that mediates calcium fluxes from internal endoplasmic reticulum calcium stores, and the other is a T-type voltage-gated calcium channel, which mediates extracellular calcium influx. These results suggest that Drosophila molecular clocks regulate ITPR and T-type channels to generate two distinct but coupled rhythms in basal calcium and in fast calcium fluctuations. It is proposed that both internal and external calcium fluxes are essential for circadian pacemaker neurons to provide rhythmic outputs and thereby, regulate the activities of downstream brain centers (Liang, 2022).

Circadian rhythms in multiple aspects of cellular physiology help organisms across taxa, from unicellular cyanobacteria to multicellular animals, adapt to environmental day-night changes. In mammals, neurons in the hypothalamic suprachiasmatic nucleus (SCN) show circadian rhythms in gene expression, intracellular calcium, neural activity, and other cellular properties. Circadian rhythms in SCN neuronal outputs coordinate circadian rhythms in other cells throughout the body and generate behavioral rhythms. The rhythms of SCN neuronal outputs can be generated cell intrinsically by the negative transcription/translation feedback loop of core clock genes as a molecular clock, which then generates 24-h oscillations in a series of genes. These gene oscillations then regulate different aspects of membrane physiology, such as the expression levels of channels for potassium, sodium, and calcium. The mechanisms by which the molecular clockworks coordinate complex membrane physiology to generate neural activity rhythms within individual circadian pacemakers remain to be defined (Liang, 2022).

Calcium signaling regulates many cellular processes, such as neural excitability, neurotransmitter release, and gene expression. Cytoplasmic calcium can be regulated from extracellular calcium influx as well as from intracellular calcium stored in the endoplasmic reticulum (ER) and mitochondria. Studies on SCN neurons in vitro and recently, in vivo measured circadian calcium rhythms (CCRs) in SCN neurons. Some studies suggested that calcium rhythms were driven by neuronal firing and voltage-gated calcium channels, while others suggested they were driven by intracellular stores via the ER channel ryanodine receptor (RyR). These alternative hypotheses may derive from the technical differences in the various studies, including the details of in vitro preparations, but also, due to a lack of single-cell resolution in the calcium measurements (Liang, 2022).

In Drosophila, circadian pacemaker neurons also show clock-driven CCRs. The dynamics can be resolved across all five major pacemaker groups (the small ventral lateral neurons [s-LNv], the large ventral lateral neurons [l-LNv], the dorsal lateral neurons [LNd], the group #1 dorsal neurons [DN1], and the group #3 Dorsal Neurons [DN3]), and each group exhibits distinct and sequential daily peak phases. Within such groups, the rhythms can be measured in single identified cells. The multihour phase diversity exhibited by this network requires a series of delays effected by environmental light and by noncell-autonomous modulation mediated by different neuropeptides. Precisely how neuropeptide signaling regulates calcium activity in pacemaker neurons over long (many-hour) durations is unknown. To begin to understand these critical mechanisms of pacemaker modulation, this study began by addressing the cellular and molecular basis of pacemaker calcium rhythms with physiological, genetic, and behavioral measures (Liang, 2022).

In this study, in vivo calcium imaging began at single-cell resolution, using a high-speed light-sheet microscope termed OCPI-2; the acronym OCPI stands for objective-coupled planar illumination. OCPI-2 represents a fundamental technical advance because it permits sampling frequencies to capture stacks of large tissue volumes, without compromising photon efficiency or spatial resolution. Whereas OCPI-1 methods permitted sampling a whole-brain volume once every 10 min across the 24-h day, OCPI-2 methods permit sampling volumetrically at rates as high as 5 Hz. Thus, both basal calcium levels and fast calcium fluctuations were simultaneously measured at single-cell resolution over entire 24-h durations. Circadian rhythmicity was found in both measures. The fast fluctuations are considered to represent events closely coupled to neuronal firing, as have previous studies conducted in much more restricted temporal durations (i.e., not circadian). In all the Drosophila pacemaker neurons studied, these two layers of calcium rhythms shared the same daily temporal pattern (i.e., they were cophasic). To gain insights into the mechanism of these patterns, the fact was exploited that in Drosophila, many calcium channels are encoded by single genes, and genetics was used to study the roles of individual channels in generating daily pacemaker calcium rhythms. This study presents results of experiments in which RNAs encoding different calcium channels were knocked down selectively in all or a subset of pacemakers. The impact of individual channels in setting both slow daily changes in basal calcium levels and in fast fluctuations was evaluated. Finally, PERIOD (PER) protein staining levels and behavior were measure to determine which channels provide feedback to the molecular clock and which are required for normal circadian output from the pacemaker network (Liang, 2022).

This study used in vivo 24-h high-frequency calcium imaging and genetic screening to study the cellular biology of daily calcium rhythms in circadian pacemaker neurons of Drosophila. It was found that the calcium rhythm is in fact a composite; it reflects daily fluctuations in both a slow component (basal levels) and a fast one (high-frequency fluctuations). Fast calcium fluctuations were interpreted as representations of calcium dynamics that occur as neurons fire single action potentials or bursts of them. While it is not sufficient to resolve single action potentials, GCaMP6-induced fluorescence is a good index of neuronal electrical activity. For individual identified pacemakers, these two calcium rhythms share the same daily pattern, yet distinct calcium sources appear to contribute differentially to these two rhythms. An extracellular calcium influx, through plasma membrane calcium channels that include the α1T subunit, is critical for the fast calcium fluctuations. In contrast, calcium fluxes from the ER via the channel ITPR are required for both the slow rhythms in the basal calcium levels and the fast ones. Importantly, both channels are essential for normal circadian behavior. Thus, the molecular clocks may drive circadian rhythms in pacemaker neuron output by regulating different calcium sources to generate coordinate but distinct rhythms in its calcium activities (Liang, 2022).

CCRs are widespread across taxa. Calcium rhythms are required for circadian pacemaker functions in both rodents and Drosophila. Studies on mammalian circadian pacemakers in the SCN are controversial regarding the temporal relationship between the CCR and rhythms in electrical activity, such as in spontaneous firing rate (SFR) and in resting membrane potential (RMP). Recordings from SCN slice cultures showed that the phases of CCR in individual pacemakers are diverse and could be different from the populational phase of SFR rhythms (16, 33). However, the populational SFR phase is composed of many diverse phases of SFR rhythms on the individual cell level; it is unclear whether SFR phases align with the phases of CCR. Imaging with both voltage sensor and calcium sensors in SCN slices, another study concluded that RMP rhythms and CCR were in phase, yet even a third study concluded that RMP rhythms and CCR were in phase in the ventral SCN but that in dorsal SCN, the CCR phase led the RMP rhythms by ~2 h. Because the voltage sensor signal measured from dorsal SCN may derive from the neural processes of ventral SCN neurons, the cellular interpretation of these results is unclear. Another source for the inconsistency might be the culture conditions; in fact, when SCN neurons were recorded in vivo by photometry, the rhythms in fast calcium activity were in phase with slow calcium rhythms. In general, comparisons of population rhythms and rhythms in single cells are not easily reconciled. Recordings that tracked pacemaker neurons from different identified groups in vivo for 24 h showed that at the single cell level, slow calcium rhythms (CCRs) were unambiguously in phase with rhythms in fast calcium fluctuations; the latter likely reflects rhythms in SFR (Liang, 2022).

To measure the fast calcium fluctuations, a high-frequency light-sheet scanning microscope termed OCPI-2 was used. OCPI-2 resolved limitations present in previous microscope designs that created a bottleneck in terms of the rate at which tissue volumes can be repeatedly scanned. Importantly, it does so without compromising photon efficiency or spatial resolution. Its use was instrumental in allowing sampling of the whole-brain volume at frequencies as high as 5 Hz periodically throughout the day. cry⁰¹ flies were used to avoid the direct light responses of pacemaker neurons, and it was found that all pacemaker groups displayed slow calcium rhythms, with patterns that were comparable with those previously reported, except for that of l-LNv. That group displayed an additional calcium peak in the early evening and another just before lights on. Because l-LNv innervates the optic lobes and receives large-scale visual inputs, it is speculated that the repeated optical scanning might anomalously activate l-LNv in the evening via visual systems. In the later experiments, when the illumination duration per hour was reduced from 31.5 s to 2.4 s, l-LNv did not show the additional evening peaks; this was true even when measured in flies wild type for cry. Shorter durations of light exposure permit all pacemaker groups to display slow calcium rhythms with phases similar to those obtained with the slow-frequency (every 10-min) recording sessions. Therefore, the concern to avoid technical artifacts when imaging from light-sensitive pacemaker appears especially acute in the case of l-LNv. Nevertheless, by carefully tuning the illumination intensity for calcium imaging, it is proposed that it is possible to monitor normal slow calcium rhythms and fast calcium fluctuations from the same individual pacemaker neurons (Liang, 2022).

The causal relationships between clock gene rhythms, calcium rhythms, and electrical activity rhythms in the SCN remain generally unresolved. Treating SCN slices with Tetrodotoxin (TTX, to block Na-dependent action potentials) diminished SFR rhythms, partially affected RMP rhythms and CCRs, and slowly affected clock gene rhythms over several days. Dispersed SCN cells in vitro showed a TTX-resistant CCR, suggesting that CCR is driven by clock gene rhythms. Thus, the variation in CCR sensitivity to TTX treatment might be caused by the degree to which clock gene rhythms in vitro become progressively dysfunctional. In Drosophila, the findings suggested that clock gene rhythms drive two components of the CCR-both basal calcium levels and fast calcium fluctuations-via circadian regulation of the ER channel ITPR and membrane voltage-gated calcium channel α1T. Both channels might then contribute to SFR and RMP rhythms. Similarly, in SCN pacemakers, pharmacologically blocking another ER channel RyR affected both CCR and SFR rhythms, suggesting that rhythms in basal calcium levels are regulated by calcium from ER and are required for fast electric activity rhythms. In addition, SCN pacemakers also showed a circadian rhythm in fast calcium activity mediated by L-type voltage-gated calcium channels. Pharmacologically blocking these membrane channels affected SFR rhythms and in some case, affected CCR. In these studies, manipulating a membrane voltage-gated calcium channel in all or a subset of pacemakers selectively affected rhythms in fast calcium fluctuations, which likely reflected SFR rhythms and thus, impaired circadian outputs; however, it did not significantly affect the slow rhythms in basal calcium levels. Manipulating the ER calcium channel IP3R in all pacemakers affected rhythms in both slow and fast calcium rhythms. Therefore, in parallel to mammalian SCN neurons, Drosophila circadian pacemakers generate calcium rhythms by regulating both ER and extracellular calcium sources. Since these results suggest little or no role for the RyR channel, the daily rhythmic regulation in fly pacemakers likely acts on a different set of ER and cytoplasmic membrane channels from those in mammalian pacemakers (Liang, 2022).

This study also presented RNAi evidence implicating the Itpr and Ca-&alpha1T genes in pacemaker cell calcium fluxes. However, for each, only a single RNAi proved effective in reducing rhythmic power and increasing percentage of arrhythmicity. Corroboration for these findings is provided by a prior report, which found that the same RNAi line used decreases Itpr RNA levels. Likewise, corroboration was found in a prior report, that described a specific Ca-alpha1T insertion line as a protein null mutation that exhibited reduced percentage of rhythmicity and power in DD but without a change in circadian period or in the daily per rhythm. They concluded that Ca-alpha1T expression does not affect the central clock mechanism and surmised instead that it likely affects the output of the circadian system, specifically via a requirement for the channel to permit normal physiological activation of pacemaker neurons. That work provides independent genetic confirmation of the RNAi results, and its conclusions align precisely with the current work (Liang, 2022).

The RNAi knockdown experiments indicate a role for the ER calcium channel SERCA in supporting slow calcium rhythms in Drosophila pacemakers and behavioral rhythms. However, SERCA knockdown also produced high rates of lethality and among survivors, strong effects on the PER molecular oscillation. It is concluded that SERCA, which maintains the ER cytoplasmic calcium gradient, is essential for the normal physiology of the cells but that these additional phenotypes precluded an assessment of its precise role in circadian rhythmicity. In contrast to the situation with SERCA, the results support a hypothesis that ITPR is a crucial actuator of the molecular clock. Previous transcriptomic analysis also supports that possibility; in circadian neurons, Itpr displays rhythmic expression, while SERCA does not. Knocking down Itpr in all circadian neurons (with tim-GAL4) generally caused stronger deficits in both behavioral rhythms and calcium rhythms than in just the PDF-positive neurons (with pdf-GAL4). One possibility might be that the RNAi expression level is lower when driven by pdf-GAL4 than when driven by tim-GAL4. However, in SI knocking down Itpr in all circadian neurons caused stronger deficits in the amplitude of calcium rhythms of non-PDF-positive neurons (LNd, DN1, and DN3) than in those of PDF-positive neurons (s-LNv and l-LNv). Hence, a difference in the vulnerability to IP3R disruption between PDF-negative and PDF-positive neurons might provide an additional or alternative explanation for why the pdf-GAL4-driven knockdown of Itpr affected neither calcium rhythms nor behavior. It is also noted that the literature provides examples, wherein a single RNAi line produces stronger behavioral effects when coupled with pdf-Gal4 than with tim-GAL4. Such findings suggest that the final result in such experiments will represent a mixture of GAL4 driver line strength and the differential contributions of the UAS:Responder gene product across different positions within an affected neural circuit (Liang, 2022).

Finally, the RNAi knockdown of the plasma membrane calcium channel α1T indicated a role for voltage-gated T-type calcium channels in the final rhythmic output of the pacemakers. Consistent with a role in the presumed output pathway, impairing the rhythm of fast calcium activity strongly affected circadian behavior but did not affect the molecular clock or the slow calcium rhythms. T-type channels play a crucial role in other pacemakers, such as the Sino-Atrial node of the mammalian heart. Their conduction in the hyperpolarized state and closure at more depolarized potentials are central to their role in generating bursting dynamics with periods much longer than the membrane time constant. Given the power spectrum of the fluctuations observed, it seems possible that α1T channels play a similar role in the fast (~0.1-Hz) fluctuations of Drosophila circadian neurons. Remarkably, the expression of α1T also displays a circadian rhythm, with distinct phases in different groups of pacemaker neurons. Collectively, these results suggest that Itpr and α1T channel activity are together critical to produce clock regulation of rhythms in both slow and fast calcium activities of key pacemaker neurons (Liang, 2022).

PINK1 and Parkin regulate IP(3)R-mediated ER calcium release

Although defects in intracellular calcium homeostasis are known to play a role in the pathogenesis of Parkinson's disease (PD), the underlying molecular mechanisms remain unclear. This study shows that loss of PTEN-induced kinase 1 (PINK1) and Parkin leads to dysregulation of inositol 1,4,5-trisphosphate receptor (IP(3)R) activity, robustly increasing ER calcium release. In addition, CDGSH iron sulfur domain 1 (CISD1, also known as mitoNEET) functions were identifed downstream of Parkin to directly control IP(3)R. Both genetic and pharmacologic suppression of CISD1 and its Drosophila homolog CISD (also known as Dosmit) restore the increased ER calcium release in PINK1 and Parkin null mammalian cells and flies, respectively, demonstrating the evolutionarily conserved regulatory mechanism of intracellular calcium homeostasis by the PINK1-Parkin pathway. More importantly, suppression of CISD in PINK1 and Parkin null flies rescues PD-related phenotypes including defective locomotor activity and dopaminergic neuronal degeneration. Based on these data, it is proposed that the regulation of ER calcium release by PINK1 and Parkin through CISD1 and IP(3)R is a feasible target for treating PD pathogenesis (Ham, 2023).

This study provides a new insight into the mechanistic connection between the dysregulation of intracellular calcium homeostasis and PD pathogenesis induced by PINK1 or Parkin deficiency. PINK1 or Parkin KO mammalian cells exhibit increased IP3R activity, leading to increased ER calcium release and cytosolic calcium levels. CISD1, a substrate of Parkin, directly controls IP3R activity and ER calcium release, indicating that PINK1, Parkin, CISD1, and IP3R all function in the same essential pathway that regulates ER and cytosolic calcium homeostasis. Loss of CISD or treatment with the CISD inhibitor pioglitazone restores the elevated ER calcium release in PINK1 and Parkin null flies and fully rescues their PD-related phenotypes. Taken together, the increased IP3R activity and ER calcium release caused by PINK1 and Parkin deficiency are key to PD pathogenesis, all of which can be rescued by suppression of CISD1 activity (Ham, 2023).

In humans, there are three isoforms of CISD, CISD1, CISD2 (also known as ERIS, Miner1, NAF-1, WFS2, and ZCD2), and CISD3 (also called as MiNT). Among the three isoforms, CISD1 and CISD2 contain a single CDGSH domain and a transmembrane domain that facilitates their anchoring to the outer membrane of mitochondria and the ER, respectively. These two isoforms form homodimers within their respective organelles. CISD3 functions as a monomer and contains two CDGSH domains. CISD3 localizes specifically to the mitochondrial matrix. However, no isoforms exist in Drosophila CISD and this single protein shows sequence similarity with both human CISD1 and CISD2. Isoform CISD1 was selected for the experiment, as CISD1 is a much better substrate for Parkin E3 ligase compared to CISD2. Furthermore, it is well known that Parkin localizes to the mitochondria upon its activation and subsequently, ubiquitinates mitochondrial protein substrates. When subcellular localization of human CISD1/2 and Drosophila CISD proteins were observed, human CISD1 and Drosophila CISD were localized in the mitochondria; however, human CISD2 was localized in the ER. Considering these points, human CISD1 was selected as the mammalian counterpart of Drosophila CISD and the experiments were performed accordingly. Interestingly, it has been demonstrated previously that CISD2 is required for BCL2 to suppress IP3R activity. Thus, this study on the regulatory mechanism of IP3R activity through CISD1 is distinct from the earlier study on the regulation of IP3R activity by CISD2. Despite the structural and functional similarities between CISD1 and CISD2, the two proteins have distinct subcellular localizations and are involved in roles independent of one another, due to their interactions with different proteins. Overall, the results of the prior and current studies suggest that both CISD1 and CISD2 are modulators of IP3R activity, but they do so via their unique mechanisms that are distinctive of each other. (Ham, 2023).

Previous studies reported that CISD1 is involved in iron homeostasis and the downregulation of CISD1 causes iron accumulation and ROS production in mitochondria. In light of these effects, ROS levels were measured in PINK1 and Parkin WT or KO mammalian cells and Drosophila, and an increase was confirmed in ROS levels in PINK1 and Parkin KO MEF cells and Drosophila. An increase was also observed in ROS levels in CISD KD and KO flies, compared to control flies. Interestingly, CISD1/CISD KD or KO in PINK1 and Parkin KO cells and Drosophila resulted in similar ROS levels compared to PINK1 and Parkin KO cells and Drosophila. Furthermore, the increased ROS levels in PINK1 or Parkin KO cells and Drosophila were not restored when CISD1/CISD was knocked down or deleted. These results implicated that the rise in ROS levels induced by loss of CISD1/CISD is not directly involved in the rescue of PD phenotypes, which was observed in CISD1/CISD loss-of-function experiments (Ham, 2023).

The Fe-S binding capability of CISD1 may play a role in its interactions with IP3R. CISD1 has been reported to interact with several proteins, including CISD2, VDAC1, and transferrin receptor (TfR). CISD249, VDAC115,50, and TfR51 proteins have been shown to interact with each other, and this interaction has been implicated in the regulation of iron homeostasis, redox signaling, and Fe-S cluster synthesis in the mitochondria. However, whether the Fe-S binding motif of CISD1 plays an essential role in protein-protein interactions is unclear. Whether the functions of CISD1 related to Fe-S binding are important to regulate IP3R activity were tested, and it was identified that the cysteine 74 residue in the Fe-S binding motif (in the CDGSH domain) of CISD1 is critical for the interaction with IP3R1. However, it was also confirmed that the Fe-S binding motif of CISD1 binds with IP3R despite C72A substitution and CDGSH pentapeptide deletion mutations. Thus, it was postulate that the structural change in the Fe-S binding motif of CISD1 does not affect the binding between CISD1 and IP3R and that pioglitazone reduces the binding of CISD1 with IP3R regardless of the stability of Fe-S binding. Altogether, the Fe-S binding ability of CISD1 is not directly related to regulating IP3R activity (Ham, 2023).

Flies with either CISD RNAi or CISD KO exhibited lower ER calcium release and cytosolic calcium levels compared to mef2-GAL4 control flies. Upon crossing with CISD RNAi or CISD KO flies, PINK1 or Parkin null flies displayed a greater reduction in ER calcium release and cytosolic calcium levels than mef2-GAL4 control flies. This observation can be explained by the varying amounts of endogenous CISD in the flies. Notably, CISD RNAi flies exhibited significantly lower endogenous CISD amounts compared to the control flies, while PINK1 or Parkin KO flies presented higher levels. The elevated amount of endogenous CISD protein in PINK1 or Parkin KO flies contributes to the increased ER calcium release observed in these flies, while the reduced endogenous CISD protein in CISD RNAi or CISD KO flies results in a more significant decrease in ER calcium release compared to the control flies. Moreover, flies resulting from the crossing of PINK1/Parkin KO with CISD RNAi/CISD KO demonstrated lower endogenous CISD levels compared to the control flies, leading to a larger reduction in ER calcium release or cytosolic calcium levels. Collectively, these findings proposed that ER calcium release and cytosolic calcium levels are modulated proportionally to the amount of endogenous CISD protein present (Ham, 2023).

Defects in ER calcium homeostasis can also have profound effects on other organelles through physical contact sites, including the ER-mitochondria interconnections known as Mitochondria-associated membranes (MAMs). MAMs are enriched with the MCU complex in the inner mitochondrial membrane and IP3R on the ER membrane. MCU and IP3R are coupled via the glucose-regulated protein 75 (Grp75), which links IP3R to the VDAC1 on the outer mitochondrial membrane, establishing connections that allow calcium exchange between the ER and mitochondria. Interestingly, previous studies show that inhibition of MCU or VDAC1 partially rescues the PD phenotypes of PINK1- and Parkin-deficient flies, suggesting that the disruption of MAMs may alleviate PD pathogenesis. Previous studies also report that the level of MAM contacts was increased in cultured human fibroblasts from PD patients carrying PINK1 or Parkin pathogenic mutations and PINK1 and Parkin null mutant flies60. In addition, our present results demonstrate that CISD1 directly binds to and regulates IP3R activity, and CISD1 is localized at MAMs and the mitochondrial outer membrane12. These data therefore suggest that CISD1 and the PINK1-Parkin pathway are crucial for the formation and maintenance of MAM structure and ER-mitochondrial calcium transduction, which in turn are critical for mitochondria-related physiology and pathologic phenotypes including calcium-dependent metabolic changes, ROS production, mitophagy, mitochondrial permeability transition, and apoptosis (Ham, 2023).

Through extensive studies, it is understood that loss of PINK1 or Parkin impairs mitophagy and that defective mitophagy is one of the potential contributing factors to the onset of PD. Furthermore, a recent study has shown increased mitophagy in thoraces and neurons of CISD KO or KD Drosophila, and the reduced mitophagy in PINK1 or Parkin KO flies was alleviated by crossing them with CISD KO or KD flies. In the current study, it was observed that loss of CISD1/CISD reduced the elevated cytosolic calcium levels observed in PINK1 or Parkin KO cells and Drosophila. Intracellular calcium signaling is an important factor in mitophagy regulation. Nix, also known as BCL2 interacting protein 3 like (BNIP3L), exhibits biological activity at both the mitochondria and the ER. At the mitochondria, Nix functions as a selective autophagy receptor, facilitating the recruitment of LC3B71. In muscle, during a mitophagy response, Nix promotes ER-dependent calcium signaling to activate the mitochondrial fission regulator dynamin-related protein 1 (DRP1), indicating the contribution of Nix to mitophagy. During hypoxia, mitochondrial Lon protein promotes FUNDC1-ULK1-mediated mitophagy at the MAMs, which depends on its binding with mitochondrial Na+/Ca2+ exchanger (NCLX). This interaction stabilizes the FUNDC1-ULK1 at the MAMs and initiates the mitophagy by regulating calcium levels between the mitochondria and cytosol. This process occurs independently of PINK1 and Parkin. Furthermore, other calcium-sensitive proteins and pathways may also contribute to PINK1-Parkin-independent mitophagy. For example, CaMKII-AMP-activated protein kinase (AMPK) pathway has been implicated in the regulation of mitophagy. Activation of AMPK by CaMKII can promote mitophagy by phosphorylating and activating proteins involved in autophagy initiation. This suggests the possibility that mitophagy could be activated by the decreased cytosolic calcium levels in CISD1/CISD KO or KD cells and Drosophila. Collectively, the regulation of mitophagy by CISD1/CISD holds the potential to alleviate PD pathogenesis caused by loss of PINK1 or Parkin. However, further investigation is required to unravel the molecular mechanism underlying mitophagy regulation by CISD1 and its interplay with intracellular calcium signaling (Ham, 2023).

Although degeneration of DA neurons is known to occur in PD, how such selective neurodegeneration occurs remains unknown. The current results show that DA neuronal loss and locomotor impairments in PINK1 and Parkin KO flies can be rescued by adjusting ER calcium release, suggesting that ER and mitochondrial calcium dysregulation may cause selective DA neuronal death. Intracellular calcium signaling in DA neurons is extremely fine-tuned as it controls many cellular processes including gene transcription, membrane excitability, dopamine neurotransmitter secretion, and synaptic plasticity. Furthermore, energy production in neurons is tightly regulated by ER and mitochondrial calcium. DA neurons promote mitochondria calcium influx from the ER to stimulate OXPHOS and the production of ATP. This bioenergetic control system is costly, as enhancing OXPHOS in the absence of strong ATP demand leads to mitochondrial hyperpolarization, retrograde electron flux through the electron transport chain, and increased production of ROS. Therefore, continuous dysregulation of calcium homeostasis in DA neurons along with exposure to risk factors (i.e., aging, mitochondrial toxins, mutations) may selectively induce metabolic stress and mitochondrial damages, leaving DA neurons more vulnerable than other neuronal populations to death (Ham, 2023).

While the importance of calcium regulation in PD pathogenesis has been recognized, previous trials of calcium-related drugs had failed to improve symptoms in PD patients. This study proposes that pioglitazone, a thiazolidinedione (TZD) and antidiabetic drug, can alleviate PD pathogenesis. Though previous clinical studies have reported mixed results on the effectiveness of pioglitazone against PD, the results clearly demonstrate that feeding pioglitazone to flies rescues PD-related phenotypes induced by PINK1 or Parkin deficiency. In addition, pioglitazone treatment reverses the increased ER calcium release and cytosolic calcium levels in PINK1 and Parkin KO MEF cells. This study thus established that pioglitazone can specifically protect PD pathogenesis caused by dysregulation of intracellular calcium homeostasis, calling for future clinical studies of pioglitazone and its analogs to be conducted specifically on PD patients that harbor PINK1 or Parkin mutations (Ham, 2023).

IP3R mediated Ca2+ release regulates protein metabolism in Drosophila neuroendocrine cells: implications for development under nutrient stress

Successful completion of animal development is fundamentally reliant on nutritional cues. Adaptations for surviving nutritional loss are coordinated in part by neural circuits. As neuropeptides secreted by neuroendocrine (NE) cells critically modulate neural circuits (see Mapping peptidergic cells in Drosophila: where DIMM fits in), this study investigated NE cell function during development under nutrient stress. Starved Drosophila larvae exhibited reduced pupariation, if either insulin signaling or IP3/Ca2+ signaling, were down-regulated in NE cells. Moreover, an IP3R (Inositol 1,4,5-trisphosphate receptor) loss-of-function mutant displayed reduced protein synthesis, which was rescued by over-expression of either InR (insulin receptor) or IP3R in NE cells of the mutant, suggesting that the two signaling pathways may be functionally compensatory. Furthermore, cultured IP3R mutant NE cells, but not neurons, exhibited reduced protein translation. Thus cell-specific regulation of protein synthesis by IP3R in NE cells influences protein metabolism. It is proposed that this regulation helps developing animals survive poor nutritional conditions (Megha, 2017).

Nutritional poverty during development has long-lasting effects on the growth and behavior of an animal. Although under-nutrition causes overall body size to decrease, the brain grows to near-normal size, a process termed 'brain sparing'. This suggests unique mechanisms in neuronal tissues to weather nutritional stress. Drosophila is an attractive model system to uncover these mechanisms because larvae subjected to nutrient restriction exhibit 'brain sparing' and nutritional effects on larval-to-pupal development are easily monitored. Additionally, growth signaling pathways activated by dietary cues such as insulin receptor (InR) and TOR signaling, are conserved in Drosophila (Megha, 2017).

When starved, larval neural stem cells (NSCs) continue to proliferate by using an InR ortholog, Alk (Anaplastic lymphoma kinase). This study focuses on neuroendocrine (NE) cells, which, unlike NSCs, are differentiated and non-dividing. Importantly, neuropeptides released by NE cells modulate neural circuits that regulate processes associated with animal physiology and behavior, eventually influencing how animals adapt to external or internal stimuli. Crucially, NE cells produce peptide hormones that regulate feeding behavior and metabolism, processes required for larvae to complete development successfully (Megha, 2017).

IP₃R (Itp-r83A in Drosophila) is an endoplasmic reticulum (ER) channel that releases stored Ca²⁺ and acts downstream of G protein-coupled receptor activation. The ER-resident protein STIM (Stromal interaction molecule) conveys loss of stored Ca²⁺ to Orai (Olf186-F in Drosophila), a plasma membrane Ca²⁺ channel, thereby enabling store-operated Ca²⁺ entry (SOCE) from the extracellular milieu. SOCE occurs in both mammals. Thus, all three molecules - IP₃R, STIM and Orai - function during stimulus-dependent elevation of cytosolic Ca²⁺ that potentiates diverse signaling outcomes, depending on the cellular context (Megha, 2017).

Loss of IP₃R and STIM leads to obesity in adult Drosophila. Importantly, adults of a hypomorphic IP₃R mutant heteroallelic combination, itpr^ka1091/ug3 (hereafter: itpr^ku) exhibit obesity, starvation resistance and hyperphagia, which are all rescued by overexpression of IP₃R in NE cells. This adult metabolic phenotype prompted an investigation of the role of IP₃R and InR in NE cells during larval development (Megha, 2017).

In summary, IP₃R-mediated Ca²⁺ signaling helps maintain normal protein translation levels in NE cells, and this activity promotes systemic protein metabolism during larval development. On a nutrient-rich diet, loss of IP₃R signaling is not detrimental, because dietary cues maintain insulin/TOR signaling, and thereby keep protein levels normal for completing development. Under starvation, dietary cues are lost. IP₃/Ca²⁺ signaling possibly provides a nutrient-independent mechanism in order to maintain protein synthesis in cells essential to surviving nutrient stress, such as NE cells in which increased levels of cell surface receptors or neuropeptides might be required for modulating relevant neural circuits. As yet, there are no receptors or neuropeptides reported to be upregulated upon starvation in dimm⁺ NE cells, but there is precedence to suggest that they might exist. For example, in starved Drosophila, the receptor for short Neuropeptide F is upregulated in the antenna, and in starved mammals levels of agouti-related peptide, which affects appetite and feeding, are increased. A recent screen identified IP₃/Ca²⁺-coupled neuropeptide receptors, on glutamatergic neurons, that are required for larval adaptation to nutrient stress. Neuropeptides from NE cells that couple to such receptors might function during starvation in this model (Megha, 2017).

By focusing on animal development, this study integrates cellular observations and organismal phenotype. Therefore, it sets the framework for the discovery of mechanistic details of how stimulus-coupled increases in cytosolic Ca²⁺ can regulate protein synthesis in a cell-specific manner, and how that consequently regulates protein metabolism in the whole animal (Megha, 2017).

Functional cooperation between the IP3 receptor and Phospholipase C secures the high sensitivity to light of Drosophila photoreceptors in vivo

Drosophila phototransduction is a model system for the ubiquitous phosphoinositide signaling. In complete darkness, spontaneous unitary current events (dark bumps) are produced by spontaneous single Gqα activation, while single-photon responses (quantum bumps) arise from synchronous activation of several Gqα molecules. Recent studies have shown that most of the spontaneous single Gqα activations do not produce dark bumps, because of a critical phospholipase Cβ (PLCβ) activity level required for bump generation. Surpassing the threshold of channel activation depends on both PLC activity and cellular [Ca(2+)], which participates in light excitation via a still unclear mechanism. This study shows that in IP3 receptor (IP₃R)-deficient photoreceptors, both light-activated Ca(2+) release from internal stores and light sensitivity were strongly attenuated. This was further verified by Ca(2+) store depletion, linking Ca(2+) release to light excitation. In IP₃R-deficient photoreceptors, dark bumps were virtually absent and the quantum-bump rate was reduced, indicating that Ca(2+) release from internal stores is necessary to reach the critical level of PLCβ catalytic activity and the cellular [Ca(2+)] required for excitation. Combination of IP₃R knockdown with reduced PLCbeta catalytic activity resulted in highly suppressed light responses that were partially rescued by cellular Ca(2+) elevation, showing a functional cooperation between IP₃R and PLCβ via released Ca(2+). These findings suggest that in contrast to the current dogma that Ca(2+) release via IP₃R does not participate in light excitation, this study shows that released Ca(2+) plays a critical role in light excitation. The positive feedback between PLCβ and IP₃R found here may represent a common feature of the inositol-lipid signaling (Kohn, 2015).

In this study, in vivo light-response suppression was accompanied by reduced Ca2+ release from IP3-sensitive stores. In addition, the rate of spontaneously produced dark bumps, which is highly sensitive to Gqα-dependent PLCβ catalytic activity and cellular Ca2+ level, was virtually abolished in IP₃R-deficient photoreceptors. This dark-bump elimination indicates that the suppressed Ca2+ release from IP3-sensitive stores underlies the suppressed catalytic activity of PLCβ, leading to suppressed light response in IP₃R-deficient photoreceptors. Further evidence that the suppressed light response arises from inhibition of Ca2+ release from IP3-sensitive stores came from blocking the Ca2+ pump by Tg, which mimicked the phenotype of the IP₃R-deficient photoreceptors in WT flies. The above findings indicate that IP₃R-mediated Ca2+ release has a critical role in light excitation of Drosophila photoreceptors. The combination of the PLCβ mutant norpAH43 with IP₃R-deficient photoreceptors, which synergistically suppressed the light response, strongly suggests that there is functional cooperation between the IP₃R and PLCβ in generation of the light response (Kohn, 2015).

It has been previously shown that an increase in cytosolic Ca2+ participates in light excitation as evidenced by enhancement of the light response following photo release of caged Ca2+ at the rising phase of the light response. The target of Ca2+ action has not been entirely resolved. PLCβ is an important target for Ca2+ action and the regulation of its catalytic activity by Ca2+ has been thoroughly investigated. These studies showed that the positive charge of Ca2+ is used to counterbalance local negative charges formed in the active site during the course of the catalytic reaction. Accordingly, Ca2+ performs electrostatic stabilization of both the substrate and the transition state, thus providing a twofold contribution to lower the activation energy of the enzyme reaction (Kohn, 2015).

The following model explains how functional cooperation between the IP₃R and PLCβ via the released Ca2+ operates and secures quantum-bump production: absorption of a single photon, which induces activation of several PLCβ molecules, is initially insufficient at resting Ca2+ levels to reach the critical level of PLCβ activity required for TRP/TRPL channel activation. Nevertheless, the IP3 molecules produced by the given PLCβ activity are able to activate the nearby IP₃Rs, mobilize Ca2+ from the stores, and elevate PLCβ activity above the threshold required for TRP/TRPL channel activation. In addition, the released Ca2+ may also reduce the threshold of TRP/TRPL channel activation and allow bump generation. According to this model, the following enzymatic reactions may explain the current findings. Each Gqα-activated PLCβ has low catalytic activity due to the relatively low (<160 nM) resting Ca2+ concentration in the cytosol. In addition, each activated PLCβ remains active for only a short (approximately several tens of milliseconds) time due to the GTPase-activating protein activity of PLCβ that causes a rapid hydrolysis of Gqα-GTP followed by inactivation of PLCβ. The initial low catalytic activity of PLCβ is apparently below the threshold required for activation of the TRP and TRPL channels, but this low activity still results in hydrolysis of PIP2 producing IP3. Since there are no IP3 buffers in the microvilli and the IP3 degradation time is relatively slow (~1 s), the produced IP3 molecules diffuse fast along the microvillus at an estimated time of ~1 ms along 1 microm long microvillus and bind to IP₃R located at the nearby submicrovillar cisternae (SMC; the photoreceptors' extensions of smooth ER). IP₃R channels residing at the SMC, which are large channels with high sensitivity for IP3 and thus can be activated at low PLCβ activity, open and release Ca2+ juxtaposed to the base of the microvillus. The released Ca2+ steeply raises the local Ca2+ concentration, probably to the microM range, because of the very small aqueous volume of the microvillus and the relatively large local Ca2+ elevation via the release mechanism (Kohn, 2015).

Accordingly, a single IP₃R channel can release ∼104 Ca2+ ions in 1 ms channel opening and Ca2+-induced Ca2+ release mechanism is a property of the IP₃R channels and of the ryanodine receptors, which reside in the ER. Ca2+ released via IP₃R of the WT SMC diffuse back toward the activated PLCβ and the TRP/TRPL channels in the microvillus. Although Ca2+ diffuses ∼20-fold slower than IP3 due to strong buffering, the diffusion constant strongly depends on Ca2+ concentration. Accordingly, at ~250 μM the Ca2+ diffusion coefficient is as large as that of IP3. Once a single TRP channel is activated, the large Ca2+ influx through this channel is sufficient to facilitate the rest of the active PLCβ molecules or reduce the threshold for TRP/TRPL channel activation in this microvillus and produce a bump that reflects activation of the entire microvillus . When there is abnormally low Ca2+ release via the IP₃R because of low IP₃R expression levels (IP₃R-RNAi), there is not enough Ca2+ to increase PLC activity or to reduce TRP activation threshold, and activated PLC in this microvillus does not produce a bump, leading to abnormally low frequency of dark bumps (Kohn, 2015).

The invasive whole-cell recording technique, which was used in previous studies and avoided Ca2+ buffering of the pipette solution, most likely resulted in abnormally elevated cytosolic Ca2+ concentration, which also allowed the Ca2+ pump to keep the stores full. This artificially elevated cytosolic [Ca2+] together with the constitutive Ca2+ leak from the full stores, bypassed the need to mobilize Ca2+ via functional IP₃R to facilitate PLCβ activity and reach its critical catalytic activity level needed to activate the TRP/TRPL channels. In the present study in the intact eye, a significant reduction in light-response amplitude was observed when the IP₃R level was reduced. Furthermore, when cellular [Ca2+] was reduced by prolonged extracellular EGTA application, the light response of the IP₃R-deficient flies was further suppressed. Moreover, when using invasive patch-clamp whole-cell recordings without Ca2+ buffering of the pipette solution, no significant difference between WT and IP₃R-deficient flies was observed, as found in the previous study. However, when pipette Ca2+ was reduced with EGTA, the phenotype of reduced light excitation was observed in both reduced quantum-bump frequency as well as in macroscopic light-response suppression. Unlike quantum bumps, dark bumps were virtually eliminated even without buffering the pipette Ca2+ in IP₃R-deficient flies, indicating that in the dark the IP₃R-deficiency led to abnormally low cytosolic [Ca2+], possibly due to reduced Ca2+ leak from stores leading to cellular [Ca2+] below the critical level required for PLC activation observed in WT flies. Alternatively, the positive feedback between the released Ca2+ and PLC may function at single PLC molecules. Hence the nominal pipette Ca2+ is not sufficient to allow PLC activity to pass the threshold of channel activation, but the released Ca2+ via IP₃R activation together with pipette Ca2+ allows PLC activity to pass this threshold and generate dark-bump (Kohn, 2015).

There is a striking functional similarity between, the cerebellar Purkinje cell (PC) proteins of the IP signaling and Drosophila photoreceptors, but the link of cerebellar mGluR1 receptor to TRPC3 activation is not clear. Interestingly, in PC neurons, stromal interaction molecule 1 (STIM1) was proved an essential regulator of Ca2+ level in neuronal endoplasmic reticulum Ca2+ stores. Accordingly, STIM1-specific deletion caused impairments in slow synaptic current and cerebellar motor behavior. Strikingly, refilling empty Ca2+ stores through increased Ca2+ level in the cytosol partially rescued the phenotype of the stim1 knock-out mice, reminiscent of the rescue of the phenotype of the IP₃R-deficient fly by artificially elevated cytosolic Ca2+. Thus, the facilitatory role of released Ca2+ on PLC in light excitation of Drosophila photoreceptors represents an essential mechanism that operates in other PI systems (Kohn, 2015).

Earlier studies of Inositol 1,4,5,-tris-phosphate receptor

The release of intracellular Ca2+ is an intermediate step in many cellular signaling processes. The soluble messenger known as inositol 1,4,5 triphosphate) acts to release intracellular Ca2+ from the intracellular store maintained within the endoplasmic reticulum. The InsP3 receptor (Itp-r83A), here called InsP3R, is a Ca2+ channel that releases intracellular Ca2+ in response to InsP3. This signal transduction pathway is used in processes as diverse as the responses to hormones, growth factors and neurotransmitters, as well as in various vertebrate sensory systems, such as olfaction, gustation and vision (Hasan, 1992 and references).

Before describing the biology of the Drosophila InsP3 receptor, a short diversion will provide a closer look at Ca2+ signaling and intracellular Ca2+ channels. There are two common motifs for Ca2+ release from intracellular storage depots into the cytosol for signal transduction, depending on whether the cells are excitable or non-excitable.

1. Nonexcitable cells: Here the slow inositol (1,4,5)-trisphosphate (InsP3)-mediated pathway predominates. Two receptor classes (the G protein-coupled receptor class of seven transmembrane-spanning receptors and the receptor tyrosine kinases), acting indirectly, release InsP3, which in turn acts as an intracellular second messenger by binding to the specialized tetrameric InsP3 receptor (InsP3R) that spans the endoplasmic reticular membrane and triggers release of Ca2+. G protein-coupled receptors activate phospholipase Cbeta while receptor tyrosine kinases stimulate phospholipase Cgamma to convert the lipid known as phosphatidylinositol (4,5)-biphosphate into the small soluble messenger InsP3 and the membrane bound lipid diacylglycerol. Diacylglycerol serves to activate another signaling cascade by activating the enzyme Protein kinase C (Clapham, 1995).

2. Excitable cells: The Ca2+ release from intracellular storage depots into the cytosol also occurs in excitable cells containing voltage-dependent Ca2+ channels that enable these cells to dramatically increase cytosolic Ca2+ levels. In excitable cells, Ca2+ entering through voltage-dependent Ca2+ channels may directly activate ryanodine receptors, the excitable cell counterparts to the InsP3 receptor, to release Ca2+ from intracellular stores (Clapham, 1995).

Molecular cloning and characterization of the Inositol 1,4,5-trisphosphate receptor in Drosophila melandogaster

The InsP3R of Drosophila (Itp-r83A) is expressed ubiquitously and is involved in multiple aspects of development: it is required for embryonic and larval development, is involved in muscle development and is implicated in chemosensory functions. The InsP3R was identified based on its homology to mammalian InsP3R. The protein is highly conserved and shows the complex structure of a transmembrane protein, with identifiable ligand binding domains, ATP binding domains, a conserved presumptive Ca2+ channel and conserved cysteine residues also found in the ryanodine receptor, but the fly protein shows no Protein kinase A phosphorylation sites, indicating that it is not regulated by the PKA pathway as is the case for the mammalian homologs (Yoshikawa, 1992).

Inositol 1,4,5-trisphosphate receptor-mediated Ca2+ signaling in the brain

Numerous modulatory signals have been postulated for mammalian InsP3R receptor: the best characterized is known as type 1 InsP3R (Insp3r1). Calmodulin, cyclic GMP-dependent protein kinase and Calcium/calmodulin dependent protein kinase II have all been proposed as modifiers of the activity of the channel protein in addition to ATP, Protein kinase A and ligand. Thus, other second messenger transduction cascades, i.e., cyclic AMP (via PKA), diacyl glycerol (via PKC), and cyclic GMP (via cyclic GMP-dependent protein kinase) all converge on a single modulatory and transducing domain found in the middle portion of the receptor. In addition, alternative splicing results in variations in regulatory regions of the protein. The protein is differentially distributed throughout the adult brain and show preferences for distinct subcellular compartments. Besides being present in cerebellar Purkinje cells, InsP3R1 is present in the olfactory tubercle, cerebral cortex, CA1 region of the hippocampus, caudate-putamen, molecular layer of the cerebellum and choroid plexis (Furuichi, 1995).

InsP3 receptor is essential for growth and differentiation but not for vision in Drosophila

Mutants in the Drosophila InsP3R gene arrest development at the second larval instar; many larval tissues are observed that have not proliferated normally in these mutants. Expression during the embryonic phase in mesoderm and sensory organ precursor cells suggests that Insp3R could be involved in embryonic development but that no embryonic phenotype is observed because of the presumed presence of a maternal transcript. If maternal transcripts play a role in embryonic development, then making germ line clones lacking all InsP3R should lead to early lethality. When such clones are generated no viable eggs or embryos are recovered. It is therefore likely that InsP3R larval lethality is a maternal-effect phenomenon due to the preponderance of maternally contributed message. It is concluded that InsP3R is essential for cell proliferation, growth and differentiation. More specific information about the roles of Insp3R in Drosophila development await analysis of mutant clones where the maternal contribution is diminished (Acharya, 1997).

Availability of Insp3R mutants has allowed an assessment of the role of InsP3R in Drosophila photoreceptor signaling. One of the many unanswered questions stimulating research in the field of invertebrate photoreceptor biology has to do with the gating mechanism of light-activated channels. In Drosophila photoreceptor neurons, light activation of rhodopsin activates a Gqalpha, which in turn activates a Phospholipase C (PLC) encoded by the norpA gene. PLC catalyzes the breakdown of PIP2 into InsP3 and diacyl glycerol; these are thought to lead to the eventual opening (and modulation) of the TRP and TRPL membrane channels and the generation of a receptor potential. It has been proposed that the TRP channel functions as a store-operated channel (opened by Ca2+ released from intracellular stores) that responds to a capacitative calcium entry signal. According to this model, light-activation of rhodopsin results in the production of InsP3, which would lead to the emptying of InsP3-sensitive internal calcium stores and the subsequent gating of plasma membrane channels. It has been thought that TRP channels and their vertebrate homologs represent examples of store-operated channels (Acharya, 1997 and references).

However, TRP (transient receptor potential) and TRPL light-activated ion channels, known to be involved in the light sensitivity of the Drosophila retina, do not localize in close proximity to the internal calcium stores, thus eliminating the possibilty of gating by protein-protein interaction via conformation coupling (Niemeyer, 1996). It has also been shown that all light-induced increases in Ca2+ are entirely dependent on the influx of extracellular Ca2+ (Ranganathan, 1994). These results suggest that internal InsP3-sensitive stores are not involved in activation of the light responses (for a contrary result see InsP3R and visual phototransduction). To catagorically test the involvement of InsP3R in light activation, clones of Insp3R negative photoreceptors were generated in an otherwise wild-type background. Mutant photoreceptors and surrounding tissues undergo retinal degeneration, consistent with defective growth of mutant larval tissue. Intracellular recordings were carried out to study light responses in control and mutant photoreceptor cells. InsP3R mutant photoreceptors are indistinguishable from wild-type controls in sensitivity, kinetics of activation and deactivation, and adaptation. Thus, no evidence was found for a role of the InsP3R in Drosophila phototransduction, and models invoking InsP3-induced calcium release in the activation of this pathway are likely to be invalid (Acharya, 1997).

REGULATION

InsP3R and visual phototransduction

Drosophila phototransduction is an important model system for studies of inositol lipid signaling. Light excitation in Drosophila photoreceptors depends on phospholipase C, because null mutants of this enzyme do not respond to light. Surprisingly, genetic elimination of the apparently single inositol tris-phosphate receptor (InsP₃R) of Drosophila has no effect on phototransduction. This led to the proposal that Drosophila photoreceptors do not use the InsP3 branch of phospholipase C (PLC)-mediated signaling for phototransduction, unlike most other inositol lipid-signaling systems. To examine this hypothesis the membrane-permeant InsP₃R antagonist 2-aminoethoxydiphenyl borate (2-APB) was applied; this has proved to be an important probe for assessing InsP₃R involvement in various signaling systems. The effects of 2-APB on Xenopus oocytes was examined. It was found that 2-APB is efficient at reversibly blocking the robust endogenous InsP3-mediated Ca2+ release and store-operated Ca2+ entry in Xenopus oocytes at a stage operating after production of InsP3 but before the opening of the surface membrane Cl- channels by Ca2+. Next it was demonstrated that 2-APB is effective at reversibly blocking the response to light of Drosophila photoreceptors in a light-dependent manner at a concentration range similar to that effective in Xenopus oocytes and other cells. It was also shown that 2-APB does not directly block the light-sensitive channels, indicating that it operates upstream in the activation of these channels. The results indicate an important link in the coupling mechanism of vertebrate store-operated channels and Drosophila TRP channels: this involves the InsP3 branch of the inositol lipid-signaling pathway (Chorna-Ornan, 2001).

To examine whether 2-APB has an effect on Drosophila phototransduction, advantage was taken of the ability to examine its effect on the intact animal using the ERG. The ERG is the sum of the electrophysiological response to light of the entire retina in vivo. Application of 2-APB to the intact eye by two pulses of pressure injections below the cornea almost abolishes the response to light ~10 min after application. The inhibitory effect is partially reversible after ~15 min and almost completely recovered after an additional 45 min (Chorna-Ornan, 2001).

To investigate whether inhibition of the ERG originates from blocking the light response of the photoreceptor cells, the effect of 2-APB as investigated using whole-cell patch clamp recordings from single photoreceptor cells. The amplitudes of the light-induced currents (LICs) are similar in all responses. 2-APB affects the response to light only slightly. A small but significant slow inward current is observed in the dark in most cells, after application of 2-APB. Additional light pulses applied during the slow inward current results in a drastic reduction in response amplitude, which eventually leads to total abolition of the response to light even when very intense white light is applied. The desensitization produced by 2-APB cannot be a secondary consequence of Ca²⁺ influx, which may accompany the slow and small inward current induced in the dark by 2-APB because 2-APB inhibits the LIC also at concentrations <50 µM, which do not induce any detectable inward current. In some experiments 2-APB was applied at zero external Ca²⁺ and it was found that application of 2-APB combined with intense light at zero external Ca²⁺ causes rapid deterioration of the response to light and spontaneous openings of the light-sensitive channels. To prevent these effects and still examine the effect of 2-APB at zero external Ca²⁺, 2-APB was applied and its effects tested using dimmer light. Under these conditions a large suppression of the LIC was observed ~13 min after application of 2-APB, thus indicating that Ca²⁺ influx cannot explain the suppression of the LIC (Chorna-Ornan, 2001).

A pronounced suppression of the response to light by 2-APB can be observed within 3 min, provided that intense light is used to test its effect. This raises the possibility that its effect is light-dependent. To test this possibility, the amplitudes of the LIC to dim and to more intense orange light pulses as a function of time was tested, during application of 2-APB to the pipette. At both test lights the amplitude of the LIC declines with time, but the decline is much faster when stronger test light is used, indicating that the effect of 2-APB is light-dependent, suggesting that inhibition by 2-ABP requires that the InsP₃R is in its activated form.When a relatively large concentration of 2-APB is used, in addition to the slow inward current mentioned above, facilitation of the response to light is observed before the blocking action is evident. This transient facilitation is not observed at low concentration of 2-APB or when dim lights are used (Chorna-Ornan, 2001).

If 2-APB is a specific inhibitor of the InsP₃R, its application would not affect the light-sensitive channels. To test this notion, activation of the light-sensitive channels directly and not via the phototransduction cascade is required. Recently, it has been found that Drosophila TRP and TRPL channels can be activated in the dark by inducing metabolic stress after elimination of NAD from the pipette solution combined with depletion of ATP caused by illumination. The mitochondrial uncoupler dinitrophenol (DNP) is also a very potent reagent for direct activation of the TRP and TRPL channels. In all cases of such activation, no significant effect of 2-APB on the constitutive current is observed (Chorna-Ornan, 2001).

Thus 2-APB is an efficient inhibitor of Drosophila phototransduction, operating both in intact cells and in isolated ommatidia, and this inhibition partially reverses when the inhibitor is removed. The great interest in 2-APB arises from its reported function as a powerful probe for assessing involvement of InsP₃ receptors in cell signaling. Indeed, the reversible inhibition of InsP₃-induced current oscillations in Xenopus oocytes strongly supports previous studies showing that 2-APB blocks Ca²⁺ release from InsP₃-sensitive Ca²⁺ stores. Furthermore, the failure of 2-APB to block the Ca²⁺-activated surface membrane Cl^- channels while it suppresses the InsP₃-induced activity indicates that the action of 2-APB is confined to the signaling stages downstream of InsP₃ production, but upstream of the Ca²⁺ release-activated processes (Chorna-Ornan, 2001).

The mode of action and the identity of the specific ER protein with which 2-APB interacts are not clear. Previous studies suggest that the action of 2-APB is on the InsP₃ branch and not the DAG branch of inositol lipid signaling, however, it has not been possible to eliminate the possibility that 2-APB targets channels other than the InsP₃ receptor. For Drosophila phototransduction a major question has been whether the InsP₃ branch of the inositol lipid signaling is necessary for excitation. The present results and previous studies on the characteristics of 2-APB inhibition provide evidence for the hypothesis that Drosophila photoreceptors use the InsP₃ branch of the inositol lipid-signaling pathway for excitation consist with previous studies on the Limulus and bee photoreceptors. In addition, the observation that a high concentration of 2-APB can release Ca²⁺ from InsP₃-sensitive stores provides further evidence that Ca²⁺ release can mediate light excitation in Drosophila. A possible explanation for the release of Ca²⁺ by 2-APB is that it binds to the open state of the InsP₃ receptor and locks it in the open state. So far, demonstration of a significant light-induced release of Ca²⁺ from ER stores, and its participation in excitation was hampered as a result of the small size of the putative InsP₃-sensitive Ca²⁺ stores of Drosophila and the difficulty of introducing exogenous chemicals to the highly compartmentalized region of these stores. Importantly, the small inward current induced in the dark by 2-APB and the transient facilitation of the LIC provide significant support for the hypothesis that Ca²⁺ release can induce excitation. Recent evidence indicates that 2-APB can indeed act as a partial activator of the InsP₃ receptor, inducing some release of Ca²⁺ (Chorna-Ornan, 2001).

The conclusion that Drosophila phototransduction uses the InsP₃ branch of the inositol-lipid-signaling pathway for light excitation is not consistent with two recent reports. The Drosophila genomic sequence identifies only one InsP₃ receptor gene in the Drosophila genome, and mutations in this gene are lethal (Acharya, 1997; Venkatesh, 1997; Raghu, 2000). However, it is possible to generate mutant photoreceptors in mosaic patches by inducing mitotic recombination in heterozygotes. Intracellular recordings from photoreceptors in such mosaic patches reveal no differences in light response from wild-type, leading the authors to conclude that the InsP₃ receptor played no role in phototransduction (Acharya, 1997). A more detailed study using mosaic eyes homozygous for a deficiency of the InsP₃ receptor of Drosophila confirmed by RT-PCR, Western blot analysis, and immunocytochemistry, has shown that the InsP₃ receptor is indeed eliminated without any effect on the response to light as tested by several functional tests using patch-clamp whole cell recordings (Raghu, 2000). In experiments on vertebrate DT40 cells, knock-out of all three known InsP₃ receptors does not prevent what appears to be normal functioning of store-operated channels (Sugawara, 1997). However, it has been suggested that these cells could be expressing an N-terminal portion of the InsP₃ receptor perhaps involved in coupling to plasma membrane entry channels but not functional as a Ca²⁺ store release channel. The reconciliation of these apparently conflicting data are likely to shed important new light on the mechanism of activation of light-sensitive channels. One possibility is that a second, still undiscovered, novel InsP₃ receptor exists because sequencing of the Drosophila genome has not been completed, the heterochromatin (about a third of the genome) has not been sequenced yet because of technical difficulties. Another possibility is that 2-APB interacts with a protein that can associate with the InsP₃ receptor but is not the InsP₃ receptor itself. Such a target may play an obligatory role in mediating the coupling process that results in activation of light-sensitive channels. It is also possible that other as yet unidentified InsP₃-responsive proteins exist that may be targets for 2-APB. The important principle finding is that 2-APB blocks activation of mammalian, Xenopus, and likely all vertebrate SOCs, and in addition it blocks activation of mammalian TRP channels as well as the TRP channels mediating the light induced current in Drosophila. However, in each case, 2-APB does not appear to directly modify channel activity. These observations have allowed the authors to conclude that there is a fundamentally conserved step in the activation process for each of these channels. In vertebrate cells, the activation appears to use input from the InsP₃ receptor, whereas in Drosophila phototransduction, the input from known InsP₃ receptors is not a requirement for channel activation. Whether a different InsP₃ binding protein mediates the inositol lipid-signaling branch in Drosophila phototransduction remains a further important question to address (Chorna-Ornan, 2001).

Compensation of inositol 1,4,5-trisphosphate receptor function by altering sarco-endoplasmic reticulum calcium ATPase activity in the Drosophila flight circuit

Ionic Ca²⁺ functions as a second messenger to control several intracellular processes. It also influences intercellular communication. The release of Ca²⁺ from intracellular stores through the inositol 1,4,5-trisphosphate receptor (InsP₃R) occurs in both excitable and nonexcitable cells. In Drosophila, InsP₃R activity is required in aminergic interneurons during pupal development for normal flight behavior. By altering intracellular Ca²⁺ and InsP₃ levels through genetic means, it is now shown that signaling through the InsP₃R is required at multiple steps for generating the neural circuit required in air puff-stimulated Drosophila flight. Decreased Ca²⁺ release in aminergic neurons during development of the flight circuit can be compensated by reducing Ca²⁺ uptake from the cytosol to intracellular stores. However, this mode of increasing intracellular Ca²⁺ is insufficient for maintenance of flight patterns over time periods necessary for normal flight. This study suggests that processes such as maintenance of wing posture and formation of the flight circuit require InsP₃ receptor function at a slow timescale and can thus be modulated by altering levels of cytosolic Ca²⁺ and InsP_3. In contrast, maintenance of flight patterns probably requires fast modulation of Ca²⁺ levels, in which the intrinsic properties of the InsP₃R play a pivotal role (Banerjee, 2006; full text of article).

The goal of this study was to understand the contribution of the intracellular Ca²⁺ release channel, the InsP₃R, in the development and function of neural circuitry. From studies in Drosophila, it has been shown that flight is critically dependent on normal activity of the InsP₃R in aminergic interneurons during pupal development suggesting that InsP₃-mediated Ca²⁺ release is required during normal development of the flight circuit. Mutants in the gene encoding the InsP₃R (itpr) in Drosophila exhibit a range of defects including altered wing posture, increased spontaneous firing, and loss of rhythmic flight patterns in response to an air puff stimulus. Together, these phenotypes contribute to the loss of flight behavior observed in itpr mutants. An obvious question that arises from these studies is whether the multiple phenotypes arise as a consequence of a single early neuronal dysfunction. Alternately, each phenotype could be attributable to independent events requiring InsP₃R activity at individual and distinct steps of flight circuit development. This study has addressed these questions by altering intracellular Ca²⁺ signals in flight-deficient itpr mutants through genetic means. In the first instance, a dominant mutant for the sarco-endoplasmic reticular Ca²⁺-ATPase (SERCA) pump was introduced into itpr mutant backgrounds. SERCA (Calcium ATPase at 60A) is responsible for pumping cytosolic Ca²⁺ into the endoplasmic reticulum (ER) store and thus maintaining the intracellular concentrations of Ca²⁺ both in the cytosol and in the ER store. The second class of mutants are in genes that encode (1) the α subunit of the heterotrimeric G-protein Gαq and (2) phospholipase Cβ (PLCβ) which generates InsP₃. These mutants are expected to reduce InsP₃ levels and thus reduce the activity of the InsP₃R, in circumstances in which InsP₃ is generated by the activation of seven transmembrane receptors. The results show that neuronal phenotypes of itpr mutants arise from at least two distinct classes of intracellular Ca²⁺ signals. One class can be modulated by the strength of InsP₃ signaling and the rate of Ca²⁺ uptake into the ER. The second class appears to depend primarily on the Ca²⁺ release properties of the InsP₃ receptor (Banerjee, 2006).

Calcium-stores mediate adaptation in axon terminals of olfactory receptor neurons in Drosophila

In vertebrates and invertebrates, sensory neurons adapt to variable ambient conditions, such as the duration or repetition of a stimulus, a physiological mechanism considered as a simple form of non-associative learning and neuronal plasticity. Although various signaling pathways, as cAMP, cGMP, and the inositol 1,4,5-triphosphate receptor (InsP3R) play a role in adaptation, their precise mechanisms of action at the cellular level remain incompletely understood. In Drosophila odor-induced Ca2+-response in axon terminals of olfactory receptor neurons (ORNs) has been shown to be related to odor duration. In particular, a relatively long odor stimulus (such as 5 s) triggers the induction of a second component involving intracellular Ca2+-stores. A recently developed in-vivo bioluminescence imaging approach was used to quantify the odor-induced Ca2+-activity in the axon terminals of ORNs. Using either a genetic approach to target specific RNAs, or a pharmacological approach, this study showed that the second component, relying on the intracellular Ca2+-stores, is responsible for the adaptation to repetitive stimuli. In the antennal lobes (a region analogous to the vertebrate olfactory bulb) ORNs make synaptic contacts with second-order neurons, the projection neurons (PNs). These synapses are modulated by GABA, through either GABAergic local interneurons (LNs) and/or some GABAergic PNs. Application of GABAergic receptor antagonists, both GABAA or GABAB, abolishes the adaptation, while RNAi targeting the GABABR (a metabotropic receptor) within the ORNs, blocks the Ca2+-store dependent component, and consequently disrupts the adaptation. These results indicate that GABA exerts a feedback control. Finally, at the behavioral level, using an olfactory test, genetically impairing the GABABR or its signaling pathway specifically in the ORNs disrupts olfactory adapted behavior. Taken together, these results indicate that a relatively long lasting form of adaptation occurs within the axon terminals of the ORNs in the antennal lobes, which depends on intracellular Ca2+-stores, attributable to a positive feedback through the GABAergic synapses (Murmu, 2011).

This study provides evidence that the bioluminescent (GFP-aequorin) Ca²⁺-sensor is sensitive enough to monitor the Ca²⁺-response following various protocols (duration and repetition-frequency) of odor application. 1 s of odor induces a response which does not significantly decrease if repeated every 5 min, whereas a longer stimulus, such as 5 s, is sufficient to induce a decrease in response following repeated odor stimulations (adaptation). Similarly, using a 5 s odor stimulation and increasing the frequency of repetition to 1-min intervals also induces, in an odor specific manner, a faster adaptation. It was also demonstrated that prolonged odor application (up to 2 min) generates a sustained Ca²⁺-response within the ORN axon terminals, indicating that the ORNs are capable of responding as long as the odor is presented, of even longer. This work also indicates that the GFP-aequorin probe is not a limiting factor for the detection of the Ca²⁺-activity. These physiological results (reduction of the Ca²⁺-activity according to prolonged/sustained odor duration and/or odor repetition) are consistent with previous studies which report that adaptation depends both on the duration of a stimulus and on the frequency of its repetition (Murmu, 2011).

Different physiological approaches, based either on fluorescence brain imaging or electrophysiological techniques have previously reported odor-induced activity in different interconnected neurons in the antennal lobes of different invertebrate model organisms, including honeybees with the goal of deciphering the neural odor code. However, except for one study performed in locusts, which indirectly described a form of adaptation, long-lasting forms of adaptation within ORNs such as that described in this study have not been reported. This is likely due to the experimental design of these previous studies, which either generally took into account the odor-induced signal solely after the response was stabilized (generally after about 5 successive odor applications), or used a shorter odor stimulation duration (< = 1 s), which as demonstrated in this study, is not sufficient to induce detectable and reliable adaptation. Additionally, others have relied on extracellular recordings of the sensillae of the antennae which reflects the activity occurring in the cell-bodies of the ORNs. In this study, monitoring the axon terminals of the ORNs, 5 s odor stimulations, repeated at 5-min intervals, induced a relatively long-lasting adaptation that resembles in term of kinetics, the long-lasting adaptation (LLA) reported in ORNs in salamanders. Indeed and interestingly, the recovery time (15 min for spearmint and octanol and 30 min for citronella) occurs over a similar time scale in salamander ORNs (which are different from the long-lasting olfactory adaptation described in C. elegans. However, in contrast to long-lasting adaptation, which was reported in isolated ORNs, the adaptation described in this study seems to rely on different mechanisms, since it is sensitive to a 'feedback control' provided by GABAergic synaptic transmission within the antennal lobes (Murmu, 2011).

In Drosophila, mutants lacking InsP₃R are defective in olfactory adaptive behavior. In vertebrates, different forms of olfactory adaptation have also been reported in the ORNs. First, this study shows in Drosophila that an adaptation mechanism occurs in axon terminal of the ORNs in the antennal lobes. Second, using two independent approaches, pharmacological and genetic, it was shown that odor-induced specific adaptation relies principally on InsP₃R and RyR. When these two different receptors are blocked or knocked-down, although some difference (variability) can be observed between different conditions, overall the odor-induced Ca²⁺-response no longer adapts or is severely affected. More specifically, it seems that the lack of adaptation is due to the non-induction of the 'second delayed and slow rising component' of the Ca²⁺-response, which is triggered in particular and specific conditions: when the duration of an odor stimulation is relatively long (1 s does not induce it, while 5 s induces an important second component. Alternatively, the second component of the response is also induced and visible particularly on the first and to a lesser extent, on the second odor applications, especially when the odor is successively repeated. This second component gradually vanishes with sequential repetition. That is, adaptation is not directly due to a decrease in the response, but rather indirectly to a defect in presynaptic Ca²⁺-increase, due to a lack of triggering release of intracellular Ca²⁺-stores, normally occurring in the first and successive responses following either a sufficiently strong (long stimulus) or repeated stimuli. These results suggest that one of the major intracellular mechanisms of adaptation depends on internal Ca²⁺-stores. In brief, the intracellular mechanism was blocked that allows the cell to adapt to long lasting or repetitive stimuli. Interestingly, in mammals, in hippocampal CA3 pyramidal neurons, intracellular Ca²⁺-stores, which are controlled by InsP₃R and/or RyR at the presynaptic terminal, have been previously implicated in neurotransmitter release as well as in synaptic plasticity (Murmu, 2011).

In vertebrates, neuronal plasticity related to odor representation occurs at the synapse between the ORNs and the second-order neurons in the olfactory bulb glomeruli, a region analogous to the invertebrate antennal lobes. At this synapse, signal transmission is modulated presynaptically by several mechanisms, a major one being via the metabotropic GABA_B receptors. This suppresses presynaptic Ca²⁺-influx and subsequently transmitter release from the receptor neurons terminal. At least two kinds of presynaptic inhibition (intra- and interglomerular) are mediated by GABA_B receptors. Intraglomerular presynaptic inhibition seems to control input sensitivity, while interglomerular presynaptic inhibition seems to increase the contrast of sensory input (although the two studies addressing this question in-vivo show contradictory results). In Drosophila, a similar mechanism seems to occur, as interglomerular presynaptic inhibition, mediated by both ionotropic and metabotropic receptors on the same axon terminal of the ORNs, mediate gain control mechanism, serving to adjust the gain of PN in response to ORN stimulation (Olsen, 2008). Yet another study has suggested that GABA_B but not GABA_A receptors are involved in presynaptic inhibition (Root, 2008) yielding a contradiction. In this study, by monitoring the Ca²⁺-release from the axon terminals of ORNs, in experimental conditions that generate a long-lasting form of adaptation, it was shown that GABAergic synaptic transmission plays a role in adaptation. Both ionotropic GABA_AR antagonists, bicuculline and picrotoxin, block partially or completely the Ca²⁺-response, while, CGP54626, a metabotropic GABA_BR antagonist, also blocks the adaptation, albeit not completely. It should be mentioned that application of picrotoxin per se induces a strong transient Ca²⁺-release within the axon terminals of the ORNs, even without odor application. This 'transient release effect' likely disturbs the resting state of the neurons, which probably accounts for the important reduction observed in the amplitude of the odor-induced response. Nevertheless, these results suggest that both types of GABA receptors (A and B) are involved in adaptation. Moreover, as proposed by the study of Olsen and Wilson (2008), it cannot be ruled-out that ORNs also express different subtypes of GABA_AR (homo- and/or heteromultimers), since the results showed that picrotoxin and particularly bicuculline, two distinct inhibitors of GABA_AR, block adaptation. Another possibility is that the effect of the two GABA_AR antagonists results from the blockage of GABA_AR on other neurons in the antennal lobes, as the LNs or certain PNs (which have not yet been demonstrated). Lastly and unfortunately, this pharmacological approach does not allow distinguishing by which precise neurons this GABAergic-dependent adaptation is mediated. With the goal of clarifying precisely in which neurons GABAergic transmission acts, the metabotropic GABA_BR (GABA_BR2-RNAi) or its signaling pathway (UAS-PTX) were blocked directly within ORNs. This yields defects in long-lasting adaptation for several conditions, seemingly in an odor specific manner. Therefore, although GABAergic effects have been described in ORNs of both Drosophila and mammals, to support 'feedback inhibition', this study reports that in different experimental conditions such as a long odor duration (5 s) and/or repetition of the stimulus, it also participates in the adaptation process. Indeed, the results suggest that GABA signaling support a positive (excitatory) feedback control instead of an inhibitory feedback, as formerly reported by other studies. Though these results seem to be contradictory, some explanations can be provided. First, as aforementioned, the experimental conditions are different: this study used a relatively high odor concentration with relatively long odor duration (5 s). In addition, recordings were taken immediately from the first odor application and the successive one, while in the experimental protocol of certain studies, the odor is generally presented several times (priming) before the beginning of recording. Consequently, it seems that these previous studies were performed on already adapted ORNs. This implies that the neuronal network in the antennal lobes was already stimulated, and therefore its dynamics was probably already modified, since as described in this study, an important effect occurs immediately after the first odor application. Moreover, GFP-aequorin allows monitoring, in continuity over a long time period, the intracellular level of calcium with high sensitivity to [Ca²⁺] (from ~ 10^-7 to 10^-3). In addition, although it is not possible to precisely assign which glomeruli are activated, this approach allows visualizing simultaneously the odor-induced Ca²⁺-activity from the entire antennal lobes (the overall depth). Therefore, the outcome of the overall response of the antennal lobes is being monitored, instead of the response from single or a few glomeruli. Finally, in vertebrates it has been reported that in certain experimental conditions, GABA could be excitatory, although this contradiction cannot yet be precisely explained. Furthermore, it seems that a given synapse can display inhibitory effects under one protocol and an excitatory effect with another. Notably, it has been reported that a short stimulation of GABA is inhibitory, while during a long stimulation, the GABA effect can switch from inhibitory to excitatory. Interestingly, this particular 'switching effect' could potentially explain the current 'contradictory' situation reported in this study: in the current experimental conditions, in which a relatively long odor stimulus (5 s) was used, GABA generates an excitatory effect, whereas in previous studies based on short (<1 s) odor stimuli, GABA seems to be inhibitor. This difference in the duration of stimuli could perhaps account for such inverted or 'switching effects' (Murmu, 2011).

To explore the behavioral and functional consequences of disturbing the GABAergic signaling pathway, flies were studied with a GABA_BR2 (RNAi) ORN-specific genetic knockdown, as well those with a component of its signaling pathway, the G-protein, blocked by the pertussis toxin. Both groups of flies present strong behavioral deficits, as adaptation-disrupted flies are not able to discern between odors and air after 5-min of exposure to odor. Interestingly, control flies reverse their choice preferring odor after a 5-min pre-exposure (adaptation) suggesting that in these experimental conditions, the meaning of the odor changes in the fly's adapted state. These results are consistent with previous studies suggesting that adaptation could serve to extend the operating range of sensory systems over different stimulus intensities. In other terms, adaptation modifies the sensitivity (threshold) to the odor, as previously reported in different organisms, such as C. elegans and vertebrates including humans. This phenomenon is similar to that in other sensory modalities, as in visual system, where light adaptation in photoreceptors sets the gain, allowing vision at both high and low light levels. As previously reported, odors could be repulsive (at high concentrations) or attractive (at low concentrations). In the current experimental conditions in control flies the odors are repulsive. However, after 5-min of preexposure, the flies adapt to this odor concentration, and when tested at the same concentration odors are then likely only weakly perceived and therefore might correspond to an attractive 'weak-odor concentration'. In a former study in similar experimental conditions, it was reported that the flies are attracted by each of these three odors for a weak odor concentration. Interestingly, reverse odor preference has already been reported in C. elegans, resulting from presynaptic changes involving a receptor-like guanylate cyclase (GCY-28) via the diacylglycerol/protein kinase C pathway. Finally, the fact that without pre-exposure all groups of flies preferred the control arm and were repelled by the odorants indicates that the odor acuity of these flies is intact. In other words, odor-adaptation and not odor-acuity is affected in each of these groups of flies. These results strengthen the idea that odor perception and adaptation are indeed two distinct and separable processes (Murmu, 2011).

This study has demonstrated that the adaptation process occurring specifically in the axon terminals of the ORNs depends on intracellular Ca²⁺-stores, through InsP₃ and ryanodine receptors. Moreover, evidence is provided that this Ca²⁺-release requires synaptic transmission, since it does not occur when the cholinergic receptors are blocked (α-bungarotoxin experiment). It also requires a feedback control through GAB A, since blocking GABA_B signaling within ORNs prevents or strongly affects adaptation, suggesting that a local neuronal network mediated by GABAergic neurons is involved (for a more complete overview, see the schematic model of synaptic interactions within the antennal lobes. In complement to the brain imaging data, knocking-down the metabotropic GABA_BR2, or its signaling pathway specifically in the ORNs, yields olfactory functional behavioral deficits. These results, combined with the results of blocking the InsP₃R or RyR suggest that a crucial olfactory integration process that can be ascribed to a form of neuronal plasticity and/or short-term memory occurs directly in the ORNs immediately after the first odor application or during a prolonged odor application. Thus, this effect could resemble the long-lasting form of odor adaptation described previously at the cellular and systems levels in vertebrates, including humans. By extension, it is hypothesized that in humans, the well-known 'odor-specific transient functional anosmia' following a prolonged odor exposure, which results from an adaptation, may also rely on intracellular Ca²⁺-stores (Murmu, 2011).

Activation of PLC by an endogenous cytokine (GBP) in Drosophila S3 cells and its application as a model for studying inositol phosphate signalling through ITPK1

Using immortalized [³H]inositol-labelled S3 cells, this study has demonstrated that various elements of the inositol phosphate signalling cascade are recruited by a Drosophila homologue from a cytokine family of so-called GBPs (growth-blocking peptides) (see The pathways of inositol phosphate metabolism). HPLC analysis revealed that dGBP (Drosophila Growth-blocking peptide) elevates Ins(1,4,5)P₃ levels 9-fold. By using fluorescent Ca²⁺ probes, it was determined that dGBP initially mobilizes Ca²⁺ from intracellular pools; the ensuing depletion of intracellular Ca²⁺ stores by dGBP subsequently activates a Ca²⁺ entry pathway. The addition of dsRNA (double-stranded RNA) to knock down expression of the Drosophila Ins(1,4,5)P₃ receptor almost completely eliminates mobilization of intracellular Ca²⁺ stores by dGBP. Taken together, the results of the present study describe a classical activation of PLC (phospholipase C) by dGBP. The peptide also promotes increases in the levels of other inositol phosphates with signalling credentials: Ins(1,3,4,5)P₄, Ins(1,4,5,6)P₄ and Ins(1,3,4,5,6)P₅. These results greatly expand the regulatory repertoire of the dGBP family, and also characterize S3 cells as a model for studying the regulation of inositol phosphate metabolism and signalling by endogenous cell-surface receptors. Therefore a cell-line (S3^ITPK1) was created in which heterologous expression of human ITPK (inositol tetrakisphosphate kinase) was controlled by an inducible metallothionein promoter. It was found that dGBP-stimulated S3^ITPK1 cells did not synthesize Ins(3,4,5,6)P₄, contradicting a hypothesis that the PLC-coupled phosphotransferase activity of ITPK1 [Ins(1,3,4,5,6)P₅+Ins(1,3,4)P₃→Ins(3,4,5,6)P₄+Ins(1,3,4,6)P₄] is driven solely by the laws of mass action. This conclusion represents a fundamental breach in understanding of ITPK1 signalling (Zhou, 2012).

Stimulus-dependent activation of PLC (phospholipase C) hydrolyses PtdIns(4,5)P₂ to generate two intracellular messengers: diacylglycerol which activates protein kinase C, and Ins(1,4,5)P₃, which binds to specific receptors {IP3R [Ins(1,4,5)P₃ receptor]; itpr in Drosophila} that gate intracellular Ca²⁺ stores. This is a ubiquitous signalling response that regulates diverse aspects of cellular biology in almost all animal cell types. As such, it is an intensively studied area of cell biology. Additionally, research into the metabolism of Ins(1,4,5)P₃ has spurred the development of a largely separate signalling industry: the study of an elaborate network of interconnected metabolites, many of which have multiple biological functions. Thus the discovery that PLC is activated by an extracellular agonist endows that agent with a large number of new signalling activities (Zhou, 2012).

Drosophila melanogaster is a genetically-tractable eukaryotic model that has proved useful in unraveling the complexities of many aspects of Ca²⁺ signalling. For example, experiments with Drosophila mutants have demonstrated that the fly's single itpr gene is important for a range of physiological response. Cultured Drosophila cells have also been used to study the receptor-dependent activation of Ins(1,4,5)P₃ production and Ca²⁺ mobilization. However, the range of opportunities offered by this model organism have not yet been fully exploited for studies into the function and agonist-regulated metabolism of the other inositol phosphate signals. Such studies, which typically rely on analysing cells that have been radiolabelled with [³H]inositol during several days of proliferation, are best suited to immortalized cell lines. Such cells have been derived from Drosophila. However, there is limited insight into the nature of peptides that act through endogenous receptors to activate PLC in these immortalized cells. The application of these cells to inositol phosphate research has therefore been restricted. To bypass this problem, some groups have heterologously expressed exogenous receptors into immortalized Drosophila cells. A more physiologically relevant model is described that utilizes endogenous receptors: the S3 imaginal-disc cell line, in which this study shows that PLC is activated by a recently discovered insect cytokine, dGBP [Drosophila GBP (growth-blocking peptide)](Zhou, 2012).

dGBP is a member of a large family of insect cytokines. These peptides range from 19-30 amino acid residues in length, and are produced upon proteolytic cleavage of longer prepropeptides. The first GBP to be identified was the factor responsible for the reduction in the rate of growth of lepidopteran larvae following their colonization by the larvae of a parasitic wasp, Costesia kariyai. The endocrinological perturbation that up-regulates GBP synthesis in the host stops it from forming the sclerotized pupal cuticle that would otherwise prevent the parasitic larvae from emerging. The GBP family also regulate morphogenesis, cell proliferation and innate immune responses, for example by stimulating plasmatocyte adhesion and spreading However, to date little progress has been made in understanding the molecular mechanisms of action of these cytokines. Recently, the discovery of dGBP has revealed that this peptide acts through c-Jun N-terminal kinase to regulate gene expression. However, in view of the multi-functionality of the GBP family, it has remained probable that these peptides recruit additional signalling pathways. The present study adds substantially to dGBP's signalling repertoire by demonstrating its ability to activate multiple facets of the PLC-dependent inositol phosphate/Ca²⁺ cascade (Zhou, 2012).

An increased insight into the molecular actions of insect cytokines such as dGBP is of interest in itself, but the importance of this field of research goes beyond the goal of expanding understanding of insect physiology. Knowledge of the roles of GBPs in immune responses in pest insects that impair human health or reduce crop yield may lead to the development of improved control programs. There is also considerable evolutionary conservation of innate immunity genes, pathways and effector mechanisms. Research into these defense mechanisms in Drosophila may ultimately improve understanding of human immune responses. Indeed, the identification of the human homologue of the Toll receptor was initially prompted by the discovery that this protein mediates antifungal immune responses in Drosophila (Zhou, 2012).

Another goal of the present study was to determine whether the discovery that dGBP activates PLC in S3 cells could be exploited to explore the signalling activities of inositol phosphate metabolizing enzymes from higher animals. In pursuit of this idea, it is noted that the Drosophila genome does not encode a homologue of the mammalian ITPK1 (inositol tetrakisphosphate 1-kinase). Therefore S3 cells offer a rare example of a model for characterizing any gain-of-function that might arise from the heterologous expression of human ITPK1. For example, the phosphorylation of Ins(1,3,4)P₃ by ITPK1 in mammals has been proposed to be the rate-limiting step in the synthesis of Ins(1,3,4,5,6)P₅ and InsP₆. However, there continue to be differences of opinion as to the relative importance of this pathway compared with the alternative ITPK1-independent route to Ins(1,3,4,5,6)P₅ and InsP₆. This study aimed to address this debate by studying the impact of ITPK1 upon Ins(1,3,4,5,6)P₅ and InsP₆ synthesis in basal and PLC-activated S3 cells. Furthermore, ITPK1 has additional significance to mammalian cell signalling because this enzyme controls the synthesis of Ins(3,4,5,6)P₄, which regulates the conductance of the Cl⁻ channel/transporter ClC-3 (chloride channel, voltage-sensitive 3). In this way, ITPK1 helps regulate a number of physiological processes, such as salt and fluid secretion, insulin secretion, and neurotransmission. However, it has proved difficult to characterize the mechanism by which Ins(3,4,5,6)P₄ synthesis is accelerated following PLC activation. To explain this phenomenon, previous studies put forward a hypothesis that is based on the laws of mass action. This study proposes that an elevated rate of phosphorylation of Ins(1,3,4)P₃ to Ins(1,3,4,6)P₄ by ITPK1 is coupled through an unusual phosphotransferase reaction to an increased rate of dephosphorylation of Ins(1,3,4,5,6)P₅ to Ins(3,4,5,6)P₄. The present study has tested this hypothesis by determining whether PLC-dependent Ins(3,4,5,6)P₄ synthesis could be heterologously introduced into S3 cells by the expression of ITPK1 (Zhou, 2012).

A central conclusion in the present study is that PLC is activated by dGBP, a member of a multi-functional family of insect cytokines. The Drosophila homologue of the GBP family was identified only quite recently, and it has been shown to stimulate transcription of antimicrobial peptides by activation of c-Jun NH₂-terminal kinase. However, the GBP family is known to regulate other diverse aspects of insect cell physiology, including larval growth, morphogenesis and cell proliferation. Therefore it was thw goal of this study to search for additional signalling pathways that dGBP might recruit, in order to gain a more complete understanding of the mechanisms that underlie the various actions of these cytokines. The demonstration that dGBP activates PLC in S3 cells opens up a number of directions for further research into insect physiology. PLC-mediated hydrolysis of PtdIns(4,5)P₂ yields diacylglycerol, which activates protein kinase C, and Ins(1,4,5)P₃, which binds itpr and gates intracellular Ca²⁺ stores. Furthermore dGBP also promotes increases in a number of other inositol phosphates, several of which have their own distinct cellular functions (Zhou, 2012).

This study used several experimental approaches to ascertain that, in S3 cells, dGBP mobilizes Ca²⁺ from intracellular Ins(1,4,5)P₃-releasable stores as a consequence of PLC activation. First, HPLC and chemical analyses were used to determine that dGBP promotes the increases in Ins(1,4,5)P₃ and related metabolites that are typical of agonist-dependent stimulation of PLC activity in mammalian cells. Secondly, fluorescent Ca²⁺ probes were used to demonstrate that dGBP promotes the biphasic Ca²⁺ response that is typically observed following the activation of PLC-coupled receptors: depletion of intracellular Ca²⁺ stores, which then drives enhanced Ca²⁺ entry. Thirdly, dsRNA was used to knock down itpr expression, whereupon the ability of dGBP to mobilize intracellular Ca²⁺ stores was almost completely eliminated (Zhou, 2012).

The various elements of the inositol phosphate cascade that are activated by dGBP have independent functions, and so may contribute to the peptide's multi-functionality. The Ins(1,4,5)P₃-mediated mobilization of Ca²⁺ probably mediates several physiological responses. The dGBP-mediated increases in Ins(1,3,4,5)P₄ levels may also have some signalling significance. Changes in Ins(1,4,5,6)P₄ levels also promise to be important, in the light of evidence that Ins(1,4,5,6)P₄ regulates gene transcription. Ins(1,3,4,5,6)P₅ levels in wild-type S3 cells were also elevated by 70% within 2 min of dGBP addition. Such a sizable Ins(1,3,4,5,6)P₅ response to PLC activation is not usually observed in mammalian cells. However, in the murine F9 teratocarcinoma cell line, PLC activation has been associated with large increases in Ins(1,3,4,5,6)P₅ levels, which regulate key aspects of the β-catenin signalling pathway. In F9 cells, Ins(1,3,4,5,6)P₅ synthesis occurs primarily through direct phosphorylation of Ins(1,4,5)P₃ by IPMK (inositol polyphosphate multikinase). This is also the case in Drosophila. Thus S3 cells may be of value for further exploring these signalling functions for Ins(1,3,4,5,6)P₅ and their contribution to the physiology of dGBP (Zhou, 2012).

The genetic tractability of Drosophila S3 cells is also an advantage when using them to explore the mechanisms of agonist-dependent regulation of the metabolism and functions of the inositol phosphate family. The present study exploited the observation (Seeds, 2004) that the Drosophila genome is itpk1-null, and so human ITPK1 was heterologously expressed in S3 cells in order to gain insight into the signalling functions of that particular kinase. These experiments yielded three significant new conclusions with regards to the actions of ITPK1 (Zhou, 2012).

First, data was obtained that are relevant to the ongoing debate concerning the degree to which ITPK1 contributes to Ins(1,3,4,5,6)P₅ synthesis in mammalian cells. Specifically, it was found that the expression of ITPK1 in S3 cells yielded several-fold increases in the steady-state levels of Ins(1,3,4,5,6)P₅ and one of its metabolites, Ins(1,4,5,6)P₄. Nevertheless, in both wild-type S3 and S3^ITPK1 cells, the ratio of Ins(1,3,4,5,6)P₅ to InsP₆ was relatively low compared with that in mammalian cells. These results suggest that factors other than ITPK1 expression itself contribute to the synthesis of the relatively large quantities of Ins(1,3,4,5,6)P₅ that are typically present in mammalian cells. For example, there is evidence that much of the mammalian cell's Ins(1,3,4,5,6)P₅ resides in a metabolically-resistant pool. The mechanism by which Ins(1,3,4,5,6)P₅ transfers into that pool in mammalian cells may be key to the accumulation of that inositol phosphate at relatively high levels (Zhou, 2012).

Secondly, the sizable accumulation of Ins(1,2,3,4,6)P₅ in PLC-activated S3^ITPK1 cells is also of interest. That result is attributed to the relatively high levels of Ins(1,3,4,6)P₄ successfully out-competing the lower levels of Ins(1,3,4,5,6)P₅ for phosphorylation by IP5K; such competition has previously been shown in vitro. Such a phenomenon could help explain a previously puzzling observation that the InsP₆ pool in rat-1 cells is largely unaffected when Ins(1,3,4,5,6)P₅ synthesis was compromised by knockdown of IPMK expression. An ITPK1-dependent alternative pathway of InsP₆ synthesis in rat-1 in cells could solve that problem: phosphorylation of Ins(1,3,4)P₃ into Ins(1,3,4,6)P₄, then into Ins(1,2,3,4,6)P₅ which then can be converted into InsP₆ (Zhou, 2012).

The third, and arguably the most important, observation to emerge from these studies with ITPK1, was the discovery that the phosphotransferase activity of ITPK1 is not sufficient by itself to recapitulate PLC-dependent synthesis of Ins(3,4,5,6)P₄, an intracellular signal that serves multiple biological roles through its regulation of Cl⁻ channel activity. Thus the results of the present study counter a previous hypothesis that Ins(3,4,5,6)P₄ levels are solely regulated by the mass-action effects of substrate supply that are exerted upon ITPK1. This outcome was unexpected, because previous structural and metabolic data had indicated that the phosphotransferase activity of human ITPK1 is inherently well-adapted to regulation by mass-action effects in vitro. Perhaps the absence of Ins(3,4,5,6)P₄ in PLC-activated S3^ITPK1 cells is because metabolic compartmentalization prevents ITPK1 from accessing Ins(1,3,4,5,6)P₅. It is also possible that there is a yet to be discovered mechanism, not active in S3 cells, by which the phosphotransferase activity of ITPK1 might be activated relative to its kinase activity. A potential mechanism might involve covalent modification of ITPK1, such as by its phosphorylation or acetylation. In any case, the present study indicates that the regulation of the Ins(3,4,5,6)P₄ signalling cascade is more complex than hitherto was appreciated. As the number of physiological responses that are regulated by Ins(3,4,5,6)P₄ increases, so the need to unravel these mechanisms becomes more urgent (Zhou, 2012).

In previous work, the heterologous expression in yeast of inositol phosphate kinases of animal and plant origin uncovered new aspects of inositol phosphate metabolism and signalling. However, the use of immortalized Drosophila cells as a host offers several advantages over the yeast model, including their more direct relevance to mammalian systems: unlike yeasts, flies have an Ins(1,4,5)P₃-releasable Ca²⁺ pool. Genetic manipulation by dsRNA is also cheap and effective in cultured fly cells. Furthermore, the demonstration that dGBP stimulates PLC adds value to S3 cells as a resource for further studies into receptor-dependent regulation of inositol phosphate function and metabolism. This work with ITPK1 offers an example of how that model can usefully be exploited. Finally, this study adds substantially to the repertoire of the GBP family by demonstrating its ability to activate multiple facets of the PLC-dependent inositol phosphate signalling cascade (Zhou, 2012).

Drosophila larval to pupal switch under nutrient stress requires IP3R/Ca2+ signalling in glutamatergic interneurons

Neuronal circuits are known to integrate nutritional information, but the identity of the circuit components is not completely understood. Amino acids are a class of nutrients that are vital for the growth and function of an organism. This study reports a neuronal circuit that allows Drosophila larvae to overcome amino acid deprivation and pupariate. Nutrient stress is sensed by the class IV multidendritic cholinergic neurons. Through live calcium imaging experiments, this study shows that these cholinergic stimuli are conveyed to glutamatergic neurons in the ventral ganglion through mAChR. IP3R-dependent calcium transients in the glutamatergic neurons convey this signal to downstream medial neurosecretory cells (mNSCs). The circuit ultimately converges at the ring gland and regulates expression of ecdysteroid biosynthetic genes. Activity in this circuit is thus likely to be an adaptation that provides a layer of regulation to help surpass nutritional stress during development (Jayakumar, 2016).

DEVELOPMENTAL BIOLOGY

Embryonic

As seen by northern analysis Ins3PR is present in 0 to 6 hour early embryos, but this expression cannot be detected by in situ hybridization. The first distinct hybridization is seen at approximately 10.5 hours as a segmental pattern in cells of the ventral epidermis lying along the intersegmental furrows and in posterior epidermal cells. In late stage-13 embryos, this expression intensifies and extends to the head region within cells of the gnathal buds, which lie ventral to the stomodeal opening. The spatiotemporal appearance of these cells in the head region suggests that they are likely to be progenitors of anterior sense organs. In stage 14 embryos, the anterior expression extends dorsally to cells in the clypeolabrum, while the lateral epidermal expression decreases to barely detectable levels. By stage 15, all detectable expression is localized to the anterior head region, presumably in primordia of anterior sense organs. As development progresses, some of these sense organs can be identified as two pairs of dorsal structures that lie along the pharynx walls and a more lateroventral pair, which is likely to be the labial organ. Finally, at stage 17, only the labial organ expresses this gene (Hasan, 1992).

Expression of the InsP3R appears in early mesoderm in stage 9 embryos. This expression is enhanced as differentiation of the mesoderm occurs into somatic and visceral layers at stage 10. Maximum expression is observed in both the somatic and visceral mesoderm at stage 13. Two rows of cardioblasts in the dorsal midline also stain strongly at stage 13. Staining of the visceral mesoderm can be observed surrounding the gut but later this disappears. This disappearance of the InsP3R can be roughly correlated with differentiation of the myoblast clusters into muscle fibers in the somatic muscles (Raghu, 1995).

In the cephalic region, anti-InsP3R antibody staining appears in the developing procephalic mesoderm at stage 9 and begins intensifying in stage 10. At stage 13 staining in the pharyngeal muscles is very distinctive in the anterior regions. Other clusters of muscles in the cephalic region also appear to stain strongly at stage 14. No InsP3R protein could be detected in any of the sense organs (Raghu, 1995).

Distribution of Ryanodine and IP₃R receptors and the endoplasmic reticulum Ca²⁺-ATPase

The biochemistry, distribution and phylogeny of Drosophila ryanodine (RyR) and inositol triphosphate (IP₃R) receptors and the endoplasmic reticulum Ca²⁺-ATPase (SERCA) have been characterized by using binding and enzymatic assays, confocal microscopy and amino acid sequence analysis. [³H]-ryanodine binding in total membranes is enhanced by AMP-PCP, caffeine and xanthine, whereas Mg²⁺, Ruthenium Red and dantrolene are inhibitors. [³H]-ryanodine binding showed a bell-shaped curve with increasing free [Ca²⁺], without complete inhibition at millimolar levels of [Ca²⁺]. [³H]-IP₃ binding is inhibited by heparin, 2-APB and xestospongin C. Microsomal Ca²⁺-ATPase activity is inhibited by thapsigargin. Confocal microscopy demonstrates abundant expression of ryanodine and inositol triphosphate receptors and abundant Ca²⁺-ATPase in Drosophila embryos and adults. Ryanodine receptor is expressed mainly in the digestive tract and parts of the nervous system (Vázquez-Martínez, 2003).

To characterize RyR, IP₃R and SERCA protein distribution in fly tissues, fluorescent compounds specific for RyR (TX-R-BODIPY-ryanodine), IP₃R (FL-Heparin), and thapsigargin-sensitive SERCA (FL-BODIPY-thapsigargin) were used. The signal associated with fluorescent ryanodine is present in practically all cells of early embryos. The label localizes mainly to the cytoplasm of cells. The fluorescent ryanodine signal observed in older embryos (stages 15-17) clearly shows RyR at higher concentrations in the digestive tract. The label associated with Drosophila SERCA and IP₃R in early embryos is also present in practically all cells. In late embryos, label is present in nearly all tissues and is distributed more homogeneously than ryanodine signals. Labeling for all three fluorescent compounds is seen in tissues derived from all germinal layers: ectoderm (epidermis), mesoderm (muscle), and endoderm (digestive tract). As seen for the ryanodine receptor, higher magnification views of cells labelled with thapsigargin and heparin also show cytoplasmic staining. Co-localization of these compounds with fluorescent ryanodine illustrates that SERCA and RyR are highly coexpressed in the digestive tract, whereas coexpression of IP₃R and RyR is evenly distributed. Label observed in these experiments is specific, since coincubation with excess ryanodine, heparin or thapsigargin abolishes labeling for ryanodine, for heparin, and for thapsigargin (Vázquez-Martínez, 2003).

Adult tissues were stained with BODIPY TR-X Ryanodine, BODIPY FL-Thapsigargin and FL-Heparin. A generalized RyR expression was observed, with higher levels in the digestive tract, muscle, and adult optic lobe and retina. Label is cytoplasmic, as in embryos. Staining is seen in tissues of ectodermal origin (nervous system, mesodermal origin (indirect flight and leg muscles), and endodermal origin (digestive tract). Staining for heparin was seen also in practically all adult tissues, and more homogeneous in levels than RyR. Most tissues show extensive colocalization of both labels. Colocalization of fluorescent ryanodine with fluorescent thapsigargin was coincidental (Vázquez-Martínez, 2003).

Published in situ hybridization data offer a very restricted expression pattern, with higher levels in late embryos in prospective antenno-maxillary complex (a complex comprising the dorsal and terminal organs) and the labial organ. In contrast, the data reveal widespread expression of the IP₃R protein: consistent with mutant defects, expression of the protein occurs at all stages and tissues. There is also high expression in the digestive tract, again consistent with the requirement for intracellular Ca²⁺ dynamics in visceral muscle function and consistent with immunocytochemistry data. Staining has less marked regional differences than RyR staining. The data support the idea that IP₃R protein, like RyR protein, is also contributed maternally and/or is expressed at levels not readily detectable by in situ hybridization at all stages and tissues. This underscores the value of examining both transcript and protein expression data, although some caution should be exercised, since heparin may label other proteins besides IP₃R protein (Vázquez-Martínez, 2003).

Larval

In both the wing and leg imaginal discs weak, though specific, staining is found associated with myoblasts. In the eye-antennal disc, however, similar staining is found to be associated with the developing photoreceptor neurons that lie just behind the morphogenetic furrow. No specific staining was found to be associated with the antennal portion of the eye-antennal disc. InsP3R staining can be detected in the dorsolongitudinal indirect flight muscles. Compared to the level of myoblast staining in the imaginal discs, these myoblasts stain more strongly. Among the myoblasts seen at this stage, the ones that lie close to the larval templates appear to stain more strongly than the migrating myoblasts. Unlike what is seen in embryonic muscles development, where the InsP3R disappears after myoblast proliferation and fusion is over, in pupal development InsP3R expression is not turned off and the antigen can be seen to be associated with the nuclei of the fused myoblasts (Raghu, 1995).

Neuronal expression of the InsP3R in developing antennae occurs after differentiation in late pupae. At 86 hours after puparium formation, well after specification of neural precursors in the antenna which occurs about 14 hours after puparium formation, InsP3R can be seen in differentiated antennal neurons. Expression is also seen in cells of nonneuronal identity. These cells form antennal muscles (Raghu, 1995).

The appearance of InsP3R in taste neurons of pupal labellar hairs was compared with the ability of the developing labellar hairs to respond to a taste stimulus. Similar to what is seen with the antennal neurons, the taste neurons at the base of the labellar hairs do not express the InsP3R until 86 hours after puparium formation. Mechanosensory response can be detected from 84 hour pupae by bending the shaft of the bristle. Only by 96 hours after puparium formation was the taste response to 0.1 M NaCl fully developed. Thus the appearance of the InsP3R in the taste neurons of the proboscis correlates well with the time when the taste hairs are developing the ability to respond to taste stimuli (Raghu, 1995).

Adult

In the adult, InsP3R mRNA can be detected in the cortex of the central brain and the antenna. Expression is also detectable in eyes and legs (Hasan, 1992).

Strong antennal staining of InsP3R protein is seen in sections through the second and third antennal segment. Staining is also observed at the base of sensory hairs present in the maxillay palp and the proboscis. The maxillary palp acts as a second olfactory organ in Drosophila, while the proboscis is primarily a taste organ. In the proboscis the staining is clearly associated with sensory neurons present at the base of taste hairs. These data suggest that IP3 may act as a second messenger during both olfactory and taste transduction in Drosophila, in a pathway similar to what has been shown to exist in vertebrates. In the brain the antigen is restricted to cell bodies and is probably nuclear-membrane-associated or perinuclear in localization. This is also the case in the eye, where the staining is associated with the nuclei of each photoreceptor cone and pigment cell. Perinuclear/nuclear-membrane staining is also seen in the muscles of the head (Raghu, 1995).

EFFECTS OF MUTATION

Histological examination of various tissues from 4-day-old InsP3R knockout mutant larvae (corresponding to late third larval instar of control larvae) reveals a pronounced defect in the growth of larval tissues and imaginal progenitor cells. For instance, brain neuroblasts and midgut progenitor cells can be recognized but do not undergo any significant proliferation. As a result, the size of the brain corresponds to that of control late-first or early-second instar larvae. Salivary glands and principal midgut epithelial cells also undergo little endoreplication of the DNA. During normal development, larval cells perform up to 10 rounds of endoreplication, resulting in large cells with highly polytenic chromosomes. Nuclei in knockout larvae undergo little endoreplication. InsP3R mutants also show dramatic defects in the development of imaginal discs; they are rudamentary at best, with little if any cell proliferation or differentiation (Acharya, 1997).

The Drosophila light-sensitive channels TRP and TRPL are prototypical members of an ion channel family responsible for a variety of receptor-mediated Ca(2+) influx phenomena, including store-operated calcium influx. While phospholipase Cbeta is essential, downstream events leading to TRP and TRPL activation remain unclear. The role of the InsP₃ receptor (InsP₃R) was examined by generating mosaic eyes homozygous for a deficiency of the only known InsP₃R gene in Drosophila. Absence of gene product was confirmed by RT-PCR, Western analysis, and immunocytochemistry. Mutant photoreceptors undergo late onset retinal degeneration; however, whole-cell recordings from young flies demonstrate that phototransduction is unaffected, since quantum bumps, macroscopic responses in the presence and absence of external Ca(2+), light adaptation, and Ca(2+) release from internal stores are all normal. Using the specific TRP channel blocker La(3+) it was demonstrated that both TRP and TRPL channel functions are unaffected. These results indicate that InsP₃R-mediated store depletion does not underlie TRP and TRPL activation in Drosophila photoreceptors (Raghu, 2000).

The inositol 1,4,5-trisphosphate (IP3) receptor is an intracellular calcium channel that couples cell membrane receptors, via the second messenger IP3, to calcium signal transduction pathways within many types of cells. IP3 receptor function has been implicated in development, but the physiological processes affected by its function have yet to be elucidated. In order to identify these processes, mutants in the IP3 receptor gene (itpr) of Drosophila were generated and their phenotype during development was studied. All itpr mutant alleles are lethal. Lethality occurs primarily during the larval stages and is preceded by delayed molting. Insect molting occurs in response to the periodic release of the steroid hormone ecdysone which, in Drosophila, is synthesized and secreted by the ring gland. The observation of delayed molting in the mutants, coupled with the expression of the IP3 receptor in the larval ring gland led the authors to examine the effect of the itpr alleles on ecdysone levels. On feeding ecdysone to mutant larvae, a partial rescue of the itpr phenotype is observed. In order to assess ecdysone levels at all larval stages, transcripts were examined of an ecdysone-inducible gene, E74; these transcripts are downregulated in larvae expressing each of the itpr alleles. Thus, disruption of the Drosophila IP3 receptor gene leads to lowered levels of ecdysone. Synthesis and release of ecdysone from the ring gland is thought to occur in response to a neurosecretory peptide hormone secreted by the brain. It is proposed that this peptide hormone requires an IP3 signaling pathway for ecdysone synthesis and release in Drosophila and other insects. This signal transduction mechanism that links neuropeptide hormones to steroid hormone secretion might be evolutionarily conserved (Venkatesh, 1997).

A role for inositol 1,4,5-trisphosphate (IP₃) as a second messenger during olfactory transduction has been postulated in both vertebrates and invertebrates. However, given the absence of either suitable pharmacological reagents or mutant alleles specific for the IP₃ signaling pathway, an unequivocal demonstration of IP₃ function in olfaction has not been possible. The role of a well-established cellular target of IP₃, the IP₃ receptor, has been studied in olfactory transduction in Drosophila. For this purpose existing viable combinations of IP₃R mutant alleles, as well as a newly generated set of viable itpr alleles, were examined for olfactory function. In all of the viable allelic combinations primary olfactory responses were found to be normal. However, a subset of itpr alleles (including a null allele) exhibit faster recovery after a strong pulse of odor, indicating that the IP₃R is required for maintenance of olfactory adaptation. Interestingly, this defect in adaptation is dominant for two of the alleles tested, suggesting that the mechanism of adaptation is sensitive to levels of the IP₃R (Despande, 2000).

Larval molting in Drosophila, as in other insects, is initiated by the coordinated release of the steroid hormone ecdysone, in response to neural signals, at precise stages during development. Using genetic and molecular methods, the roles played by two major signaling pathways in the regulation of larval molting have been examined in Drosophila. Previous studies have shown that mutants for the Inositol 1,4,5-trisphosphate receptor gene (Itpr) are larval lethals. In addition, they exhibit delays in molting that can be rescued by exogenous feeding of 20-hydroxyecdysone. Mutants for adenylate cyclase (rut) synergize, during larval molting, with Itpr mutant alleles, indicating that both cAMP and InsP3 signaling pathways function in this process. The two pathways act in parallel to affect molting, as judged by phenotypes obtained through expression of dominant negative and dominant active forms of protein kinase A (PKA) in tissues that normally express the InsP3 receptor. Furthermore, these studies predict the existence of feedback inhibition through protein kinase A on the InsP3 receptor by increased levels of 20-hydroxyecdysone (Venkatesh, 2001).

An understanding of the signaling pathways that control insect molting has come primarily from pharmacological and biochemical studies on lepidopterans with similar studies extending to Drosophila. These studies have shown that neural factors, which include the PTTH, stimulate the prothoracic gland (a part of the ring gland in higher Dipterans including Drosophila) to synthesize and secrete ecdysone, which is subsequently converted to its active form of 20-hydroxyecdysone in other tissues. Biochemical and molecular analyses of PTTH isolated from lepidopterans and Drosophila have shown that the peptide hormone is quite different in the two classes of insects, indicating that signaling downstream of PTTH in the prothoracic gland may also differ. In fact, while extracellular calcium is required for secretion of ecdysone in both systems, cAMP has been demonstrated to be a second messenger only in lepidopterans. Molecular identification of other key players, such as the PTTH receptor and the channel for entry of extracellular calcium, has not yet been determined. The first indication that insect larval molting is regulated by InsP₃ signaling came from analysis of Drosophila mutants for the InsP₃ receptor gene. Data presented in this study now implicate, in addition, the cAMP pathway in control of larval molting in Drosophila. Since exogenous 20-hydroxyecdysone can rescue the molting delays caused by disruption of either pathway, it is likely that both pathways control 20-hydroxyecdysone levels during molting. Due to technical difficulties associated with measuring 20-hydroxyecdysone levels in Drosophila larvae, these measurements could not be carried out directly. Instead, transcript levels of an ecdysone-inducible gene, E74, were used as an indirect measure of 20-hydroxyecdysone levels (Venkatesh, 2001).

Interestingly, steroid secretion by the adrenal fasciculata-reticularis cells of mammalian adrenal glands in response to adrenocorticotrophic hormone occurs through the cAMP pathway, while InsP₃-mediated Ca²⁺ release is required for the steroidogenic action of Angiotensin II on adrenal glomerulosa cells. An increase in cytosolic Ca²⁺ levels is thought to affect multiple steps in mammalian steroid biosynthesis, including one crucial step that requires the transfer of endogenous cholesterol from the outer to the inner mitochondrial membrane. The data presented in this study support a similar model in which 20-hydroxyecdysone levels are regulated through activation of both InsP₃ and cAMP signals. The presence of multiple genes encoding adenylate cyclases allows rut mutant alleles to proceed through molting normally. Presumably, however, activity of the alternate adenylate cyclase(s) is dependent on InsP₃ receptor function since removal of the Itpr gene in rut mutant backgrounds leads to phenotypes that are synergistic. Activation of the two second messenger pathways probably occurs in the ring gland via PTTH and other as yet unidentified neural factors. Alternate explanations whereby InsP₃ and/or cAMP signaling are required for PTTH release from neurons or during conversion of 20-hydroxyecdysone precursors to 20-hydroxyecdysone cannot be ruled out at this stage. In either event the two pathways act in parallel to maintain 20-hydroxyecdysone levels perhaps via nonoverlapping downstream targets (Venkatesh, 2001).

Since ecdysone secretion occurs as tightly regulated peaks, preceding each molt, inherent in the system should be a mechanism that inhibits ecdysone secretion once the peak level has been reached. On the basis of data from the UAS-mC* transgene (coding for a dominant active form of PKA), it is suggested that increased levels of 20-hydroxyecdysone in the hemolymph initiate a negative feedback loop that requires PKA activation and inhibition of the InsP₃ receptor. Thus the activated PKA phenotype is not rescued by increased levels of 20-hydroxyecdysone, but is rescued by increased levels of the itpr transgene. Interestingly, the effect of the UAS-mC* transgene on molting is also lost when Itpr gene levels are reduced as in larvae of the genotype UAS-mC*/+; 1664GAL4/itpr^90B0. This observation supports the idea that the Itpr gene is downstream of the UAS-mC* effect, and in addition suggests that the negative feedback is highly sensitive to levels of the Itpr gene. While these results demonstrate interactions between the two signaling pathways, the molecular basis of these interactions is unknown as yet. Since mammalian InsP₃ receptors can be directly phosphorylated by PKA, the possibility exists that a similar mechanism might operate in the negative feedback step predicted from these results. However, both predicted isoforms of the Drosophila InsP₃ receptor, which are present in larval tissues and derive from two known splice variant forms of the itpr cDNA, lack putative PKA phosphorylation sites as determined by Prosite analysis. It is possible that a low-abundance isoform of the InsP₃ receptor exists in specific larval cells that may be directly regulated by PKA. Additionally, there are almost certainly other unidentified players in this system that this study has not revealed. It should be possible to identify some or all of these factors using suitable genetic interaction screens in the future (Venkatesh, 2001).

Phototransduction in Drosophila is compromised by Gal4 expression but not by InsP3 receptor knockdown or mutation

Drosophila phototransduction is mediated by phospholipase C, leading to activation of transient receptor potential (TRP) and TRP-like (TRPL) channels by mechanisms that are unresolved. A role for InsP3 receptors (IP3Rs) had been excluded because IP3R mutants (itpr) appeared to have normal light responses; however, this was recently challenged by Kohn et al. ("Functional cooperation between the IP3 receptor and phospholipase C secures the high sensitivity to light of Drosophila photoreceptors in vivo," Journal of Neuroscience 35:2530), who reported defects in phototransduction after IP3R-RNAi knockdown. They concluded that InsP3-induced Ca2+ release plays a critical role in facilitating channel activation, and that previous failure to detect IP3R phenotypes resulted from trace Ca2+ in electrodes substituting for InsP3-induced Ca2+ release. In an attempt to confirm this, electroretinograms, whole-cell recordings, and GCaMP6f Ca2+ imaging were performed from both IP3R-RNAi flies and itpr-null mutants. Like Kohn et al., this study used GMRGal4 to drive expression of UAS-IP3R-RNAi, but controls expressing GMRGal4 alone were also used. Several GMRGal4 phenotypes are described suggestive of compromised development, including reductions in sensitivity, dark noise, potassium currents, and cell size and capacitance, as well as extreme variations in sensitivity between cells. However, no effect of IP3R RNAi or mutation was found on photoreceptor responses or Ca2+ signals, indicating that the IP3R plays little or no role in Drosophila phototransduction (Bollepalli, 2017).

Genetic dissection of itpr gene function reveals a vital requirement in aminergic cells of Drosophila larvae

Signaling by the second messenger inositol 1,4,5-trisphosphate is thought to affect several developmental and physiological processes. Mutants in the inositol 1,4,5-trisphosphate receptor (itpr) gene of Drosophila exhibit delays in molting while stronger alleles are also larval lethal. In a freshly generated set of EMS alleles for the itpr locus single point mutations in seven mutant chromosomes have been sequenced and identified. The predicted allelic strength of these mutants matches the observed levels of lethality. They range from weak hypomorphs to complete nulls. Interestingly, lethality in three heteroallelic combinations has a component of cold sensitivity. The temporal focus of cold sensitivity lies in the larval stages, predominantly at second instar. Coupled with the observation that an itpr homozygous null allele dies at the second instar stage, it appears that there is a critical period for itpr gene function in second instar larvae. The focus of this critical function is shown to lie in aminergic cells, by rescue with UAS-itpr and DdCGAL4. However, this function does not require synaptic activity, suggesting that InsP₃-mediated Ca²⁺ release regulates the neurohormonal action of serotonin (Joshi, 2004).

To identify the tissue/cells that contribute to lethality observed in itpr mutants, tissue- and cell-specific GAL4 strains were used to drive expression of the UAS-itpr transgene in ug3/sv35 organisms, the majority of which die as second instars. Expression of the InsP₃ receptor in larvae is known to be in the brain and ring gland complex, among other tissues. Consequently, attempts were made to rescue lethality with lines that express in the central nervous system, peripheral nervous system, and ring gland. Complete rescue of second instar lethality was obtained with elavGAL4, including a normal transition from second to third instar larvae. elavGAL4-rescued organisms also pupate but are unable to eclose as adults. Expression of the elav gene is known to occur in all postmitotic neurons. In addition, significant expression of elavGAL4 is seen in cells of the ring gland, including the corpora cardiaca in second and third instar larvae. Several GAL4 lines were tested that were known to express in subsets of larval neurons and expression was looked for in cells of the ring gland. Among the GAL4 lines, which could rescue second to third instar lethality to varying levels, were DdCGAL4 and c929. In the ug3/sv35 strain, 6.3 ± 0.6 third instar larvae were seen at 152-160 hr AEL. GAL4 expression in these strains occurs in all dopamine and serotonin neurons (DdCGAL4) and in all peptidergic neurons (c929). Similar to elavGAL4, both strains also have significant GAL4 expression in the corpora cardiaca region of second and third instar larval ring glands. Since the corpora cardiaca is the only tissue of overlap between DdCGAL4 and C929, the modest rescue observed with c929 is attributed to these cells. The higher level of rescue observed in DdCGAL4-containing organisms indicates that in addition to the corpora cardiaca, dopamine and serotonin neurons in the larval brain also require itpr gene function. Of these two neurotransmitters, serotonin immunoreactivity has also been reported in the corpora cardiaca. This was confirmed by immunostaining second and third instar ring glands with a polyclonal antiserum to serotonin. Taken together, these data point toward a critical requirement for the InsP₃ receptor in serotonin-containing cells of second instar larvae (Joshi, 2004).

To obtain an understanding of the cellular function performed by the InsP₃ receptor in DdCGAL4 positive cells, synaptic function in these cells was inhibited by expression of the UAS-tetanus toxin transgene (UAS-TNT. Expression of tetanus toxin in Drosophila neurons is known to specifically block evoked neurotransmitter release and to reduce the frequency of spontaneous quantal release. DdCGAL4-driven expression of UAS-TNT did not affect larval viability or molting. In contrast, when TNT expression is under control of c929 in peptidergic neurons, there is a significant loss of viability in second instar larvae. Therefore, the TNT transgene used in this work is functional and capable of affecting viability, which is dependent on the neurons where it is expressed. Complete loss of DdCGAL4-expressing cells, however, is critical for larval viability, since expression of a cell death gene (UAS-hid) in these cells resulted in a high level of larval lethality (Joshi, 2004).

Analysis of larval lethality with the newly generated InsP₃ alleles described in this study has provided new insights into InsP₃ receptor function in Drosophila. The observations are consistent with the existence of a physiological process in second instar larvae, which requires a critical level of activity from the zygotically derived InsP₃ receptor, the absence of which leads to lethality. This requirement occurs prior to the InsP₃ receptor's role in regulating larval molting as suggested by the following observation. While feeding of 20-hydroxyecdysone can rescue molting delays in a nonlethal allele, it is not able to rescue lethality of any allelic combination. However, the focus of both these defects in itpr mutants could lie in serotonin cells. This idea is supported by the fact that 5-HT immunoreactive fibers have been seen extending to the prothoracic gland and corpora allata. A role for serotonin in larval molting has been proposed in other insects (Joshi, 2004).

From the rescue profiles obtained with elavGAL4 and DdCGAL4 it is also clear that there is a pupal phase of lethality, which is rescued effectively by UAS-itpr expression in the domains of neurGAL4 and prosGAL4. To understand the cause of pupal lethality, the expression of these strains in pupae needs further investigation (Joshi, 2004).

Both serotonin and dopamine are best known in their roles as neurotransmitters. However, serotonin is also known to have an essential role during gastrulation of Drosophila embryos when it is thought to trigger changes in cell adhesiveness by as-yet-unknown cellular mechanisms. In the context of neurons, serotonin can act as a neurotransmitter or as a neurohormone. On the basis of the results of experiments with expression of UAS-TNT in DdCGAL4-positive cells, it is proposed that serotonin's action as a neurohormone is critical for larval viability. The partial rescue of second instar lethality by UAS-itpr expression in serotonin-positive neurohemal cells of the corpora cardiaca (c929GAL4), supports this idea. Neurohemal cells have no synaptic activity and their function is to secrete either stored or freshly synthesized neurohormones. Serotonin-positive fibers and varicosities extend from the corpora cardiaca to other regions of the ring gland, the aorta wall, and the surface of the gut. The effect of serotonin release from these fibers thus could be on the physiological function of any of these tissues. A direct test of this hypothesis would be to see the effect of inhibiting secretory pathways required for neurohormonal release (unrelated to neurotransmission) in serotonin cells, such as the one described recently in Drosophila neurons (Murthy, 2003). However, at present the possibility that a neurohormone other than serotonin is secreted by DdCGAL4-expressing cells, that might be the critical factor in the observed lethality, cannot be ruled out (Joshi, 2004).

The observations related to cold sensitivity in ug3/ka1091 also support the idea of the InsP₃ receptor's role in regulating neurohormonal release from aminergic cells. Exposure to cold temperatures is known to have an inhibitory effect on secretory pathways described in CHO cells and in chromaffin cells. Moreover, certain exocytosis mutants in yeast are cold sensitive (Joshi, 2004).

Further investigation is required to understand the nature of extracellular signals that activate the InsP₃ receptor in DdCGAL4-positive cells. It is known that several neurosecretory neurons send their processes to the corpora cardiaca. Serotonin and dopamine cells of the larval brain and ventral ganglion are mostly interneurons and probably receive inputs from many types of neurons. A combination of cellular and genetic studies will be required to understand the nature of these signals (Joshi, 2004).

Loss of flight and associated neuronal rhythmicity in Inositol 1,4,5-trisphosphate receptor mutants of Drosophila

Coordinated flight in winged insects requires rhythmic activity of the underlying neural circuit. Drosophila mutants for the inositol 1,4,5-trisphosphate (InsP₃) receptor gene (itpr) are flightless. Electrophysiological recordings from thoracic indirect flight muscles show increased spontaneous firing accompanied by a loss of rhythmic flight activity patterns normally generated in response to a gentle puff of air. In contrast, climbing speed, the jump response, and electrical properties of the giant fiber pathway are normal, indicating that general motor coordination and neuronal excitability are much less sensitive to itpr mutations. All mutant phenotypes are rescued by expression of an itpr⁺ transgene in serotonin and dopamine neurons. Pharmacological and immunohistochemical experiments support the idea that the InsP₃ receptor functions to modulate flight specifically through serotonergic interneurons. InsP₃ receptor action appears to be important for normal development of the flight circuit and its central pattern generator (Banerjee, 2004 ).

This study shows that InsP₃R is required for air puff-induced flight and associated neuronal rhythmicity. This requirement is in the domain of DdCGAL4 (serotonin and dopamine)-positive neurons during the first 48 hr of pupal development. From pharmacological and anatomical experiments, it appears likely that the InsP₃R is required in serotonergic neurons, although a requirement in dopaminergic cells cannot be ruled out at this stage (Banerjee, 2004).

Changes in intracellular calcium levels as a consequence of InsP₃R function are likely to affect diverse aspects of neuronal physiology. The itpr alleles studied here are viable and hence their behavioral and physiological phenotypes probably reflect neuronal functions most sensitive to InsP₃-mediated Ca²⁺ release. Interestingly, the foci of these functions lie in aminergic cells that release the neurotransmitters serotonin or dopamine. Furthermore, despite the fact that the observed phenotypes are behavioral and physiological, a significant proportion of these arise from a developmental requirement for the InsP₃R. Thus, normal formation and functioning of the air puff-stimulated flight circuit in adults requires the itpr gene during its formation and growth in pupae (Banerjee, 2004).

It is known that motoneurons innervating the indirect flight muscles undergo dendritic and axonal remodeling during pupariation. From several studies in both invertebrates and vertebrates, it appears that among the biogenic amines, serotonin can modulate axon outgrowth. In serotonin-positive cerebral giant cells of Lymnaea stagnalis, release of 5-HT autoregulates axon growth by inducing growth cone collapse. The cerebral giant cells form part of the neural circuit that controls rhythmic feeding behavior in these molluscs. In Drosophila, inhibition of serotonin synthesis has been shown to cause excessive branching of serotonergic axon terminals during embryonic and larval development. More recently, serotonin has been shown to affect development of the swimming circuit in zebrafish, and a role for serotonergic interneurons has been described during development of the left-right coordination of rhythmic motor activity in rat spinal cord. Together with these studies, it is reasonable to postulate that itpr mutants affect development of the flight circuit through modulating serotonin release. The observation that the flight deficit (~45%) in DdCGAL4/UAS-TNT organisms (in which tetanus toxin is expressed in the pattern of the the Ddc promoter) is less than that of a majority of the itpr mutants (>80%) suggests that a component of this release is neurohormonal and not entirely because of evoked neuronal activity. A role for the InsP₃R in neurohormonal secretion is also apparent in DdCGAL4-positive neurons and neurohemal cells of second instar Drosophila larvae. These data raise the possibility that release of serotonin is particularly sensitive to intracellular Ca²⁺ levels regulated through the InsP₃R. This idea needs to be tested rigorously by additional experiments (Banerjee, 2004).

Both serotonin and dopamine have been shown to modulate the acute activity of motor circuits. Increased serotonin release has been shown to increase the postsynaptic response of flight motoneurons in locust. Recently, serotonin was shown to alter motoneuron firing in Drosophila larvae in a biphasic manner. The increased frequency of spontaneous firing and the loss of air puff induced flight in itpr mutants suggest that disruption of InsP₃R function could alter normal firing of motor neurons, which innervate the DLMs. The required InsP₃R activity does not reside in the motor neurons but rather in aminergic (possibly serotonergic) neurons innervating and controlling motor neuron activity. One explanation is that inhibitory connections that modulate flight motor neurons have been lost or weakened during development. However, as adult feeding of PCPA results in lowered synthesis of serotonin and resembles the blocking effects of release of serotonin by TNT from these interneurons and also partially phenocopies itpr phenotype, it is possible that the InsP₃R continues to play a central role in flight coordination (Banerjee, 2004).

Anatomical analysis has shown that cell bodies positive for serotonin and DdcGAL4 exist in the thoracic ganglia: no arborization from these cell bodies toward flight motoneurons was detected. Instead, the data suggest that 5-HT-positive varicosities on flight motor neurons arise from serotonin and DdcGAL4-positive axon tracts that descend from the brain. This observation argues for a serotonergic modulation of flight motor neuron function from neurons in the brain. Localization of the InsP₃R to these neurons and their axon tracts was not feasible, because immunohistochemistry with existing antisera to the Drosophila InsP₃R gives a cross-reaction with nonspecific antigen(s) in adult CNS preparations. The existing itprGAL4 line could not be used either, because it represents a subset of the itpr gene expression domain and does not express in the adult nervous system. Very likely, the physiological defects observed are attributable to a combination of inappropriate circuit formation during development and altered neuronal activity in adults. Although development has a significant contribution, the existing tools do not allow investigation of an adult-specific requirement in an unambiguous manner. However, given the perdurance of the InsP₃R in adults (as judged by the expression of a UAS-itprGFP transgene induced in pupae by the same heat shock regime as that for UAS-itpr⁺), a contribution to acute flight signaling remains a distinct possibility (Banerjee, 2004).

Tbese studies show that the InsP₃R is essential for development of the neural circuit that probably functions as the central pattern generator for air puff-induced flight. The function of the InsP₃R is in aminergic cells, indicating that at a cellular level, it functions in the release of serotonin and/or dopamine. Earlier, Ca²⁺ release from vesicular InsP₃Rs in chromaffin cells, acinar cells, and islet cells has been proposed to release Ca²⁺ required for secretion. The profound developmental and functional actions of InsP₃R, through release of biogenic amines, is a novel finding and could have relevance for vertebrate systems, where serotonin has been shown to affect neural circuit development. Significantly, knock-outs for the InsP₃R1 in mice exhibit ataxia and motor discoordination at birth (Banerjee, 2004).

Ectopic expression of a Drosophila InsP(3)R channel mutant has dominant-negative effects in vivo

The inositol 1,4,5-trisphosphate (InsP₃) receptor is a tetrameric intracellular calcium channel. It is an integral component of the InsP₃ signaling pathway in multicellular organisms, where it regulates cellular calcium dynamics in many different contexts. In order to understand how the primary structure of the InsP₃R affects its functional properties, the kinetics of Ca²⁺-release in vitro from single point mutants of the Drosophila InsP₃R have been determined. Among these, the Ka901 mutant in the putative selectivity-filter of the pore is of particular interest. It is non-functional in the homomeric form whereas it forms functional channels (with altered channel properties) when co-expressed with wild-type channels. Due to its changed functional properties the Ka901 mutant protein has dominant-negative effects in vivo. Cells expressing Ka901:WT channels exhibit much higher levels of cytosolic Ca²⁺ upon stimulation as compared with cells over-expressing just the wild-type DmInsP₃R, thus supporting in vitro observations that increased Ca²⁺ release is a property of heteromeric Ka901:WT channels. Furthermore, ectopic expression of the Ka901 mutant channel in aminergic cells of Drosophila alters electrophysiological properties of a flight circuit and results in defective flight behavior (Srikanth, 2005).

Intracellular Ca²⁺ signaling and store-operated Ca²⁺ entry are required in Drosophila neurons for flight

Neuronal Ca²⁺ signals can affect excitability and neural circuit formation. Ca²⁺ signals are modified by Ca²⁺ flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca²⁺ stores and their release to neuronal processes is poorly understood. This study shows, by neuron-specific siRNA depletion, that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca²⁺ store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP₃R) can partially compensate for their loss of flight. Ca²⁺ measurements show that Orai gain-of-function contributes to the quanta of Ca²⁺-release through mutant InsP₃Rs and elevates store-operated Ca²⁺ entry in Drosophila neurons. These data show that replenishment of intracellular store Ca²⁺ in neurons is required for Drosophila flight (Venkiteswaran, 2009).

Several aspects of neuronal function are regulated by ionic calcium (Ca²⁺). Specific attributes of a Ca²⁺ 'signature' such as amplitude, duration, and frequency of the signal can determine the morphology of a neural circuit by affecting the outcome of cell migration, the direction taken by a growth-cone, dendritic development, and synaptogenesis. Ca²⁺ signals also determine the nature and strength of neural connections in a circuit by specifying neurotransmitters and receptors. Most of these Ca²⁺ signals have been attributed to the entry of extracellular Ca²⁺ through voltage-operated channels or ionotropic receptors. However, other components of the 'Ca²⁺ tool-kit' coupled to Ca²⁺ release from intracellular Ca²⁺ stores are also present in neurons. These molecules include the store-operated Ca²⁺ (SOC) channel, encoded by the Orai gene, identified in siRNA screens for molecules that reduce or abolish Ca²⁺ influx from the extracellular milieu after intracellular Ca²⁺ store depletion (Feske, 2006; Vig, 2006b; Zhang, 2006). Several reports have confirmed its identity as the pore forming subunit of the Ca²⁺-release activated Ca²⁺ (CRAC) channel (Prakriya, 2006; Vig, M., 2006b; Yeromin, 2006). Activation of this CRAC channel is mediated by the endoplasmic reticulum (ER) resident protein STIM (stromal interaction molecule), also identified in an RNAi screen for molecules that regulate SOC influx (Liou, 2005; Zhang, 2005). STIM1 is a Ca²⁺ sensor that activates CRAC channels and migrates from the Ca²⁺ store to the plasma membrane. Nature 437: 902-905). STIM senses the drop in ER Ca²⁺ levels, and interacts with Orai by a mechanism which is only just being understood (Yuan, 2009). Orai and STIM function in conjunction with the sarco-endoplasmic reticular Ca²⁺-ATPase pump (SERCA) to maintain ER store Ca²⁺ and basal Ca²⁺. The importance of intracellular Ca²⁺ homeostasis and SOC entry (SOCE) in neural circuit formation and in neuronal function and physiology remains to be elucidated (Venkiteswaran, 2009).

This study reports how Orai and STIM mediated Ca²⁺ influx and Ca²⁺ homeostasis in Drosophila neurons contribute to cellular and systemic phenotypes. Reduced SOCE, measured in primary neuronal cultures, is accompanied by a range of defects in adults, including altered wing posture, increased spontaneous firing, and loss of rhythmic flight patterns. These phenotypes mirror the spontaneous hyperexcitability of flight neuro-muscular junctions and loss of rhythmic flight patterns observed in Drosophila mutants of the inositol 1,4,5-trisphosphate receptor (InsP₃R, itpr gene). The InsP₃R is a ligand gated Ca²⁺-channel present on the membranes of intracellular Ca²⁺ stores. It is thought to be critical for various aspects of neuronal function. Mutants in the gene coding for the mouse InsP₃R1 are ataxic. Cerebellar slices from InsP₃R1 knockout mice show reduced long-term depression, indicating that altered synaptic plasticity of the cognate neural circuits could underlie the observed ataxia (Venkiteswaran, 2009).

To understand the temporal and spatial nature of intracellular Ca²⁺ signals required during flight circuit development and function, dOrai (CG11430) and dSERCA (encoded by CaP-60A gene, CG3725) function was modulated by genetic means in itpr mutants [using a dominant mutant allele (Kum¹⁷⁰) for the gene (Ca-P60A) encoding the SERCA]. This modulation can restore flight to flightless adults, by altering several parameters of intracellular Ca²⁺ homeostasis including SOCE. These results suggest that components of the central pattern generator (CPG) required for maintenance of normal rhythmic flight in adults have a stringent requirement for SOCE after InsP₃R stimulation (Venkiteswaran, 2009).

This study shows that SOC entry through the Orai/STIM pathway and the rate of clearance of cytoplasmic Ca²⁺ by SERCA together shape intracellular Ca²⁺ response curves in Drosophila larval neurons. The phenotypic changes associated with altering Orai/STIM function on their own and in itpr mutant combinations suggest that these Ca²⁺ dynamics are conserved through development among neurons in pupae and adults. The development and function of the flight circuit appears most sensitive to these cellular Ca²⁺ dynamics, changes in which have a profound effect on its physiological and behavioral outputs. Direct measurements of Ca²⁺ in flight circuit neurons are necessary in future to understand why these cells are more sensitive to changes in intracellular Ca²⁺ signaling. Other circuits such as those required for walking, climbing and jumping remain unaffected. Possible effects of altering intracellular Ca²⁺ homeostasis on higher order neural functions have yet to be determined (Venkiteswaran, 2009).

The flow of information in a neural circuit goes through multiple steps within and between cells. Suppression experiments, such as the ones described in this study, present a powerful genetic tool for understanding the mechanisms underlying both the formation of such circuits and their function. The correlation observed between adult phenotypes and Ca²⁺ dynamics in populations of larval neurons from the various genotypes supports the following conclusions. Out-spread wings, higher spontaneous firing, and initiation of rhythmic firing on air-puff delivery in itpr^ku (a heteroallelic mutant combination of itpr) are suppressed by either increasing the quanta (through hypermorphic alleles of dOrai and by dOrai⁺ overexpression) or by increasing the perdurance (through mutant Kum¹⁷⁰) of the intracellular Ca²⁺ signal (Kum is ). Flight ability and maintenance of flight patterns requires SOCE in addition to increased quanta and perdurance of the Ca²⁺ signals, suggesting that SOCE in neurons contributes to recurring Ca²⁺ signals essential for flight maintenance (Venkiteswaran, 2009).

The signals that trigger InsP₃ generation in Drosophila neurons and the nature of the downstream cellular response remain to be investigated. Previous work has shown that rescue of flight and related physiological phenotypes in itpr mutants require UASitpr⁺ expression in early to midpupal stages, indicating the InsP₃R activity is necessary during development of the flight circuit (Banerjee, 2004). Due to perdurance of the InsP₃R, its requirement in adults was not established. This study found that a basal level of dOrai⁺ expression through development followed by ubiquitous overexpression in adults can help initiate flight in itpr^ku, indicating a requirement for SOCE in adult neurons that probably constitute the CPG for flight. The precise neuronal circuit and neurons in the flight CPG are under investigation (Frye, 2004). Aminergic, glutamatergic, and insulin producing neurons could assist in development and/or directly constitute the circuit. Similar patterns of neuronal activity in the flight circuit of itpr mutants, either by generating different combinations of Ca²⁺ fluxes (as shown in this study), or by UASitpr⁺ expression in nonoverlapping neuronal domains supports the idea that different aspects of neuronal activity can compensate for each other to maintain constant network output (Venkiteswaran, 2009).

Precisely how hypermorphic dOrai alleles modify itpr^ku function to increase the quanta of Ca²⁺ release remains to be investigated. The ability of itpr^ku to maintain elevated [Ca²⁺]_ER at 25 °C suggests a possible interaction between this heteroallelic combination and Orai/STIM. The mutated residue in itpr^ka1091 (Gly to Ser at 1891) lies in the modulatory domain, whereas in itpr^ug3, it lies in the ligand binding domain (Ser to Phe at 224); both residues are conserved in mammalian InsP₃R isoforms. The mutant residues could directly affect InsP₃R interactions with a store Ca²⁺ regulating molecule like STIM. Recent reports (Redondo, 2006) also demonstrate the formation of macromolecular assemblies of InsP₃R, SERCA, and SOC channels, suggesting specific functional interactions between them (Venkiteswaran, 2009).

Last, these results suggest new ways of treating diseases where altered intracellular Ca²⁺ signaling or homeostasis has been suggested as a causative agent. Perhaps, the best documented of these diseases are spino-cerebellar ataxia 15, which arises by heterozygosity of the mammalian IP₃R1 gene, severe combined immunodeficiency due to a mutation in Orai1, and Darier's disease from a mutation in SERCA2. Based on the underlying changes in intracellular Ca²⁺ properties in these genetic diseases, this study suggests ways of deciding appropriate combination of drugs that might target the causative gene products and their functionally interacting partners (Venkiteswaran, 2009 and references therein).

Loss of IP3 receptor function in neuropeptide secreting neurons leads to obesity in adult Drosophila

Intracellular calcium signaling regulates a variety of cellular and physiological processes. The inositol 1,4,5 trisphosphate receptor (IP3R) is a ligand gated calcium channel present on the membranes of endoplasmic reticular stores. Previous work has shown that Drosophila mutants for the IP3R (itprku) become unnaturally obese as adults with excessive storage of lipids on a normal diet. While the phenotype manifests in cells of the fat body, genetic studies suggest dysregulation of a neurohormonal axis. This study shows that knockdown of the IP3R, either in all neurons or in peptidergic neurons alone, mimics known itpr mutant phenotypes. The peptidergic neuron domain includes, but is not restricted to, the medial neurosecretory cells as well as the stomatogastric nervous system. Conversely, expression of an itpr+ cDNA in the same set of peptidergic neurons rescues metabolic defects of itprku mutants. Transcript levels of a gene encoding a gastric lipase CG5932 (magro), which is known to regulate triacylglyceride storage, can be regulated by itpr knockdown and over-expression in peptidergic neurons. Thus, the focus of observed itpr mutant phenotypes of starvation resistance, increased body weight, elevated lipid storage and hyperphagia derive primarily from peptidergic neurons. The present study shows that itpr function in peptidergic neurons is not only necessary but also sufficient for maintaining normal lipid metabolism in Drosophila. These results suggest that intracellular calcium signaling in peptidergic neurons affects lipid metabolism by both cell autonomous and non-autonomous mechanisms (Subramanian, 2013).

Interaction between cAMP and intracellular Ca-signaling pathways during odor-perception and adaptation in Drosophila

Binding of an odorant to olfactory receptors triggers cascades of second messenger systems in olfactory receptor neurons (ORNs). The transduction mechanism at ORNs is mediated by cAMP and/or inositol,1,4,5-triphosphate (InsP3)-signaling pathways in an odorant-dependent manner. This study used interfering-RNAi to disrupt the level of cAMP alone or in combination with the InsP3-signaling pathway cellular targets, InsP3 receptor (InsP3R) or ryanodine receptor (RyR) in ORNs, and quantify at ORN axon terminals in the antennal lobe, the odor-induced Ca2+-response. In-vivo functional bioluminescence Ca2+-imaging indicates that a single 5s application of an odor increased Ca2+-transients at ORN axon terminals. However, compared to wild-type controls, the magnitude and duration of ORN Ca2+-response was significantly diminished in cAMP-defective flies. In a behavioral assay, perception of odorants was defective in flies with a disrupted cAMP level suggesting that the ability of flies to correctly detect an odor depends on cAMP. Simultaneous disruption of cAMP level and InsP3R or RyR further diminished the magnitude and duration of ORN response to odorants and affected the flies' ability to detect an odor. In conclusion, this study provides functional evidence that cAMP and InsP3-signaling pathways act in synergy to mediate odor processing within the ORN axon terminals, which is encoded in the magnitude and duration of ORN response (Murmu, 2016).

Drosophila wing imaginal discs respond to mechanical injury via slow InsP3R-mediated intercellular calcium waves

Calcium signalling is a highly versatile cellular communication system that modulates basic functions such as cell contractility, essential steps of animal development such as fertilization and higher-order processes such as memory. This study probed the function of calcium signalling in Drosophila wing imaginal discs through a combination of ex vivo and in vivo imaging and genetic analysis. Wing discs were found display slow, long-range intercellular calcium waves (ICWs) when mechanically stressed in vivo or cultured ex vivo. These slow imaginal disc intercellular calcium waves (SIDICs) are mediated by the inositol-3-phosphate receptor, the endoplasmic reticulum (ER) calcium pump SERCA and the key gap junction component Inx2. The knockdown of genes required for SIDIC formation and propagation negatively affects wing disc recovery after mechanical injury. These results reveal a role for ICWs in wing disc homoeostasis and highlight the utility of the wing disc as a model for calcium signalling studies (Restrepo, 2016).

Spontaneous Ca2+ influx in Drosophila pupal neurons is modulated by IP3-receptor function and influences maturation of the flight circuit

Inositol 1,4,5-trisphosphate receptors (IP3R) are Ca2+ channels on the neuronal endoplasmic reticulum (ER) membrane. They are gated by IP3, produced upon external stimulation and activation of G protein-coupled receptors on the plasma membrane (PM). IP3-mediated Ca2+ release, and the resulting depletion of the ER store, triggers entry of extracellular Ca2+ by store-operated Ca2+ entry (SOCE). Mutations in IP3R attenuate SOCE. Compromised IP3R function and SOCE during pupal development of Drosophila leads to flight deficits and mimics suppression of neuronal activity during pupal or adult development. To understand the effect of compromised IP3R function on pupal neuronal calcium signaling, the effects were examined of mutations in the IP3R gene (itpr) on Ca2+ signals in cultured neurons derived from Drosophila pupae. Increased spontaneous Ca2+ influx across was observed the PM of isolated pupal neurons with mutant IP3R and also a loss of SOCE. Both spontaneous Ca2+ influx and reduced SOCE were reversed by over-expression of dOrai and dSTIM, which encode the SOCE Ca2+ channel and the ER Ca2+-sensor that regulates it, respectively. Expression of voltage-gated Ca2+ channels (cac, Ca-alpha1D and Ca-alphaT) was significantly reduced in itpr mutant neurons. However, expression of trp mRNAs and transient receptor potential (TRP) protein were increased, suggesting that TRP channels might contribute to the increased spontaneous Ca2+ influx in neurons with mutant IP3R. Thus, IP3R/SOCE modulates spontaneous Ca2+ influx and expression of PM Ca2+ channels in Drosophila pupal neurons. Spontaneous Ca2+ influx compensates for the loss of SOCE in Drosophila itpr mutant neurons (Chakraborty, 2017).

EVOLUTIONARY HOMOLOGS

Sequence analysis of Drosophila RyR, IP₃R and SERCA

Sequence analysis of RyR, IP₃R and SERCA was performed as a way to address similarities to and differences from their vertebrate counterparts and within themselves. Computer-generated alignments of 15 RyRs, 13 IP₃Rs and 21 SERCAs were analyzed. The extent of identity between Drosophila RyR and other RyRs considered in this study was the lowest and ranged from 37% (with Homo sapiens RyR type 1) to 45% (with Caenorhabditis elegans unique RyR isoform). It was thus not possible by this means to recognize an accentuated identity among Drosophila RyR and any of the three vertebrate isoforms (Vázquez-Martínez, 2003).

The identity detected between Drosophila IP₃R and other IP₃Rs was intermediate and ranged from 36% (with Caenorhabditis elegans unique IP₃R isoform) to 57% (with Panulirus argus unique IP₃R isoform). In contrast with RyR isoforms, vertebrate IP₃R type 1 isoform showed a slightly higher percentage of identity with the fruit fly receptor (56%), than IP₃R type 2 (53%) and type 3 (50%). SERCA enzymes had the highest percentage of identity within themselves. The range went from 67% (with both Rattus norvergicus and Homo sapiens SERCA type 3) to 81% (with the Procambarus clarkii unique SERCA isoform). Drosophila SERCA had a slightly higher identity with vertebrate type 1 and 2 SERCAs (71-73%) than with type 3 (67-69%) (Vázquez-Martínez, 2003).

Equal-weight ('unrooted') Parsimony and Neighbor Joining analyses of the sequences were performed for RyRs, IP₃Rs and SERCAs. Both programs yielded virtually identical topologies suggesting, as expected, that the three Drosophila proteins grouped together with all other invertebrate genes. Both types of calcium release channels were separated in the phylogram tree very clearly. Drosophila RyR is sister to C. elegans RyR, and both are in a different node from vertebrate RyRs. Type 2 and 3 RyRs were co-segregated in one group and separated from type 1 RyRs (Vázquez-Martínez, 2003).

Drosophila IP₃R was sister to crustacean P. argus IP₃R, whereas the receptor of C. elegans split from all other IP₃Rs. Vertebrate type 1 and 2 IP₃Rs shared a common node. Vertebrate and invertebrate RyRs and IP₃Rs form distinct clades within each type of calcium release channel. Drosophila SERCA grouped with other invertebrates and was closer to vertebrate type 1 SERCAs. Vertebrate type 3 SERCAs were the more distal proteins compared with invertebrate SERCAs (Vázquez-Martínez, 2003).

The homology between the Drosophila RyR and other RyRs, like the C. elegans gene and the three vertebrate ones, was in all cases around 40%. Thus, it is not possible to deduce relationships between the invertebrate receptors and their vertebrate counterparts from sequence data. However, inspection of the phylogram and cladogram indicates that the Drosophila and C. elegans RyRs form a separate group from the vertebrate isoforms. It might seems premature to assign the Drosophila and C. elegans RyRs as type 1 based only in their conspicuous muscular localization, but biochemical data support such a tenet. Taken together, evidence points to a closer relationship between vertebrate RyR type 1 and invertebrate RyR (Vázquez-Martínez, 2003).

IP₃Rs are different: the homology between the Drosophila IP₃R and the IP₃R from C. elegans was smaller (36%) than with the vertebrate isoforms 2 (53%), 3 (50%) and 1 (56%), or the lobster IP₃R (57%). The data may suggest that the Drosophila IP₃R is closer to the vertebrate IP₃R type 1 than the other 2 isoforms. One can speculate that this state of affairs is due to evolutionary divergence since the last common ancestor between nematodes and arthropods/vertebrates happened earlier in time than the split between arthropods and vertebrates. Once the vertebrate lineage split from the arthropods, several duplication events in the vertebrate lineage gave rise to the current three isoforms. It can be added that similarities in homology values among all RyRs considered could be explained by somewhat different structural constraints in RyRs compared to IP₃Rs (Vázquez-Martínez, 2003).

RyRs and IP₃Rs are homologous proteins sharing 30-35% homology at the amino acid level. However, there are three regions where the homology is higher: (1) the first 600 amino acids (numbering based on RyRs sequences), (2) the central region between amino acids 1500 and 2600, and (3) the C-terminal domain starting from residue 3900, containing the transmembrane domains. Both the phylogram and the cladogram show that the RyRs and IP₃Rs from invertebrates are grouped separately from vertebrate isoforms. Vertebrate isoforms could have diverged because they are specialized to fulfill physiological requirements of determined tissues; for example, RyR type 1 allows the excitation-contraction coupling of skeletal muscle, whereas RyR type 2 does the same in cardiac muscle. What is the strategy used in invertebrates? There are three possibilities: (1) that in Drosophila and other invertebrates the specialized roles of each receptor can be accomplished by alternatively spliced forms of the RyR and IP₃R genes; (2) that the intrinsic molecular properties of each receptor enable them to carry out all the different functions encompassed by their vertebrate counterparts, or (3) that owing to the different nature of tissues in vertebrates and invertebrates, such specialized roles are not required (Vázquez-Martínez, 2003).

InsP3R mutation

Signaling through an epidermal growth factor receptor in the nematode C. elegans stimulates a Ca2+ release pathway that is independent of Ras. Activity of LET-23, the C. elegans homolog of the epidermal growth factor receptor (see Drosophila Egf receptor), is required in multiple tissues. RAS activation is necessary and sufficient for certain LET-23 functions. Two sets of experiments suggest that LIN-3 (a C. elegans homolog of the epidermal growth factor) and LET-23 function in the hermaphrodite gonad are RAS independent. (1) Mutations in components of the RAS pathway capable of rescuing defects in vulval development and viability caused by reduction-of-function let-23 mutations are unable to rescue the sterility caused by such mutations. (2) Mutational analysis of the let-23 gene suggests that distinct domains of the receptor mediate LET-23 function in the vulva, versus the hermaphrodite gonad. In particular, while certain putative binding sites for Src Homology-2 domain-containing proteins are required for LET-23 function in viability and vulval induction, a distinct pair of sites is necessary and sufficient for function in the hermaphrodite gonad (Clandinin, 1998).

Mutations in two loci, lfe-1/itr-1 and lfe-2 are tissue-specific suppressors of reduced LIN-3/LET-23-mediated signaling. Mutations in lfe-1/itr-1 are likely to be gain-of-function while a suppressing mutation in lfe-2 is likely loss-of-function. lfe-1/itr-1 and lfe-2 appear to function downstream of let-23 for hermaphrodite fertility because genetic epistasis tests using both a reduction-of-function allele and a null allele of let-23 demonstrate that mutations in lfe-1/itr-1 or lfe-2 can bypass LET-23 function in the gonad. In addition to their suppression phenotype, the activities of lfe-1/itr-1 and lfe-2 appear to be involved in normal hermaphrodite fertility because lfe-1/itr-1(gf); lfe-2(lf) double-mutant animals display ovulation defects similar to those observed in animals bearing reduction of function mutations in either let-23 or lin-3. It is believed that the fertility function of these two genes lies in the adult somatic gonad, based on an analysis of LFE-2. In particular, LFE-2 is expressed in the adult spermatheca, and misexpression of LFE-2 in adult animals is sufficient to induce defects in spermathecal function (Clandinin, 1998).

Molecular characterization of LFE-1/ITR-1 and LFE-2 argues strongly that LET-23 regulates intracellular calcium levels in the spermatheca. LFE-1/ITR-1 encodes a C. elegans homolog of the mammalian IP3R, an established effector of intracellular calcium release. LFE-2 encodes a nematode homolog of IP3 kinase, whose in vivo function in mammalian cells is unknown but which is very likely to play a regulatory role in calcium release based on its substrate specificity. Thus, an inositol trisphosphate receptor can act as a RAS-independent, tissue-specific positive effector of LET-23. An inositol trisphosphate kinase negatively regulates this transduction pathway. Signals transduced by LET-23 control ovulation through changes in spermathecal dilation, possibly dependent upon calcium release regulated by the second messengers IP3 and IP4. It is likely that LET-23 functions by activation of phospholipase Cgamma, which promotes release of intracellular calcium through production of IP3. These results demonstrate that one mechanism by which receptor tyrosine kinases can evoke tissue-specific responses is through activation of distinct signal transduction cascades in different tissues (Clandinin, 1998).

Type 1 InsP3 receptor (InsP3R1) is the major neuronal member of the InsP3R family in the central nervous system, especially enriched in cerebellar Purkinje cells but also concentrated in neurons in the hippocampal CA1 region, caudate-putamen, and cerebral cortex. Most InsP3R1-deficient mice generated by gene targeting die in utero; animals surviving to birth have severe ataxia and tonic or tonic-clonic seizures and die before weaning. An electroencephalogram shows that they suffer from epilepsy, indicating that InsP3R1 is essential for proper brain function. However, observation by light microscope of the hematoxylin-eosin staining of the brain and peripheral tissues of InsP3R1-deficient mice shows no abnormality, and the unique electrophysiological properties of the cerebellar Purkinje cells of InsP3R1-deficient mice are not severely impaired (Matsumoto, 1996).

Domain structure of InsP3R

In an attempt to define structural regions of the type I inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) involved in its intracellular targeting to the endoplasmic reticulum (ER), the use of green fluorescent protein (GFP) was employed to monitor the localization of a truncated InsP3R mutant containing just the putative transmembrane spanning domain and the C-terminal cytoplasmic domain (amino acids 2216-2749), termed InsP3R(ES). A chimeric GFP-InsP3R(ES) fusion protein was expressed in Xenopus laevis oocytes, and fluorescent confocal microscopy was used to monitor its intracellular localization. Intense fluorescence shows up in the perinuclear region and in a reticular-network under the animal pole of the oocyte, consistent with the targeting of expressed GFP-InsP3R(ES) to perinuclear ER and ER under the animal pole. These findings are consistent with the intracellular localization of the endogenous Xenopus InsP3R shown previously. Electron microscopy data indicate that expressed GFP-InsP3R(ES) is in fact targeted to the ER. Sodium carbonate extraction of microsomal membranes and cross-linking experiments indicate that the expressed chimeric protein is in fact membrane anchored and able to form a homotetrameric complex. These data provide evidence that InsP3R(ES) constitutes the membrane spanning domain of the InsP3R and is able to mediate homotetramer formation, without the need for the large N-terminal cytoplasmic domain. The localization of GFP-InsP3R(ES) on the ER indicates that an ER retention/targeting signal is contained within the transmembrane spanning domain of the inositol trisphosphate receptor (Sayers, 1997).

Inositol 1,4,5-trisphosphate receptor (InsP3R) is an inositol InsP3-gated Ca2+ release channel. Type 1 InsP3R (InsP3R1) is the neuronal member of the IP3R family in the CNS and is predominantly expressed in cerebellar Purkinje cells. To elucidate the molecular mechanisms responsible for coupling gene expression to neuronal InsP3/Ca2+ signaling, the structure and function of the 5'-flanking region of the mouse InsP3R1 gene has been studied. The cloned 5'-flanking region has several sequences sharing identity with motifs for known transcriptional regulation. 5'-flanking regions 1N (from -528 to +169) and 4N (from -4,187 to +169) were fused to a beta-galactosidase gene (lacZ) as a reporter marker and their in vivo gene expression characterized. Both 1N and 4N fusion genes function as strong promoters in a neuroblastoma-glioma hybrid cell line NG108-15. Moreover, both 1N and 4N transgenic mouse lines carrying these 1N and 4N fusion genes show characteristic patterns of beta-galactosidase activity in the CNS that are almost consistent with that of the endogenous InsP3R1 protein, thereby suggesting that the 1N region from -528 to +169 contains sequence elements responsible for regulating gene expression in neurons and for specifying predominant expression in cerebellar Purkinje cells (Furutama, 1996).

The amino acid sequence responsible for the calmodulin (CaM)-binding ability of mouse type 1 InsP3 receptor (InsP3R1) was determined. Various fragments of InsP3R1 were examined for their CaM-binding ability. The sequence stretching from Lys-1564 to Arg-1585 is necessary for the binding. The full-length InsP3R1 with replacement of Trp-1576 by Ala has lost its CaM-binding ability. Antibody against residues 1564-1585 of InsP3R1 inhibits cerebellar InsP3R1 from binding CaM. The fluorescence spectrum of the peptide that corresponds to residues 1564-1585 shifts when Ca(2+)-CaM is added. From the change in the fluorescence spectrum, the dissociation constant (KD) between the peptide and CaM was estimated to be 0.7 microM. The submicromolar value of KD suggests an actual interaction within cells takes place between CaM and InsP3R1. The CaM-binding ability of other types of InsP3Rs was also examined. A part of the type 2InsP3R, including the region showing sequence identity with the CaM-binding domain of IP3R1, also binds CaM, while the expressed full-length type 3 InsP3R does not (Yamada, 1995).

To study the Ca2+ regulation of the inositol 1,4,5-trisphosphate receptor (InsP3R) at the molecular level, the mouse type I InsP3R was expressed as a glutathione S-transferase fusion protein. Both cytosolic and luminal Ca2+ binding sites exist. The luminal Ca2+ binding site maps to the nonconserved acidic subregion of the luminal loop between amino acids 2463 and 2528. The cytosolic Ca2+ binding site is localized in a region just preceding the transmembrane domain M1. The Ca2+ binding site maps to a 23-amino acid stretch between amino acids 2124 and 2146. This cytosolic region showed a single high affinity site for Ca2+. Neither of the identified Ca2+ binding regions contain an EF-hand motif. It is concluded that the type I InsP3R has at least two quite distinct types of Ca2+ binding sites, localized in different structural regions of the protein (Sienaert, 1996).

Structural and functional analyses were used to investigate the regulation of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) by Ca2+. To define the structural determinants for Ca2+ binding, cDNAs encoding GST fusion proteins that cover the complete linear cytosolic sequence of the InsP3R-1 were expressed in bacteria. The fusion proteins were screened for Ca2+ and ruthenium red binding through the use of 45Ca2+ and ruthenium red overlay procedures. Six new cytosolic Ca2+-binding regions were detected on the InsP3R, in addition to the one described earlier. Strong 45Ca2+ and ruthenium red binding domains are found in the N-terminal region of the InsP3R as follows: two Ca2+-binding domains are located within the InsP3-binding domain, and three Ca2+ binding stretches are located in a 500-amino acid region just downstream of the InsP3-binding domain. A sixth Ca2+-binding stretch is detected in the proximity of the calmodulin-binding domain. Evidence for the involvement of multiple Ca2+-binding sites in the regulation of the InsP3R was obtained from functional studies on permeabilized A7r5 cells, in which the effects of Ca2+ and Sr2+ on the EC50 were examined, as well as the cooperativity of the InsP3-induced Ca2+ release. The activation by cytosolic Ca2+ is due to a shift in EC50 toward lower InsP3 concentrations; this effect is mimicked by Sr2+. The inhibition by cytosolic Ca2+ is caused by a decrease in cooperativity and by a shift in EC50 toward higher InsP3 concentrations. The effect on cooperativity occurs at lower Ca2+ concentrations than the inhibitory effect on the EC50. Sr2+ mimics the effect of Ca2+ on the cooperativity but not the inhibitory effect on the EC50. The different [Ca2+] and [Sr2+] dependencies suggest that three different cytosolic interaction sites are involved. Luminal Ca2+ stimulates the release without affecting the Hill coefficient or the EC50, excluding the involvement of one of the cytosolic Ca2+-binding sites. It is concluded that multiple Ca2+-binding sites are localized on the InsP3R-1 and that at least four different Ca2+-interaction sites may be involved in the complex feedback regulation of the release by Ca2+ (Sienaert, 1997).

To define the structural determinants for inositol 1,4, 5-trisphosphate (InsP3) binding of the type 1 inositol 1,4, 5-trisphosphate receptor (InsP3R1), a means of expressing the N-terminal 734 amino acids of InsP3R1 (T734) that contain the InsP3 binding region was developed in Escherichia coli. The T734 protein expressed in E. coli exhibits a similar binding specificity and affinity for InsP3 as the native InsP3R from mouse cerebellum. Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduces InsP3 binding activity. However, when deleted a little more toward the C terminus (to residues 220, 223, and 225), the binding activity is retrieved. Further N-terminal deletions over the first 228 amino acids completely abolish it again. C-terminal deletions up to residue 579 do not affect the binding activity, whereas those up to residue 568 completely abolish it. In addition, the expressed 356-amino acid polypeptide (residues 224-579) exhibits specific binding activity. Taken together, residues 226-578 are sufficient and close enough to the minimum region for the specific InsP3 binding, and thus form an InsP3 binding "core." Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amino acids of T734. Single amino acid substitutions for 10 residues, which are widely distributed within the binding core and conserved among all members of the InsP3R family, significantly reduce the binding activity. Among these residues three (Arg-265, Lys-508, and Arg-511) are critical for the specific binding, and Arg-568 is implicated in the binding specificity for various inositol phosphates. It is suggested that some of these 10 residues form a basic pocket that interacts with the negatively charged phosphate groups of InsP3 (Yoshikawa, 1996).

Subtypes of the type-1 inositol 1,4,5-trisphosphate (InsP3) receptor differ at the mRNA level in two small variably spliced segments. Segment SI encodes for a sequence within the InsP3-binding domain, thus its presence or absence could affect the functions of the receptor. Anti-peptide antibodies were used to confirm the existence of different subtypes of the InsP3 receptor (InsP3R) protein. The antibody against residues 322-332 within the SI region recognizes a 260 kDa polypeptide in membranes prepared from rat cerebellum or cerebral cortex. The cerebellum contains a few percent of the InsP3R protein having the SI region, whereas the cerebral cortex contained a high proportion of receptors with the SI region. These two tissues are representative of both isoforms, SI- or SI+, and display the same [3H]InsP3-binding characteristics. Thus, the SI region is not involved in the basic properties of the receptor. Deletion of the peptide 316-352 containing the SI segment greatly reduces InsP3 binding. Antibodies against the SI region or against residues 337-349 do not modify the binding of [3H]InsP3 in the cortical membranes rich in the SI+ isoform or in cerebellar membranes. These results suggest that the SI region is not part of the binding site. The subcellular distribution of these two isoforms was investigated in rat liver. The two isoforms are identified in different membrane fractions and they follow the same subcellular distribution. It is suggested that the domain with the SI region may be involved in a function other than InsP3-induced Ca2+ release (Lievremont, 1996).

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of intracellular Ca2+ channels that exist as homo- or hetero-tetramers. In order to determine whether the N-terminal ligand-binding domain is in close physical proximity to the C-terminal pore domain, microsomal membranes were prepared from COS-7 cells expressing recombinant type I and type III IP3R isoforms. Trypsin digestion followed by cross-linking and co-immunoprecipitation of peptide fragments suggest an inter-subunit N- and C-terminal interaction in both homo- and hetero-tetramers. This observation is further supported by the ability of in vitro translated C-terminal peptides to interact specifically with an N-terminal fusion protein. Using a ⁴⁵Ca2+ flux assay, functional evidence that the ligand-binding domain of one subunit can gate the pore domain of an adjacent subunit is provided. It is concluded that common structural motifs are shared between the type I and type III IP3Rs and it is proposed that the gating mechanism of IP3R Ca2+ channels involves the association of the N-terminus of one subunit with the C-terminus of an adjacent subunit in both homo- and heterotetrameric complexes (Boehning, 2000).

Many important cell functions are controlled by Ca2+ release from intracellular stores via the inositol 1,4,5-trisphosphate receptor (IP3R), which requires both IP3 and Ca2+ for its activity. Due to the Ca2+ requirement, the IP3R and the cytoplasmic Ca2+ concentration form a positive feedback loop, which has been assumed to confer regenerativity on the IP3-induced Ca2+ release and to play an important role in the generation of spatiotemporal patterns of Ca2+ signals such as Ca2+ waves and oscillations. Glutamate 2100 of rat type 1 IP3R (IP3R1) is a key residue for the Ca2+ requirement. Substitution of this residue by aspartate (E2100D) results in a 10-fold decrease in the Ca2+ sensitivity without other effects on the properties of the IP3R1. Agonist-induced Ca2+ responses are greatly diminished in cells expressing the E2100D mutant IP3R1, particularly the rate of rise of the initial Ca2+ spike is markedly reduced and the subsequent Ca2+ oscillations are abolished. These results demonstrate that the Ca2+ sensitivity of the IP3R is functionally indispensable for the determination of Ca2+ signaling patterns (Miyakawa, 2001).

In a variety of cells, the Ca2+ signalling process is mediated by the endoplasmic-reticulum-membrane-associated Ca2+ release channel, inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Being ubiquitous and present in organisms ranging from humans to Caenorhabditis elegans, InsP3R has a vital role in the control of cellular and physiological processes as diverse as cell division, cell proliferation, apoptosis, fertilization, development, behavior, memory and learning. Mouse type I InsP3R (InsP3R1), found in high abundance in cerebellar Purkinje cells, is a polypeptide with three major functionally distinct regions: the amino-terminal InsP3-binding region, the central modulatory region and the carboxy-terminal channel region. A 2.2-Å crystal structure of the InsP3-binding core of mouse InsP3R1 is presented in complex with InsP3. The asymmetric, boomerang-like structure consists of an N-terminal ß-trefoil domain and a C-terminal alpha-helical domain containing an 'armadillo repeat'-like fold. The cleft formed by the two domains exposes a cluster of arginine and lysine residues that coordinate the three phosphoryl groups of InsP3. Putative Ca2+-binding sites are identified in two separate locations within the InsP3-binding core (Bosanac, 2002).

The importance of a direct coupling between InsP3 and Ca2+ binding to the cytoplasmic portion of InsP3R in the regulation of Ca2+ release channel activity has already been established. Truncation mutagenesis studies have identified two Ca2+-binding fragments of InsP3R, one encoding residues 304-381 and the other corresponding to residues 378-450. Residues E425, D426, E428, D442 and D444 have been shown to be essential for Ca2+ coordination. These residues are part of the two surface acidic clusters identified in the present crystal structure of mouse InsP3R1c. The first site, Ca-I, is located in the ß-domain and consists of residues E246, E425, D426 and E428. The second site, Ca-II, located across the two domains, is composed of residues E283, E285, D444 and D448. Interestingly, Ca2+-binding site Ca-II overlaps with the conserved region P-II, suggesting that the binding of Ca2+ to this site is conformationally coupled with the protein-protein interaction involving other protein domain(s). This finding, together with electron microscope studies of InsP3R and biochemical studies, leads to a tempting speculation on the Ca2+-InsP3 coupling mechanism required for channel activation. The role of binding of InsP3 to the core domain (residues 226-576) might include the release of a conformational constraint that prevents Ca2+ from binding to the receptor. The N-terminal InsP3 binding suppressor region (residues 1-225) might be directly involved in this negative regulation of Ca2+ binding to the receptor, in addition to the modulation of InsP3 binding affinity20. It is equally possible that some other part of the InsP3R or an unidentified cellular protein is involved in this InsP3-Ca2+ coupling mechanism (Bosanac, 2002).

Expression and function of multiple InsP3R isoforms

The expression of inositol 1,4,5-trisphosphate receptor type 1 (InsP3R1) in the mouse central nervous system (CNS) was studied by in situ hybridization. The receptor mRNAs are widely localized throughout the CNS, predominantly in the olfactory tubercle, cerebral cortex, CA1 pyramidal cell layer of the hippocampus, caudate putamen, and cerebellar Purkinje cells, where phosphoinositide turnover is known to be stimulated by various neurotransmitter receptors. In the Purkinje cells where InsP3R1 mRNA is most abundantly expressed, transcript appears to be translocated to the distal dendrites, since a strong hybridization density is observed in the molecular layer of the cerebellum. The preliminary hybridization data using probes for three distinct InsP3R subtypes show preferential expression of InsP3R1 in many parts of the CNS. The expression level of other receptor subtypes (InsP3R2 and InsP3R3) is quantitatively lower, suggesting that a homotetramer formed of InsP3R1 subtype may play a central part in InsP3/Ca2+ signaling in the neuronal function, whereas a homotetramer of other subtypes and a possible heterotetramer among subtypes may be involved in differential InsP3/Ca2+ signalling (Furuichi, 1993).

The distribution of the type 1 InsP3R (InsP3R1) was studied during development. In brain, InsP3R1 is expressed in neurons from very early in development; low levels of expression are first detected after the neurons have migrated to their final positions, when they start to differentiate and begin axonal growth. Increasing levels of expression are observed later in development, during the time of synaptogenesis and dendritic contact. Glial cells do not express InsP3R1, except for a transient period of expression, probably by oligodendrocytes in developing fiber tracts during the onset of myelination. In contrast in the brain, both grey and white matter of the spinal cord express InsP3R1 throughout development, and it remains present in the adult spinal cord. InsP3R1 is expressed in the peripheral nervous system. Strong labelling is observed in the dorsal root ganglia; during development this expression seems to coincide with the onset of axonogenesis. These results suggest that InsP3 may be involved in the regulatory mechanism controlling Ca2+ levels in neurons during the periods of cell differentiation, axonogenesis and synaptogenesis (Dent, 1996).

The human type 1 inositol 1,4,5-trisphosphate receptor contains the S2 splice site, which appears to be the region most divergent between rat and human. An additional alternatively spliced region 9 amino acids long (termed S3) is found in the coupling domain. Alternatively spliced forms are found in both human and rat. PCR analysis of brain and peripheral tissues from human and rat shows both transcripts of the type 1 InsP3R in all tissues. The long form predominates in most brain regions (except the cerebellum) while the short form predominates in peripheral tissues. The sequence of the longer form in human appears to create an additional consensus protein kinase C phosphorylation site (Nucifora, 1995).

Three types of InsP3 receptors have been identified in mammals. The three receptor types are encoded by homologous genes and are structurally similar, suggesting two alternative hypotheses about the biological significance of multiple InsP3 receptors: (1) the different InsP3 receptors could have similar functions as InsP3-gated Ca2+ channels, and the presence of multiple genes could then serve as a mechanism to allow tissue-specific differential expression of receptors; or (2) the different receptors are co-expressed in cells but have distinct biological roles in these cells. To test these hypothesis, the similarities and differences between the expression, alternative splicing, and ligand binding of different receptors were investigated. Co-expression of different InsP3 receptors occurs in almost all tissues and cell lines tested. Although all receptor types exhibit a similar specificity for inositol phosphates, the different receptors have different affinities for InsP3, with a relative order of affinities of type 2 > type1> type 3. These findings suggest that the presence of multiple InsP3-sensitive Ca2+ pools with differential responsiveness to InsP3 may be a general property of all cells mediated by the presence of multiple types of InsP3 receptors (Newton, 1994).

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ release channel responsible for mobilizing stored Ca2+. Three different receptor types have been molecularly cloned, and their genes have been classified into a family. The gene for the type 1 receptor (InsP3R1) is predominantly expressed in cerebellar Purkinje neurons, but its gene product is localized widely in a variety of tissues. There is, however, little information on what types of cells express the other two receptor types, type 2 and type 3 (InsP3R2 and InsP3R3, respectively). Compared with InsP3R1, the levels of expression of InsP3R2 and InsP3R3 mRNAs are low in all of the tissues tested. InsP3R2 mRNA is localized in the intralobular duct cells of the submandibular gland, the urinary tubule cells of the kidney, the epithelial cells of epididymal ducts and the follicular granulosa cells of the ovary, while the InsP3R3 mRNA is distributed in gastric cells, salivary and pancreatic acinar cells and the epithelium of the small intestine. All of these cells that express either InsP3R2 or InsP3R3 mRNA are known to have a secretory function in which InsP3/Ca2+ signaling has been shown to be involved, and thus either InsP3R2 or InsP3R3 may be a prerequisite to secretion in these cells (Fujino, 1995).

There are at least three types of InsP3R subunits, designated type 1, type 2, and type 3. The levels of expression of InsP3R subunits in various cell lines were investigated by Western blot analysis using type-specific antibodies against 15 C-terminal amino acids for each InsP3R subunit. All the three types of InsP3R subunits are expressed in each cell line examined, but their levels of expression vary. The distinct types of InsP3R subunits assemble to form heterotetramers in CHO-K1 cells. Heterotetramers were also found in rat liver, in which InsP3R type 1 and type 2 are expressed abundantly (Monkawa, 1995).

A distinctive feature of many endothelia is an abundant population of noncoated plasmalemmal vesicles, or caveolae. Caveolae have been implicated in many important cellular processes, including transcytosis, endocytosis, potocytosis, and even signal transduction. Because caveolae have not been purified from endothelial cell surfaces, little is known directly about their structure and function in the endothelium. To delineate the transport role of these caveolae, they have been purified from isolated luminal endothelial plasma membranes of rat lung. The rat lung luminal endothelial cell surfaces were isolated after coating them, in situ, with positively charged colloidal silica. The caveolae were then separated from these coated membranes and purified to yield a homogeneous population of morphologically distinct vesicles enriched in the structural protein caveolin. As with caveolae found on the endothelial cell surface in vivo, these highly purified caveolae contain the plasmalemmal Ca(2+)-ATPase and inositol 1,4,5-trisphosphate surface receptors. By contrast, other plasma membrane proteins excluded from the caveolae include angiotensin-converting enzyme, beta-actin, and band 4.1. The purified caveolae appear to represent specific microdomains of the cell surface with their own unique molecular topography (Schnitzer, 1995).

Stimulation of B-cell antigen receptor (BCR) induces a rapid increase in cytoplasmic free calcium due to the release of calcium from intracellular stores and an influx from the extracellular environment. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ligand-gated channels that release intracellular calcium stores in response to the second messenger, inositol 1,4,5-trisphosphate. Most hematopoietic cells, including B cells, express at least two of the three different types of IP3R. B cells in which a single type of IP3R has been deleted still mobilize calcium in response to BCR stimulation, whereas this calcium mobilization is abrogated in B cells lacking all three types of IP3R. Calcium mobilization by a transfected G protein-coupled receptor (muscarinic M1 receptor) is also abolished in only triple-deficient cells. Capacitative Ca2+ entry, stimulated by thapsigargin, remains unaffected by loss of all three types of IP3R. These data establish that IP3Rs are essential and functionally redundant mediators for both BCR- and muscarinic receptor-induced calcium mobilization, but not for thapsigargin-induced Ca2+ influx. BCR-induced apoptosis is significantly inhibited by loss of all three types of IP3R, suggesting an important role for Ca2+ in the process of apoptosis (Sugawara, 1997).

The potential role of IP3 receptors during nuclear envelope assembly in vitro was investigated using Xenopus egg extracts. Previous work suggests that Ca2+ mobilization is required for nuclear vesicle fusion and implicates IP3 receptor activity. To test the involvement of IP3 receptors using selective reagents, three distinct polyclonal antibodies were obtained to the type 1 IP3 receptor. Pretreatment of membranes with two of the antibodies inhibits IP3-stimulated Ca2+ release in vitro and also inhibits nuclear vesicle fusion. One inhibitory serum is directed against 420 residues within the "coupling" domain, which includes several potential regulatory sites. The other inhibitory serum is directed against 95 residues near the C terminus and identifies an inhibitory epitope(s) in this region. The antibodies have no effect on receptor affinity for IP3. Because nuclear vesicle fusion is inhibited by antibodies that block Ca2+ flux, it is concluded that the activation of IP3 receptors is required for fusion. The signal that activates the channel during fusion is unknown (Sullivan, 1995).

InsP3 is extensively metabolized through a network of phosphorylation and dephosphorylation steps to products with potential second messenger function. Inositol 1,3,4,5-tetrakisphosphate [InsP4(1,3,4,5)], the direct metabolite of InsP3, has also been associated with Ca2+ signaling, but whether InsP4(1,3,4,5) acts in combination with InsP3 or whether it regulates Ca2+ signaling directly and independently is unclear, particularly in neurons. Olfactory receptor neurons in the lobster (Panulirus argus) express an InsP4(1,3,4,5) receptor in the plasma membrane that is a functional channel. The channel differs in conductance, kinetics, and voltage sensitivity from two plasma membrane InsP3-gated channels previously reported in these neurons. In close spatial proximity, the InsP4(1,3,4,5)-and InsP3-gated channels interact reciprocally to alter the channels' open probabilities in what may be a novel mechanism for regulating Ca2+ entry in neurons (Fadool, 1994).

Inositol 1,4,5-trisphosphate (IP3) plays a key role in Ca2+ signaling. Ca2+ signaling generates a variety of spatio-temporal patterns that control important cell functions. Multiple subtypes of IP3 receptors (IP3R-1, -2 and -3) are expressed in a tissue- and development-specific manner and form heterotetrameric channels through which stored Ca2+ is released, but the physiological significance of the differential expression of IP3R subtypes is not known. The Ca2+-signaling mechanism was studied in genetically engineered B cells that express either a single or a combination of IP3R subtypes. Ca2+-signaling patterns depend on the IP3R subtypes, which differ significantly in their response to agonists, i.e. IP3, Ca2+ and ATP. IP3R-2 is the most sensitive to IP3 and is required for the long lasting, regular Ca2+ oscillations that occur upon activation of B-cell receptors. IP3R-1 is highly sensitive to ATP and mediates less regular Ca2+ oscillations. IP3R-3 is the least sensitive to IP3 and Ca2+, and tends to generate monophasic Ca2+ transients. Functional interactions are shown between coexpressed subtypes. These results demonstrate that differential expression of IP3R subtypes helps to encode IP3-mediated Ca2+ signaling (Miyakawa, 1999).

InsP3 receptors: Requirement for store-operated Ca2+ channels

Receptor-induced Ca2+ signals comprise two interdependent components -- rapid Ca2+ release from Ca2+ stores in the endoplasmic reticulum (ER) and Ca2+ entry through slowly activating plasma membrane (PM) store-operated channels (SOCs). The trigger for SOC activation is decreased Ca2+ in the ER lumen. The coupling mechanism between ER calcium ion stores and PM store-operated channels SOCs is crucial to Ca2+ signaling but has eluded detection. Direct coupling between ER and PM has been hypothesized, and evidence indicates that physical docking of ER with the PM is involved in SOC activation. The mammalian TRP family of receptor-operated ion channels has been suggested to share some operational parameters with SOCs. Evidence has been provided that human TRP3 channel activation results from interaction with InsP3Rs. However, other evidence indicates that diacylglycerol (DAG), not InsP3, is the phospholipase C (PLC) product that mediates activation of TRP3 channels and that TRP3 channels operate independent of stores (Ma, 2000 and references therein).

SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells have revealed that TRP3 channels differ by being store independent. However, condensed cortical F-actin prevents activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevents both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs (Ma, 2000).

InsP3 receptors: their regulation, and calcium dynamics

The ability of cAMP-dependent protein kinase (PKA) to phosphorylate type I, II, and III inositol 1,4,5-trisphosphate (InsP3) receptors was examined. The receptors either were immunopurified from cell lines and then phosphorylated with purified PKA or were phosphorylated in intact cells after activating intracellular cAMP formation. The type I receptor is a good substrate for PKA, whereas type II and III receptors are phosphorylated relatively weakly. Despite these differences, each of the receptors is phosphorylated in intact cells in response to forskolin or activation of neurohormone receptors. Detailed examination of SH-SY5Y neuroblastoma cells, which express type I receptor, reveal that minor increases in cAMP concentration are sufficient to cause maximal phosphorylation. Thus, VIP and pituitary adenylyl cyclase activating peptide (acting through Gs-coupled pituitary adenylyl cyclase activating peptide-I receptors) are potent stimuli of type I receptor phosphorylation, and remarkably, even slight increases in cAMP concentration induced by carbachol (acting through Gq-coupled muscarinic receptors) or other Ca2+ mobilizing agents are sufficient to cause phosphorylation. Finally, PKA enhances InsP3-induced Ca2+ mobilization in a range of permeabilized cell types, irrespective of whether the type I, II, or III receptor is predominant. In summary, these data show that all InsP3 receptors are phosphorylated by PKA, albeit with marked differences in stoichiometry. The ability of both Gs- and Gq-coupled cell surface receptors to effect InsP3 receptor phosphorylation by PKA suggests that this process is widespread in mammalian cells and provides multiple routes by which the cAMP signaling pathway can influence Ca2+ mobilization (Wojcikiewicz, 1998).

Cyclic nucleotide-regulated phosphorylation of the neuronal type I inositol 1,4,5-trisphosphate (IP3) receptor immunopurified from rat cerebellar membranes was examined in vitro and in rat cerebellar slices in situ. The isolated IP3 receptor protein is phosphorylated by both cAMP- and cGMP-dependent protein kinases on two distinct sites as determined by thermolytic phosphopeptide mapping, phosphopeptide 1, representing Ser-1589, and phosphopeptide 2, representing Ser-1756 in the rat protein. Phosphopeptide maps show that cAMP-dependent protein kinase (PKA) labels both sites with the same time course and same stoichiometry, whereas cGMP-dependent protein kinase (PKG) phosphorylates Ser-1756 with a higher velocity and a higher stoichiometry than Ser-1589. Synthetic decapeptides corresponding to the two phosphorylation sites [peptide 1, AARRDSVLAA (Ser-1589), and peptide 2, SGRRESLTSF (Ser-1756)] were used to determine kinetic constants for the phosphorylation by PKG and PKA, and the catalytic efficiencies were in agreement with the results obtained by in vitro phosphorylation of the intact protein. In cerebellar slices prelabeled with [32P]orthophosphate, activation of endogenous kinases by incubation in the presence of cAMP/cGMP analogs and specific inhibitors of PKG and PKA induces in both cases a 3-fold increase in phosphorylation of the IP3 receptor. Thermolytic phosphopeptide mapping of in situ labeled IP3 receptor by PKA shows labeling on the same sites (Ser-1589 and Ser-1756) as in vitro. In contrast to the findings in vitro, PKG preferentially phosphorylates Ser-1589 in situ. Because both PKG and the IP3 receptor are specifically enriched in cerebellar Purkinje cells, PKG may be an important IP3 receptor regulator in vivo (Haug, 1999).

In the central nervous system, release of Ca2+ from intracellular stores contributes to numerous functions, including neurotransmitter release and long-term potentiation and depression. The developmental profile and the regulation of inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) was examined in primary cultures of cerebellar granule cells. The expression of both receptor types increases during development. Whereas the expression of type 1 IP3R appears to be regulated by Ca2+ influx through L type channels or N-methyl-D-aspartate (NMDA) receptors, RyR levels increase independent of Ca2+. The main target of Ca2+-influx-regulating IP3R expression is the Ca2+ calmodulin-dependent protein phosphatase calcineurin, because pharmacological blockade of this protein abolishes IP3R expression. Although calcineurin has been shown to regulate the phosphorylation state of the IP3R, the effect described here is at the transcriptional level because IP3R mRNA changes in parallel with protein levels. Thus, calcineurin plays a dual role in IP3R-mediated Ca2+ signaling: it regulates IP3R function by dephosphorylation in the short-term time scale and IP3R expression over more extended periods (Genazzani, 1999).

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP3), and pure cerebellar InsP3 receptors were characterized by using a scintillation proximity assay. In the absence of Ca2+, 125I-labeled calmodulin reversibly binds to multiple sites on InsP3 receptors and Ca2+ increases the binding by 190% +/- 10%; the half-maximal effect occurs when the Ca2+ concentration is 184 +/- 14 nM. In the absence of Ca2+, calmodulin causes a reversible, concentration-dependent (IC50 = 3.1 +/- 0.2 microM) inhibition of [3H]InsP3 binding by decreasing the affinity of the receptor for InsP3. This effect is similar at all Ca2+ concentrations, indicating that the site through which calmodulin inhibits InsP3 binding has similar affinities for calmodulin and Ca2+-calmodulin. Calmodulin (10 microM) inhibita the Ca2+ release from cerebellar microsomes evoked by submaximal (but not by maximal) concentrations of InsP3. Tonic inhibition of InsP3 receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to InsP3 and limit spontaneous activation of InsP3 receptors in the dendritic spines. Inhibition of InsP3 receptors by calmodulin at all cytosolic Ca2+ concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca2+, suggests that calmodulin may also allow both feedback control of InsP3 receptors and integration of inputs from other signaling pathways (Patel, 1997).

The second messenger inositol-1,4,5-trisphosphate (InsP3) releases Ca2+ from intracellular Ca2+ stores by activating specific receptors on the membranes of these stores. In many cells, InsP3 is a global signalling molecule that liberates Ca2+ throughout the cytoplasm. However, in neurons the situation might be different, because synaptic activity may produce InsP3 at discrete locations. InsP3 signaling was characterized in postsynaptic cerebellar Purkinje neurons, which have a high level of InsP3 receptors. Repetitive activation of the synapse between parallel fibers and Purkinje cells causes InsP3-mediated Ca2+ release in the Purkinje cells. This Ca2+ release is restricted to individual postsynaptic spines, where both metabotropic glutamate receptors and InsP3 receptors are located, or to multiple spines and adjacent dendritic shafts. Focal photolysis of caged InsP3 in Purkinje cell dendrites also produces Ca2+ signals that spread only a few micrometers from the site of InsP3 production. Uncaged InsP3 produces a long-lasting depression of parallel-fiber synaptic transmission that is limited to synapses where the Ca2+ concentration is raised. Thus, in Purkinje cells InP3 acts within a restricted spatial range that allows it to regulate the function of local groups of parallel-fiber synapses (Finch, 1999).

The inositol 1,4,5-trisphosphate receptor (InsP3R) is the main calcium (Ca2+) release channel in most tissues. Three isoforms have been identified, but only types I and II InsP3R have been characterized. The functional properties of the type III InsP3R were examined because this receptor is restricted to the trigger zone from which Ca2+ waves originate and it has distinctive InsP3-binding properties. Type III InsP3R forms Ca2+ channels with single-channel currents that are similar to those of type I InsP3R; however, the open probability of type III InsP3R isoform increases monotonically with increased cytoplasmic Ca2+ concentration, whereas the type I isoform has a bell-shaped dependence on cytoplasmic Ca2+. The properties of type III InsP3R provide positive feedback as Ca2+ is released; the lack of negative feedback allows complete Ca2+ release from intracellular stores. Thus, activation of type III InsP3R in cells that express only this isoform results in a single transient, but global, increase in the concentration of cytosolic Ca2+. The bell-shaped Ca2+-dependence curve of type I InsP3R is ideal for supporting Ca2+ oscillations, whereas the properties of type III InsP3R are better suited to signal initiation (Hagar, 1998).

Calcium ions are released from intracellular stores in response to agonist-stimulated production of inositol 1,4,5-trisphosphate (InsP3), a second messenger generated at the cell membrane. Depletion of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+ across the plasma membrane. The influx of Ca2+ can be recorded as store-operated channels (SOC) in the plasma membrane or as a current known as the Ca2+-release-activated current [I(crac)]. A critical question in cell signaling is how SOC and I(crac) sense and respond to Ca2+-store depletion: in one model, a messenger molecule is generated that activates Ca2+ entry in response to store depletion; in an alternative model, InsP3 receptors in the stores are coupled to SOC and I(crac). The mammalian Htrp3 protein forms a well defined store-operated channel and so provides a suitable system for studying the effect of Ca2+-store depletion on SOC and I(crac). Htrp3 channels stably expressed in HEK293 cells are in a tight functional interaction with the InsP3 receptors. Htrp3 channels present in the same plasma membrane patch can be activated by Ca2+ mobilization in intact cells and by InsP3 in excised patches. This activation of Htrp3 by InsP3 is lost on extensive washing of excised patches but is restored by addition of native or recombinant InsP3-bound InsP3 receptors. These results provide evidence for the coupling hypothesis, in which InsP3 receptors activated by InsP3 interact with SOC and regulate I(crac) (Kiselyov, 1998).

The interaction of intracellular free calcium ([Ca2+]i) and cAMP signaling mechanisms was examined in intact single megakaryocytes by using a combination of single-cell fluorescence microscopy to measure [Ca2+]i and flash photolysis of caged Ca2+, inositol 1,4, 5-trisphosphate (IP3), and/or cAMP in order to rapidly elevate the concentration of these compounds inside the cell. Photolysis of caged IP3 stimulates Ca2+ release from an IP3-sensitive store. The cAMP-elevating agent carbacyclin inhibits this IP3-induced rise in [Ca2+]i but does not affect the rate of Ca2+ removal from the cytoplasm after photolysis of caged Ca2+. Photolysis of caged cAMP during ADP-induced [Ca2+]i oscillations causes the [Ca2+]i oscillation to transiently cease without affecting the rate of Ca2+ uptake and/or extrusion. It has been concluded that the principal mechanism of cAMP-dependent inhibition of Ca2+ mobilization in megakaryocytes appears to be inhibition of IP3-induced Ca2+ release and not stimulation of Ca2+ removal from the cytoplasm. Two inhibitors of cAMP-dependent protein kinase, a specific peptide inhibitor of the catalytic subunit of cAMP protein kinase and KT5720, block the inhibitory effect of carbacyclin, indicating that the inhibition of IP3-induced Ca2+-release by carbacyclin is mediated by cAMP-dependent protein kinase. These results imply that cAMP protein kinase is required for inhibition of IP3-induced Ca2+-release, although it is not clear whether this inhibition requires phosphorylation of only the IP3 receptor, or if the phosphorylation of other proteins is involved (Tertyshnikova, 1998).

Repetitive transient increases in cytosolic calcium concentration (calcium spikes or calcium oscillations) are a common mode of signal transduction in receptor-mediated cell activation. Repetitive calcium spikes are initiated by phospholipase C-mediated production of inositol 1,4,5-trisphosphate (InsP3) and are thought to be generated by a positive feedback mechanism in which calcium potentiates its own release, a negative feedback mechanism by which calcium release is terminated, and a slow recovery process that defines the time interval between calcium spikes. The molecular mechanisms that terminate each calcium spike and define the spike frequency are not yet known. In intact rat basophilic leukemia cells calcium responses induced by InsP3 are diminished for a period of 30-60 s following an InsP3-induced calcium spike. The sensitivity of calcium release for InsP3 was probed by UV laser-mediated photorelease of InsP3, and calcium responses were monitored by fluorescence calcium imaging. A maximal loss in sensitivity (desensitization) is observed for InsP3 increases that result in a near maximal calcium spike and is expressed as an 80-100% reduction in the calcium response to an equal amount of InsP3, released 10 s after the first UV pulse. When the amount of released InsP3 in the second pulse is increased 2-3-fold, desensitization is overcome and a second calcium response of equal amplitude to the first is produced. A power dependence of 3.2 was measured between the amount of released InsP3 and the amplitude of the triggered calcium response, explaining how a small decrease in InsP3 sensitivity can lead to a nearly complete reduction in the calcium response. Desensitization is abolished by the addition of the calcium buffers BAPTA and EGTA and can be induced by microinjection of calcium, suggesting that it is a calcium-dependent process. Half-maximal desensitization is observed at a free calcium concentration of 290 nM and increases with a power of 3.7 with peak calcium concentration. These studies suggest that reversible desensitization of InsP3-induced calcium release serves as a "saw-tooth" parameter that controls the termination of each spike and the frequency of calcium spikes (Oancea, 1996).

Activation of intracellular Ca2+ channels by inositol 1,4,5-trisphosphate (InsP3) represents the initial Ca2+ mobilization step in response to many extracellular signals. InsP3-induced channel activation in permeabilized hepatocytes is followed by a time-dependent inactivation, which is a direct consequence of ligand binding. The inactivation by InsP3 parallels the quantal character of channel opening, giving rise to a unique process of incremental inactivation whereby discrete channel populations are inhibited at each InsP3 dose. InsP3 can induce inactivation in the absence of stored Ca2+, but the inactivation rate is enhanced by increases of cytosolic Ca2+. The inhibitory effect of InsP3 can be reversed by InsP3 washout, or by chelation of cytosolic Ca2+. Thus, InsP3 and Ca2+ act as coinhibitors of the InsP3-sensitive Ca2+ channel. Inactivation is an inherent consequence of InsP3-induced channel opening that can terminate increases of cytosolic Ca2+ (Hajnoczky, 1994).

T cell receptor stimulation triggers a physical association between the nonreceptor protein tyrosine kinase Fyn and the InsP3R, which induces tyrosine phosphorylation of the InsP3R. Fyn activates an InsP3-gated calcium channel in vitro, and tyrosine phosphorylation of the InsP3R during T cell activation is reduced in thymocytes from fyn-/- mice. Thus, activation of the InsP3R by tyrosine phosphorylation may play a role in regulating intracellular calcium (Jayaraman, 1996).

Transient rise in nuclear calcium concentration is implicated in the regulation of events controlling gene expression. Various mechanisms by which calcium is transported to the nucleus are the subject of vehement debate. Inositol 1,4,5-trisphosphate (InsP3) and inositol-1,3,4,5-tetrakisphosphate (InsP4) receptors have been located to the nucleus and their role in nuclear calcium signaling has been proposed. Outer nuclear membrane was separated from the inner membrane. The two membrane preparations were, as best as possible, devoid of cross contamination as attested by marker enzyme activity, Western blotting with antilamin antibody, and electron microscopy. InsP4 receptor and Ca(2+)-ATPase were located to the outer nuclear membrane. InsP3 receptor was located to the inner nuclear membrane. ATP or InsP4 induces nuclear calcium uptake. External free calcium concentration, in the medium bathing the nuclei, determines the choice for ATP or InsP4-mediated calcium transport. A mechanistic model is presented for nuclear calcium transport. According to this model, calcium can reach the nucleus envelope either by the action of ATP or InsP4. However, the calcium release from the nucleus envelope to the nucleoplasm is mediated by InsP3 through the activation of InsP3 receptor, which is located to the inner nuclear membrane. The action of InsP3 in this process is instantaneous and transient and is sensitive to heparin (Humbert, 1996).

The immunophilin FK506 binding protein 12 (FKBP12) is associated with and modulates the ryanodine receptor calcium release channel of skeletal muscle. Ryanodine receptor has amino acid homology and functional similarity with another intracellular Ca2+ release channel, the inositol 1,4,5-trisphosphate receptor (InsP3R). Highly purified preparations of InsP3R contain FKBP12. The complex of these two proteins is disrupted by the immunosuppressants FK506 and rapamycin, both of which are known to bind FKBP12 with high affinity. Disrupting the InsP3R-FKBP12 interaction increases Ca2+ flux through InsP3R, an effect that is reversed by added FKBP12. FKBP12 appears to be physiologically linked to InsP3R, regulating its Ca2+ conductance (Cameron, 1995).

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor leads to activation of phospholipase C gamma isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5-10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 microM; maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4-2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36(M3R) cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4-2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacities to release Ca2+ when a subsequent stimulus results in production of InsP3 (Wilson, 1998).

During early embryonic development, IP₃-Ca²⁺ signaling transduces ventral signaling at the time of dorsoventral axis formation. To identify molecules functioning upstream in this signal pathway, effects were measured of a panel of inhibitory antibodies against Galphaq/11, Galphas/olf, or Galphai/o/t/z. While all these antibodies show direct inhibition of their targets, their effects varied in terms of the redirection of ventral mesoderm to a dorsal fate. Anti-Galphas/olf antibody shows strong induction of dorsal fate; anti-Galphai/o/t/z antibody does so weakly, and anti-Galphaq/11 antibody is without effect. Injection of betaARK, a Gbetagamma inhibitor, mimics the dorsalizing effect of anti-Galphas/olf antibody, whereas injection of adenylyl cyclase inhibitors at a concentration that inhibits Galphas-coupled cAMP increase does not do so. The activation of Galphas-coupled receptor gives rise to Ca²⁺ transients. All these results suggest that activation of the Galphas-coupled receptor relays dorsoventral signal to Gbetaggamma, which then stimulates PLCbeta and then the IP₃-Ca²⁺ system. This signaling pathway may play a crucial role in transducing ventral signals (Kume, 2000).

In Xenopus, patterning of the body axis occurs by sequential inductive events. Maternal activation of the Wnt pathway is required for the initiation of axis formation, by creating a Nieuwkoop center and mediating the dorsalizing function of the Nieuwkoop center. The Spemann organizer of the Xenopus embryo can be subdivided into two discrete activities: trunk organizer and head organizer. The molecular mechanism of trunk organizer formation involves several factors secreted from the blastopore lip that act by repressing signaling by bone morphogenetic proteins (BMPs), which antagonize the organizer. The finding that anti-Galphas/olf antibody or betaARK injection induces trunk but not head organizer is similar to a previous result using anti-IP3R inhibitory antibodies. These results correlate with the facts that the secondary axes induced by Noggin, Chordin, and truncated BMP receptors often lack the anteriormost structures and that head induction requires simultaneous repression of BMP and Wnt signaling in Xenopus. The IP₃-Ca²⁺ signaling system may crosstalk with the BMP pathway, by means of a mechanism which still remains unknown (Kume, 2000).

There are other candidates for upstream factors that may activate the IP₃-Ca²⁺ signaling system. Zygotic activation of the Wnt pathway is suggested to be required for ventro-lateral mesoderm formation. Members of the Wnt-5a class of proteins do not induce ectopic dorsal axis duplication, unless coexpressed with certain members of the frizzled family, yet they do decrease cell adhesion and perturb morphogenetic movement during gastrulation in Xenopus embryos. There is evidence that the Wnt-5a class can function in a non-cell-autonomous manner to block the ability of members of the Wnt-1 class to induce a secondary axis. Overexpression of Xwnt-5a with rat frizzled-2 increases the frequencies of Ca²⁺ spikes in zebrafish. Activation of Galphas/olf-coupled receptor elicits Ca²⁺ transients of an interval that resembles one reported for Xwnt-5a. It will be of interest to determine whether Xwnt-5a is the endogenous upstream ligand that activates IP₃-Ca²⁺ signaling during embryonic development (Kume, 2000).

The downstream targets of the Ca²⁺ transients during early embryonic axis formation remain largely unknown. There is evidence that varying the frequency or intensity of Ca²⁺ transients can alter the physiological output. One well-known example of molecules modulated by frequency of Ca²⁺ is calmodulin-dependent kinase II, which regulates other enzymes dependent on Ca²⁺. The enzyme is activated to varying degrees depending on the frequency of Ca²⁺ oscillations. Varying the frequency or intensity of the Ca²⁺ rise can contribute to activation of different subsets of developmental genes. Examinations of the types of IP₃-Ca²⁺ signaling that activate separate sets of genes and, in turn, lead to a specific developmental program, are expected to elucidate the mechanism underlying the early developmental events (Kume, 2000).

A novel function of the presenilins (PS1 and PS2) in governing capacitative calcium entry (CCE), a refilling mechanism for depleted intracellular calcium stores, is reported. Abrogation of functional PS1, by either knocking out PS1 or expressing inactive PS1, markedly potentiates CCE, suggesting a role for PS1 in the modulation of CCE. In contrast, familial Alzheimer's disease (FAD)-linked mutant PS1 or PS2 significantly attenuates CCE and store depletion-activated currents. While inhibition of CCE selectively increases the amyloidogenic amyloid ß peptide (Aß42), increased accumulation of the peptide has no effect on CCE. Thus, reduced CCE is most likely an early cellular event leading to increased Aß42 generation associated with FAD mutant presenilins. These data indicate that the CCE pathway is a novel therapeutic target for Alzheimer's disease (Yoo, 2000).

It is suggested that autosomal dominant FAD mutant presenilins exert a gain of function by downregulating CCE while increasing IP₃-mediated release from the ER store, leading to diminished luminal Ca²⁺ concentration ([Ca²⁺]_ER). It is interesting to note that changes in [Ca²⁺]_ER influence a number of cellular functions, including chaperone activities and gene expression. Therefore, it is tempting to speculate that reduced CCE may also be an upstream event leading to other molecular phenotypes associated with FAD mutant presenilins, including altered unfolded protein response. Interestingly, in transgenic mice harboring spinocerebellar ataxia type 1 (SCA1) mutant gene products, TRP3, SERCA2, and IP₃-R (all components of CCE), are specifically downregulated. This suggests the potential contribution of CCE dysregulation in other neurodegenerative diseases in addition to AD. CCE involves direct physical interaction between the ER and plasma membrane constituents. According to this conformational coupling mechanism, a conformational change of the IP₃ receptor (IP₃-R) upon agonist stimulation and subsequent release of Ca²⁺ leads to the formation of a molecular complex containing IP₃-R bound to molecular constituents in the plasma membrane harboring CCE channels. This then allows extracellular Ca²⁺ to replenish the ER store. It has been postulated that the presenilins modulate the gamma-secretase activity via few possible mechanisms: the presenilins might be the gamma-secretases themselves, and serve as essential cofactors for the gamma-secretase action, or regulate intracellular trafficking of a putative gamma-secretase to the target site where relevant substrates are localized. Given a role for presenilins in governing CCE, the presenilins may also modulate proteolytic processing of APP and Notch at or near the cell surface at sites of ER-plasma membrane coupling. It is conceivable that the presenilins may regulate or directly mediate the cleavage of protein(s) involved in modulating CCE. In any event, a gain in the biological activity of the presenilins, owing to autosomal dominant FAD mutations, may attenuate CCE while increasing gamma-secretase activity. Further experimentation will be necessary to elucidate this connection. Finally, augmentation of CCE, through the identification of agonists of plasma membrane store-operated Ca²⁺ channels (e.g., TRP or as yet undiscovered CCE channels) that mediate CCE, could potentially be employed to reduce PS-associated gamma-secretase activity, and the generation of Aß as a novel therapeutic means for preventing or treating AD (Yoo, 2000).

Agonist-evoked [Ca²⁺]_i oscillations have been considered a biophysical phenomenon reflecting the regulation of the IP₃ receptor by [Ca²⁺]_i. [Ca²⁺]_i oscillations are a biochemical phenomenon emanating from regulation of Ca2+ signaling by the regulators of G protein signaling (RGS) proteins. [Ca²⁺]_i oscillations evoked by G protein-coupled receptors require the action of RGS proteins. Inhibition of endogenous RGS protein action disrupts agonist-evoked [Ca²⁺]_i oscillations by a stepwise conversion to a sustained response. Based on these findings and the effect of mutant RGS proteins and anti-RGS protein antibodies on Ca2+ signaling, it is proposed that RGS proteins within the G protein-coupled receptor complexes provide a biochemical control of [Ca²⁺]_i oscillations (Luo, 2001).

A possible model for the biochemical control of [Ca²⁺]_i oscillation by RGS proteins is based on the present work and on the regulation of RGS protein function by PIP₃ and Ca²⁺-calmodulin (CaM). In the resting state, RGS proteins in signaling complexes are active and exert tonic inhibition by converting all spontaneously generated alphaq·GTP to alphaq·GDP. The findings that (1) all recombinant RGS proteins that interact with Galphaq tested to date are potent inhibitors of Ca²⁺ signaling when infused into cells and (2) activation of IP₃-dependent signaling by the K/Q mutant, and activation of Ca²⁺ signaling by the anti-RGS protein Abs show that RGS proteins are indeed active in resting cells to exert tonic inhibition of Ca²⁺ signaling. The agonist-activated receptor simulates the GDP/GTP exchange reaction to increase the rate of generation and the steady-state concentration of alpha·GTP. At this stage, the rate of alpha·GTP generation is faster than the rate of RGS protein-assisted GTP hydrolysis, resulting in the activation of PLCbeta, hydrolysis of PIP₂ to generate IP₃, and initiation of Ca²⁺ (step 1). In the first of the four steps described here, the activated receptor may not only activate Galpha, but may also stabilize the inactive state of RGS proteins by promoting the formation of PIP₃. The rate and extent of Galpha·GTP production and inhibition of RGS protein GAP activity is a function of agonist concentration. The Galphaq·PLCbeta complex continues to produce IP₃ and release Ca²⁺ until all stores exposed to IP₃ are depleted. At low concentration of agonist, only the stores in the vicinity of signaling complexes are exposed to IP₃. At increasing agonist concentration and, thus, IP₃ production, an increased fraction of the stores is discharged by IP₃ (Luo, 2001).

At the end of Ca²⁺ release and at the peak of [Ca²⁺]_i increase, high [Ca²⁺]_i in the vicinity of the IP₃ pore inhibits the channel to reduce the Ca²⁺ permeability of the stores' membrane. To terminate the stimulated state, the activity of RGS proteins has to be restored. This can be by formation of sufficient [Ca²⁺-CaM] to relieve the inhibition of RGS protein GAP activity by PIP₃ (step 2). This will lead to binding of the Ca²⁺/CaM/RGS protein to alphaq·GTP, acceleration of GTP hydrolysis, and inhibition of PLCbeta and IP₃ production. The reduction in [IP₃] together with reduced IP₃R channel activity, reloads the stores with Ca²⁺ (step 3). It is proposed that subsequent dissociation of Ca²⁺-CaM from RGS proteins stabilizes the RGS protein conformation that binds PIP₃ (or another inhibitor) to block RGS GAP activity (step 4). An important feature of the transition between step 3 and step 4 is that Galphaq cannot activate PLCbeta until Ca²⁺-CaM dissociates from RGS proteins. This provides a plausible mechanism for regulation of oscillation frequency. For many calmodulin-regulated proteins, regulation of enzymatic activity by calmodulin shows a hysteresis behavior. Upon Ca²⁺ increase, binding of calmodulin to target proteins and their activation is fast, whereas upon reduction of Ca²⁺, dissociation of Ca²⁺-CaM and termination of the active state is slow. Such behavior may also be a feature of the interaction of Ca²⁺-CaM with RGS proteins and determines the duration of the delay between [Ca²⁺]_i spikes. At low agonist concentrations, the rates of Galphaq activation and, possibly, dissociation of Ca²⁺-CaM from RGS proteins are slow, resulting in a low frequency oscillation in [IP₃] and [Ca²⁺]_i. Increasing agonist concentration can increase [Ca²⁺]_i oscillation frequency by increasing the rates of Galphaq activation and/or dissociation of Ca²⁺-CaM from RGS proteins. At very high agonist concentration, the rate of Galphaq activation is maximal, and RGS proteins remain in the inactive, PIP₃-bound state. This would, of course, result in a sustained response (Luo, 2001).

Biochemical control of Ca²⁺ oscillations by RGS proteins can explain several features of the oscillations. (1) The mode of activation of Galphaq by the receptor and inhibition of Galphaq by Ca²⁺-CaM bound RGS proteins can generate the receptor-specific patterns of [Ca²⁺]_i spiking observed in many cell types. In fact, RGS proteins differentially interact with the muscarinic, CCK, and bombesin receptors in pancreatic acini. (2) The model can explain how several receptors respond to increasing agonist concentration with increased oscillation frequency without affecting spike amplitude. Increased rates of Galphaq activation of the same signaling complexes will result in constant amplitude but increased oscillations frequency. (3) In many cells, including pancreatic acinar cells, the shape of individual Ca²⁺ spikes is receptor specific. The shape of individual [Ca²⁺]_i spikes can be determined by the rates of Galphaq activation and periodic activation and inactivation of RGS GAP activity. (4) Slow dissociation of Ca²⁺-CaM from RGS proteins can explain oscillations in [IP₃] and maintain low [IP₃] between [Ca²⁺]_i spikes. Reduction in [IP₃] between Ca²⁺ spikes can explain how Ca²⁺ release remains inactive for long periods of time between the spikes (Luo, 2001).

It is important to note that the biochemical (by RGS proteins) and biophysical (by Ca²⁺-dependent regulation of IP₃R) regulation of [Ca²⁺]_i oscillations are not mutually exclusive. Rather, it is likely that both regulatory events determine the final shape of the oscillations. However, the results suggest that the primary oscillator is the receptor·Galphaq·RGS protein complex. Regulation of IP₃R channel activity and, probably, the Ca²⁺ pumps is necessary to aid the receptor complex in controlling the oscillations. For example, inhibition of the IP₃R channel by high local Ca²⁺ will start reuptake of Ca²⁺ into the store by the SERCA pumps prior to complete reduction in [IP₃]. By placing the oscillator at the receptor complex, the receptor governs regulation of [Ca²⁺]_i oscillations. Furthermore, in this manner the oscillations can be precisely controlled by the state of occupancy of the receptor with ligands. In other words, the mode of occupancy of the receptor with agonist will determine the type of signal transduced into the cell interior (Luo, 2001).

The most common form of Ca²⁺ signaling by Gq-coupled receptors entails activation of PLCβ2 by Gαq to generate IP₃ and evoke Ca²⁺ release from the ER. Another form of Ca²⁺ signaling by G protein-coupled receptors involves activation of Gi to release Gβγ, which activates PLCβ1. Whether Gβγ has additional roles in Ca²⁺ signaling is unknown. Introduction of Gβγ into cells activates Ca²⁺ release from the IP₃ Ca²⁺ pool and Ca² oscillations. This can be due to activation of PLCβ1 or direct activation of the IP₃R by Gβγ. Gβγ potently activates the IP₃ receptor. Thus, Gβγ-triggered [Ca²⁺]_i oscillations are not affected by inhibition of PLCβ. Coimmunoprecipitation and competition experiments with Gβγ scavengers suggest binding of Gβγ to IP₃ receptors. Furthermore, Gβγ inhibits IP₃ binding to IP₃ receptors. Notably, Gβγ activates single IP₃R channels in native ER as effectively as IP₃. The physiological significance of this form of signaling is demonstrated by the reciprocal sensitivity of Ca²⁺ signals evoked by Gi- and Gq-coupled receptors to Gβγ scavenging and PLCβ inhibition. It is proposed that gating of IP₃R by Gβγ is a new mode of Ca²⁺ signaling with particular significance for Gi-coupled receptors (Zeng, 2003).

Ca2+ signals in neurons use specific temporal and spatial patterns to encode unambiguous information about crucial cellular functions. To understand the molecular basis for initiation and propagation of inositol 1,4,5-trisphosphate (InsP3)-mediated intracellular Ca2+ signals, the subcellular distribution of components of the InsP3 pathway was correlated with measurements of agonist-induced intracellular Ca2+ transients in cultured rat hippocampal neurons and pheochromocytoma cells. Specialized domains were found with high levels of phosphatidylinositol-4-phosphate kinase (PIPKI) and chromogranin B (CGB), proteins acting synergistically to increase InsP3 receptor (InsP3R) activity and sensitivity. In contrast, Ca2+ pumps in the plasma membrane (PMCA) and sarco-endoplasmic reticulum as well as buffers that antagonize the rise in intracellular Ca2+ were distributed uniformly. By pharmacologically blocking phosphatidylinositol-4-kinase and PIPKI or disrupting the CGB-InsP3R interaction by transfecting an interfering polypeptide fragment, major changes were produced in the initiation site and kinetics of the Ca2+ signal. This study shows that a limited number of proteins can reassemble to form unique, spatially restricted signaling domains to generate distinctive signals in different regions of the same neuron. The finding that the subcellular location of initiation sites and protein microdomains was cell type specific will help to establish differences in spatiotemporal Ca2+ signaling in different types of neurons (Jacob, 2005; full text of article).

Mechanism of Ca2+ disruption in Alzheimer's disease by presenilin regulation of InsP3 receptor channel gating

Mutations in presenilins (PS) are the major cause of familial Alzheimer's disease (FAD) and have been associated with calcium (Ca2+) signaling abnormalities. FAD mutant PS1 (M146L)and PS2 (N141I) interact with the inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+ release channel and exert profound stimulatory effects on its gating activity in response to saturating and suboptimal levels of InsP3. These interactions result in exaggerated cellular Ca2+ signaling in response to agonist stimulation as well as enhanced low-level Ca2+ signaling in unstimulated cells. Parallel studies in InsP3R-expressing and -deficient cells revealed that enhanced Ca2+ release from the endoplasmic reticulum as a result of the specific interaction of PS1-M146L with the InsP3R stimulates amyloid beta processing, an important feature of AD pathology. These observations provide molecular insights into the 'Ca2+ dysregulation' hypothesis of AD pathogenesis and suggest novel targets for therapeutic intervention (Cheung, 2008).

Inositol 1,4,5-trisphosphate receptor and [Ca2+]i oscillations after fertilization

Fertilization in the ascidians triggers an activation wave of calcium release followed by intracellular calcium oscillations synchronous with periodic membrane potential excursions during the completion of the meiotic cell cycle. Fertilization also causes a fast decrease in the egg plasma membrane depolarization-activated calcium current and a large increase in capacitance, thought to represent membrane addition to the egg surface. The temporal and causal relationships among these changes have been analyzed in the eggs of Phallusia mammillata. The role of ryanodine receptor (RyR) and InsP3 receptor (InsP3R) have been defined during fertilization and meiosis by looking at the effects of InsP3, cyclic ADP ribose (cADPR), and ryanodine in perfused oocytes. InsP3 is able to trigger sustained oscillations in intracellular calcium concentration in unfertilized oocytes, resembling those recorded in fertilized egg completing meiosis. The sustained oscillations resulting from InsP3 perfusion in unfertilized oocytes are sufficient to cause the emission of both polar bodies. In contrast, ryanodine or cADPR never trigg er detectable calcium signal in perfused oocytes. Instead, nanomolar concentrations of ryanodine or cADPR cause a capacitance change, implying a net insertion of membrane to the oocyte surface, and the triggering of a fast decrease in the depolarization-activated calcium current. Both changes are similar to the changes in conductance and capacitance naturally observed following fertilization. These effects, although not associated with measurable calcium signals, are abolished by coperfusion of the calcium chelator BAPTA. In contrast to ryanodine or cADPR, sustained perfusion of the oocyte with nanomolar concentrations of InsP3 causes no capacitance change and only a slow to moderate decrease in calcium current. These observations on inseminated patch-clamped eggs further indicate that membrane insertion, which starts 15-20 sec after the onset of the membrane conductance, changes at fertilization, and can be altered by interfering with the RyR. These results imply that, in ascidians, as in some mammals, RyR and InsP3R play distinct roles during fertilization (Albrieux, 1997).

Elevation of intracellular Ca2+ at fertilization is essential for the initiation of development in the Xenopus egg, but the pathway between sperm-egg interaction and Ca2+ release from the egg's endoplasmic reticulum is not well understood. Injection of an inhibitory antibody against the type I IP(3) receptor reduces Ca2+ release at fertilization, indicating that the Ca2+ release requires IP(3). Xenopus eggs were injected with specific inhibitors of the activation of two phospholipase C isoforms, PLCgamma and PLCbeta to see if these proteins are involved in initiation of IP(3) production. The Src-homology 2 (SH2) domains of PLCgamma were used to inhibit SH2-mediated activation of PLCgamma, and an antibody against G(q) family G-proteins was used to inhibit G(q)-mediated activation of PLCbeta. Though the PLCgamma SH2 domains inhibit platelet-derived growth factor (PDGF)-induced Ca2+ release in eggs with exogenously expressed PDGF receptors, these domains do not inhibit the Ca2+ rise at fertilization. Similarly, the G(q) family antibody blocks serotonin-induced Ca2+ release in eggs with exogenously expressed serotonin 2C receptors, but not the Ca2+ rise at fertilization. A mixture of PLCgamma SH2 domains and the G(q) antibody also do not inhibit the Ca2+ rise at fertilization. These results indicate that Ca2+ release at fertilization of Xenopus eggs requires type I IP(3)-gated Ca2+ channels, but not SH2 domain-mediated activation of PLCgamma or G(q)-mediated activation of PLCbeta (Runft, 1999).

Fertilization in mammals stimulates a series of Ca2+ oscillations that continue for 3-4 h. Cell-cycle-dependent changes in the ability to release Ca2+ are one mechanism that leads to the inhibition of Ca2+ transients after fertilization. The downregulation of InsP3Rs at fertilization may be an additional mechanism for inhibiting Ca2+ transients. The mechanism of this InsP3R downregulation has been examined. Three observations suggest that neither egg activation nor Ca2+ transients are necessary or sufficient for the stimulation of InsP3R downregulation: (1) parthenogenetic activation fails to stimulate downregulation; (2) downregulation persists when fertilization-induced Ca2+ transients and egg activation are inhibited using BAPTA; (3) downregulation can be induced in immature oocytes that do not undergo egg activation. Other than fertilization, the only stimulus that downregulates InsP3Rs is microinjection of the potent InsP3R agonist adenophostin A. InsP3R downregulation is inhibited by the cysteine protease inhibitor ALLN but MG132 and lactacystin are not effective. Maturing oocytes were injected with adenophostin A and metaphase II eggs depleted of InsP3Rs were produced. Sperm-induced Ca2+ signaling is inhibited in such InsP3R-depleted eggs. These data show that InsP3R binding is sufficient for downregulation and that Ca2+ signaling at fertilization is mediated via the InsP3R (Brind, 2000).

Sperm entry in mammalian eggs initiates oscillations in the concentration of free calcium ([Ca2+]i). In mouse eggs, oscillations start at metaphase II (MII) and conclude as the zygotes progress into interphase and commence pronuclear (PN) formation. The inositol 1,4,5-trisphosphate receptor (IP3R-1), which underlies the oscillations, undergoes degradation during this transition, suggesting that one or more of the eggs' Ca2+-releasing machinery components may be regulated in a cell cycle-dependent manner, thereby coordinating [Ca2+]i responses with the cell cycle. To ascertain the site(s) of interaction, oscillations were initiated at different stages of the cell cycle in zygotes with different IP3R-1 mass. In addition to sperm, two other agonists were used: porcine sperm factor (pSF), which stimulates production of IP3, and adenophostin A, a non-hydrolyzable analogue of IP3. None of the agonists tested induced oscillations at interphase, suggesting that neither decreased IP3R-1 mass nor lack of production or excessive IP3 degradation can account for the insensitivity to IP3 at this stage. Moreover, the releasable Ca2+ content of the stores did not change by interphase, but it did decrease by first mitosis. More importantly, experiments revealed that IP3R-1 sensitivity and possibly IP3 binding were altered at interphase, and these data demonstrate stage-specific IP3R-1 phosphorylation by M-phase kinases. Accordingly, increasing the activity of M-phase kinases restores the oscillatory-permissive state in zygotes. It is therefore proposed that the restriction of oscillations in mouse zygotes to the metaphase stage may be coordinated at the level of IP3R-1 and that this involves cell cycle stage-specific receptor phosphorylation (Jellerette, 2004).

Fertilization in the female reproductive tract depends on intercellular signaling mechanisms that coordinate sperm presence with oocyte meiotic progression. To achieve this coordination in C. elegans, sperm release an extracellular signal, the major sperm protein (MSP), to induce oocyte meiotic maturation and ovulation. MSP binds to multiple receptors, including the VAB-1 Eph receptor protein-tyrosine kinase on oocyte and ovarian sheath cell surfaces. Canonical VAB-1 ligands called ephrins negatively regulate oocyte maturation and MPK-1 mitogen-activated protein kinase (MAPK) activation. MSP and VAB-1 regulate the signaling properties of two Ca2+ channels that are encoded by the NMR-1 N-methyl D-aspartate type glutamate receptor subunit and ITR-1 inositol 1,4,5-triphosphate receptor. Ephrin/VAB-1 signaling acts upstream of ITR-1 to inhibit meiotic resumption, while NMR-1 prevents signaling by the UNC-43 Ca2+/calmodulin-dependent protein kinase II (CaMKII). MSP binding to VAB-1 stimulates NMR-1-dependent UNC-43 activation, and UNC-43 acts redundantly in oocytes to promote oocyte maturation and MAPK activation. These results support a model in which VAB-1 switches from a negative regulator into a redundant positive regulator of oocyte maturation upon binding to MSP. NMR-1 mediates this switch by controlling UNC-43 CaMKII activation at the oocyte cortex (Corrigan, 2005).

InsP3R interaction with Myosin II

Molecular and physiological studies of cells implicate interactions between the cytoskeleton and the intracellular calcium signaling machinery as an important mechanism for the regulation of calcium signaling. However, little is known about the functions of such mechanisms in animals. A key component of the calcium signaling network is the intracellular release of calcium in response to the production of the second messenger inositol 1,4,5-trisphosphate (IP3), mediated by the IP3 receptor (IP3R). C. elegans IP3Rs, encoded by the gene itr-1, interact directly with myosin II. The interactions between two myosin proteins, UNC-54 and MYO-1, and ITR-1 were identified in a yeast two-hybrid screen and subsequently confirmed in vivo and in vitro. The interaction sites on both the IP3R and MYO-1 have been defined. To test the effect of disrupting the interaction in vivo interacting fragments of both proteins were overexpressed in C. elegans. This decreases the animal's ability to upregulate pharyngeal pumping in response to food. This is a known IP3-mediated process. Other IP3-mediated processes, e.g., defecation, were unaffected. Thus it appears that interactions between IP3Rs and myosin are required for maintaining the specificity of IP3 signaling in C. elegans and probably more generally (Walker, 2002).

InsP3R interaction with Trp channels

Homologs of Drosophila Trp (transient receptor potential) form plasma membrane channels that mediate Ca2+ entry following the activation of phospholipase C by cell surface receptors. Among the seven Trp homologous found in mammals, Trp3 has been shown to interact with and respond to IP3 receptors (IP3Rs) for activation. Trp4 and other Trp proteins also interact with IP3Rs. The IP3R-binding domain also interacts with calmodulin (CaM) in a Ca2+-dependent manner with affinities ranging from 10 nM for Trp2 to 290 nM for Trp6. In addition, other binding sites for CaM and IP3Rs are present in the alpha but not the ß isoform of Trp4. In the presence of Ca2+, the Trp-IP3R interaction is inhibited by CaM. However, a synthetic peptide representing a Trp-binding domain of IP3Rs inhibits the binding of CaM to Trp3, -6, and -7 more effectively than that to Trp1, -2, -4, and -5. In inside-out membrane patches, Trp4 is activated strongly by calmidazolium, an antagonist of CaM, and a high (50 µM) but not a low (5 µM) concentration of the Trp-binding peptide of the IP3R. These data support the view that both CaM and IP3Rs play important roles in controlling the gating of Trp-based channels. However, the sensitivity and responses to CaM and IP3Rs differ for each Trp (Tang, 2001).

Members of the Snail family of zinc finger transcription factors are known to play critical roles in neurogenesis in invertebrates, but none of these factors has been linked to vertebrate neuronal differentiation. Expression of a mammalian Snail family member is restricted to the nervous system. Human and murine Scratch (Scrt) share 81% and 69% identity to Drosophila Scrt and the Caenorhabditis elegans neuronal antiapoptotic protein, CES-1, respectively, across the five zinc finger domain. Expression of mammalian Scrt is predominantly confined to the brain and spinal cord, appearing in newly differentiating, postmitotic neurons and persisting into postnatal life. Additional expression is seen in the retina and, significantly, in neuroendocrine (NE) cells of the lung. In a parallel fashion, hScrt expression is detected in lung cancers with NE features, especially small cell lung cancer. hScrt shares the capacity of other Snail family members to bind to E-box enhancer motifs, which are targets of basic helix-loop-helix (bHLH) transcription factors. hScrt directly antagonizes the function of heterodimers of the proneural bHLH protein achaete-scute homolog-1 and E12, leading to active transcriptional repression at E-box motifs. Thus, Scrt has the potential to function in newly differentiating, postmitotic neurons and in cancers with NE features by modulating the action of bHLH transcription factors critical for neuronal differentiation (Nakakura, 2001).

Like other Snail family members, hScrt is a nuclear protein that functions as a transcriptional repressor. Repressor activity resides within the N-terminal non-zinc finger region. However, the conserved N-terminal eight amino acids that hScrt shares with other SNAG domain containing proteins are not required for repressor function. This observation is in contrast to other reports ascribing important repressor function to the N-terminal twenty amino acids of the SNAG domain of vertebrate Snail, Slug, Smuc, and Gfi1 proteins. Though modest nuclear targeting activity has been described for this N-terminal region of Gfi1, effective nuclear localization depends on the full-length protein, including the zinc finger domain. The N-terminal non-zinc finger region of hScrt is not sufficient for proper expression in the nucleus; conversely, information within the zinc finger domain is necessary and sufficient to effect nuclear localization (Nakakura, 2001).

During vertebrate development, Mash1 and Neurogenin1 and -2 are transiently expressed in proliferating neurons of the nervous system and exhibit determination and differentiation functions. mScrt is expressed in an adjacent layer characteristic of newly differentiating neurons and hScrt can repress hASH1-E12-mediated reporter transactivation. Taken together, Scrt may modulate the effects of ASH1-E12 on common target genes, thereby potentially affecting neuronal determination and differentiation. Because Scrt expression is more widespread than Mash1, it is possible that Scrt may interact functionally with other bHLH transcription factors, such as the Neurogenins. Functional interactions occur between Escargot and Scute-Daughterless, as well as Smuc and MyoD-E12. Thus, interactions between Snail family and bHLH factors may be a common theme in development. Further studies of Scrt should provide insight into programs of neural differentiation that appear conserved in normal and neoplastic tissues (Nakakura, 2001).

Mammalian homologs of Drosophila Trp form plasma membrane channels that mediate Ca2+ influx in response to activation of phospholipase C and internal Ca2+ store depletion. Human Trp3 is activated by inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) and interacting domains, one on Trp and two on IP3R. Trp3 binds Ca2+-calmodulin (Ca2+/CaM) at a site that overlaps with the IP3R binding domain. Using patch-clamp recordings from inside-out patches, it has been shown that Trp3 has a high intrinsic activity that is suppressed by Ca2+/CaM under resting conditions. Trp3 is activated by the following: a Trp-binding peptide from IP3R that displaces CaM from Trp3, a myosin light chain kinase Ca2+/CaM binding peptide that prevents CaM from binding to Trp3, and calmidazolium, an inactivator of Ca2+/CaM. It is concluded that inhibition of the inhibitory action of CaM is a key step of Trp3 channel activation by IP3Rs (Zhang, 2001).

BANK regulates BCR-induced calcium mobilization by promoting tyrosine phosphorylation of IP(3) receptor

B-cell activation mediated through the antigen receptor is dependent on activation of protein tyrosine kinases (PTKs) such as Lyn and Syk and subsequent phosphorylation of various signaling proteins. This study reports on the identification and characterization of the B-cell scaffold protein with ankyrin repeats (BANK: Drosophila homolog - Stumps), a novel substrate of tyrosine kinases. BANK is expressed in B cells and is tyrosine phosphorylated upon B-cell antigen receptor (BCR) stimulation; the phosphorylation is mediated predominantly by Syk. Overexpression of BANK in B cells leads to enhancement of BCR-induced calcium mobilization. It was found that both Lyn and inositol 1,4,5-trisphosphate receptor [IP(3)R] associate with the distinct regions of BANK and that BANK promotes Lyn-mediated tyrosine phosphorylation of IP(3)R. Given that IP(3)R channel activity is up-regulated by its tyrosine phosphorylation, BANK appears to be a novel scaffold protein regulating BCR-induced calcium mobilization by connecting PTKs to IP(3)R. Because BANK expression is confined to functional BCR-expressing B cells, BANK-mediated calcium mobilization may be specific to foreign antigen-induced immune responses rather than to signaling required for B-cell development (Yokoyama, 2003)

InsP3 receptors: Cell division and cell cycle

Recent studies suggest that mammalian preimplantation development may also be regulated by the release of Ca2+ from intracellular stores. The rate of cavitation and cell division is accelerated after a transient elevation of intracellular Ca2+ levels is induced in morulae by exposure to ethanol or ionomycin. Embryos exposed to BAPTA-AM, a chelator of intracellular Ca2+, exhibit a brief dose-dependent reduction in basal Ca2+ levels, a temporal inhibition of ionophore-induced Ca2+ signaling and a subsequent delay in blastocoel formation. BAPTA-AM at 0.5 microM does not significantly alter the basal intracellular calcium level, but chelates Ca2+ that is released after ethanol exposure and thereby attenuates the ethanol-induced acceleration of cavitation. BAPTA-AM also inhibits cell division to the 16-cell stage in a dose-dependent manner, which correlates with the inhibition of cavitation. Thimerosal and inositol 1,4,5-trisphosphate significantly elevate the intracellular Ca2+ concentration in mouse morula-stage embryos, providing evidence for the existence of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores. Although caffeine fails to release intracellular Ca2+, ryanodine induces a small biphasic release of Ca2+, suggesting that ryanodine-sensitive Ca2+ stores may also exist in mouse embryos. Morulae exposed to the calmodulin (See Drosophila Calmodulin) inhibitor W-7 exhibit a dose-dependent delay in blastocoel formation. A 4 hour exposure to 10 microM W-7 does not significantly alter cavitation, but attenuates the ionophore-induced stimulation of blastocoel formation. This finding suggests that the developmental effects produced through Ca2+ signaling are mediated by calmodulin. These results demonstrate that Ca2+ release in mouse morulae occurs predominantly through the inositol 1,4,5-trisphosphate receptor, and that alteration of intracellular Ca2+ levels can accelerate or delay embryonic growth and differentiation, providing a mechanistic link between the regulation of oocyte and embryonic development (Stachecki, 1996).

At fertilization a transient increase in intracellular Ca2+ concentration occurs in eggs of all species studied to date, as a trigger signal for egg activation. The causitive agent is as yet unknown. Hamster sperm extract (SE) possessing Ca2+ oscillation-inducing activity was microinjected into the peripheral or central region of mouse eggs, and the first increase in intracellular Ca2+ concentration ([Ca2+]i), together with the spread of fluorescence-labeled SE in the ooplasm, was investigated by imaging with confocal microscopy. Injection into the periphery always induces a Ca2+ wave that starts from the injection site after a delay of 5 to 30 s, depending on the concentration of SE. The diluted SE causes a wave of two-step [Ca2+]i rises, which is always observed at fertilization. Injection into the center can induce a radial Ca2+ wave with a relatively high dose of SE, but a lower dose of SE causes a [Ca2+]i rise after a longer delay, which is initiated synchronously over the ooplasm or is preceded in a peripheral area. Injection of diluted SE remarkably prolongs the delay time and reduces the rate of [Ca2+]i rise. The critical concentration of SE needed to induce [Ca2+]i rise is significantly lower in the periphery. These results indicate that the sensitivity to SE is higher in the cortex. SE-induced [Ca2+]i rises are blocked by an antibody against the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R). The cortex is substantially more sensitive to injected InsP3 induction of Ca2+ release than is the center. It is suggested that the cortex of mouse eggs may involve a functionally specialized organization of InsP3Rs and Ca2+ pools in which a cytosolic sperm factor(s) could act upon sperm-egg fusion to cause Ca2+ release, leading to the Ca2+ wave at fertilization (Oda, 1999).

The cleavage signal transferred to the future cleavage cortex during anaphase has been proposed as 'cleavage stimulus', but no signal has proved to induce cleavage furrows. The local Ca2+ transient along the cleavage furrow has been reported, but the Ca2+ source has remained unknown. To address these questions, functions of Ca2+ stores in dividing newt eggs have been studied. Microinjection of the Ca2+ store-enriched microsome fraction to the dividing newt egg induces a local extra-cleavage furrow at the injection site in 64-67% of the injected newt eggs while coinjection with inositol 1,4, 5-trisphosphate receptor [IP(3)R] antagonists heparin or anti-type 1-IP(3)R antibody clearly suppress this induction (5% and 11% in induction rates, respectively). Injection of cerebellar microsomes from the type 1-IP(3)R-deficient mice induces extracleavage furrows albeit at a low rate (19%). These observations strongly suggest that Ca2+ stores along with IP(3)R induce and position a cleavage furrow via IP(3)-induced Ca2+ release (IICR), that functions as Ca(2+)-releasing machinery and as the putative cleavage stimulus itself (Mitsuyama, 1999).

Inositol 1,4,5-tris-phosphate (IP3) binding to its receptors (IP3R) in the endoplasmic reticulum (ER) activates Ca2+ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca2+ concentration signals, including temporal oscillations and propagating waves. IP3-mediated Ca2+ release is also controlled by cytoplasmic Ca2+ concentration with both positive and negative feedback. Single-channel properties of the IP3R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP3R on cytoplasmic Ca2+ and IP3 concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca2+ concentration ([Ca2+]i) response centered at approximately 300 nM-1 microM, the open probability remains elevated in the presence of saturating levels of IP3, even as [Ca2+]i is raised to high concentrations, displaying two distinct types of functional Ca2+ binding sites: activating sites and inhibitory sites. These results demonstrate that Ca2+ is a true receptor agonist, whereas the sole function of IP3 is to relieve Ca2+ inhibition of IP3R. Allosteric tuning of Ca2+ inhibition by IP3 enables the individual IP3R Ca2+ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca2+ concentration signaling and quantal Ca2+ release (Mak, 1999).

Vertebrate oocytes proceed through meiosis I before undergoing a cytostatic factor (CSF)-mediated arrest at metaphase of meiosis II. Exit from MII arrest is stimulated by a sperm-induced increase in intracellular Ca2+. This increase in Ca2+ results in the destruction of cyclin B1, the regulatory subunit of cdk1 that leads to inactivation of maturation promoting factor (MPF) and egg activation. Progression through meiosis I also involves cyclin B1 destruction, but it is not known whether Ca2+ can activate the destruction machinery during MI. Ca2+-induced cyclin destruction was investigated in MI and MII by using a cyclin B1-GFP fusion protein and measurement of intracellular Ca2+. No evidence was found for a role for Ca2+ in MI since oocytes progress through MI in the absence of detectable Ca2+ transients. Furthermore, Ca2+ increases induced by photorelease of InsP3 stimulate a persistent destruction of cyclin B1-GFP in MII but not MI stage oocytes. In addition to a steady decrease in cyclin B1-GFP fluorescence, the increase in Ca2+ stimulated a transient decrease in fluorescence in both MI and MII stage oocytes. Similar transient decreases in fluorescence imposed on a more persistent fluorescence decrease were detected in cyclin-GFP-injected eggs undergoing fertilization-induced Ca2+ oscillations. The transient decreases in fluorescence were not a result of cyclin B1 destruction since transients persisted in the presence of a proteasome inhibitor and were detected in controls injected with eGFP and in untreated oocytes. It is concluded that increases in cytosolic Ca2+ induce transient changes in autofluorescence. Also, the pattern of cyclin B1 degradation at fertilization is not stepwise but exponential. Furthermore, this Ca2+-induced increase in degradation of cyclin B1 requires factors specific to mature oocytes, and that to overcome arrest at MII, Ca2+ acts to release the CSF-mediated brake on cyclin B1 destruction (Marangos, 2004).

Developmental expression of InsP3 receptors

A study of the temporal-spatial localization of Xenopus IP3 receptor (XIP3R) was made in order to elucidate the role of inositol 1,4,5-trisphosphate (IP3) receptors during early embryogenesis in Xenopus. XIP3R protein is enriched in the animal hemisphere of early cleavage stage embryos, becoming localized in the ectoderm and involuted mesoderm in gastrula stage embryos. Up to tailbud stages, expression of XIP3R is observed in the mesodermal tissues and in most subregions of the central nervous system. A quantitative analysis of endogenous IP3 mass during normal early embryogenesis reveals an increase in IP3 mass first observed at early gastrula stage (10.5 h), with enrichment in the ectoderm throughout the gastrula stages, implying a potential role during gastrulation (Kume, 1997a).

The inositol 1,4,5-trisphosphate (IP3) receptor is a calcium ion channel involved in the release of free Ca2+ from intracellular stores. For analysis of the role of IP3-induced Ca2+ release (IICR) on patterning of the embryonic body, monoclonal antibodies that inhibit IICR were produced. Injection of these blocking antibodies into the ventral part of early Xenopus embryos induces modest dorsal differentiation. A close correlation between IICR blocking potencies and ectopic dorsal axis induction frequency suggests that an active IP3-Ca2+ signal may participate in the modulation of ventral differentiation. The fact that antibody to IP3R cannot induce head mesoderm suggests that the IP3R system is sufficient for dorsal respecification to take place, but not for the induction of a full spectrum of organizer-specific genes (Kume, 1997b).

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ channel that releases Ca2+ from internal Ca2+ stores in response to InsP3. Although InsP3R is highly expressed in various regions of the mammalian brain, the functional role of this receptor has not been clarified. Cerebellar slices prepared from mice with a disrupted InsP3R type 1 gene, which is predominantly expressed in Purkinje cells, completely lack long-term depression (LTD), a model of synaptic plasticity in the cerebellum. Moreover, a specific antibody against InsP3R1, introduced into wild-type Purkinje cells through patch pipettes, blocks the induction of LTD. These data indicate that, in addition to Ca2+ influx through Ca2+ channels on the plasma membrane, Ca2+ release from InsP3R plays an essential role in the induction of LTD, suggesting a physiological importance for InsP3R in Purkinje cells (Inoue, 1998).

A role of InsP3 receptors in Wingless signaling

In Drosophila, members of the frizzled family of tissue-polarity genes (see Drosophila Frizzled and Frizzled 2) encode proteins that are likely to function as cell-surface receptors of the type known as Wnt receptors, and to initiate signal transduction across the cell membrane. Stimulation of a G-protein-linked receptor initiates the hydrolysis of a membrane-bound inositol lipid, generating at least two second messengers: diacylglycerol and inositol-1,4,5-trisphosphate (InsP3). Diacylglycerol stimulates protein kinase C while InsP3 promotes the release of intracellular calcium (see Drosophila InsP3 receptor). The rat protein Frizzled-2 causes an increase in the release of intracellular calcium, which is enhanced by Xwnt-5a, a member of the Wnt family. Pertussis toxin (PTX) (which is a specific inhibitor of G alpha0 and G alphai subunits of G proteins that act by preventing the catalysis of GDP-GTP exchange stimulated by receptors) inhibits rat protein Frizzled-2 modulation of calcium flux. A nonhydrolysable GDP analog that irreversibly inactives G-protein-coupled events, inhibits rat FZ-2 induced Ca2+ transients. The release of intracellular calcium is suppressed by an inhibitor of the enzyme inositol monophosphatase, and hence of the phosphatidylinositol signaling pathway. This suppression can be rescued by injection of the compound myo-inositol, which overcomes the decrease in this intermediate caused by the inhibitor. These results indicate that some Wnt proteins work through specific Frizzled homologs to stimulate the phosphatidylinositol signalling pathway via heterotrimeric G-protein subunits, and that FZ-2 stimulates the phosphatidylinositol cycle through the betagamma subunits of pertussis-toxin-sensitive G proteins, leading to release of intracellular Ca2+ and diverse cellular responses. Since Gbetagamma subunits also activate protein kinase C, which may be involved in Wnt signaling, the responses by cells and embryos to signaling through Frizzled homologs could involve the stimulation of multiple cytoplasmic pathways. In early vertebrate embryos, regulation of the phosphatidylinositol pathway may be important for establishing the embryonic mesoderm and in other processes (Slusarski, 1997).

It is thought that inositol-1,4,5-trisphosphate (Ins(1,4,5)P₃)-Ca²⁺ signalling has a function in dorsoventral axis formation in Xenopus embryos; however, the immediate target of free Ca²⁺ is unclear. The secreted Wnt protein family comprises two functional groups, the canonical Wnt and Wnt/Ca²⁺ pathways. The Wnt/Ca²⁺ pathway interferes with the canonical Wnt pathway, but the underlying molecular mechanism is poorly understood. The complementary DNA coding for the Xenopus homolog of nuclear factor of activated T cells (XNF-AT) has been cloned. A gain-of-function, calcineurin-independent active XNF-AT mutation (CA XNF-AT) inhibits anterior development of the primary axis, as well as Xwnt-8-induced ectopic dorsal axis development in embryos. A loss-of-function, dominant negative XNF-AT mutation (DN XNF-AT) induces ectopic dorsal axis formation and expression of the canonical Wnt signalling target molecules siamois and Xnr3. Xwnt-5A induces translocation of XNF-AT from the cytosol to the nucleus. These data indicate that XNF-AT functions as a downstream target of the Wnt/Ca²⁺ and Ins(1,4,5)P₃-Ca²⁺ pathways, and has an essential role in mediating ventral signals in the Xenopus embryo through suppression of the canonical Wnt pathway (Saneyoshi, 2002).

Since injected myo-inositol blocks the effect of dominant negative GSK3ß-induced secondary axis formation, these findings support the idea that there is cross-talk between phosphatidylinositide cycle signalling and the canonical Wnt pathway. The tyrosine kinase-linked receptor signalling pathway also activates Ins(1,4,5)P₃-Ca²⁺ signalling through phospholipase Cgamma activation. Gain of function of Ins(1,4,5)P₃-Ca²⁺ signalling on the dorsal side of the embryo leads to a dorso-anterior structure deficiency, whereas loss of function on the ventral side induces a partial ectopic dorsal axis. These findings suggest that the Ins(1,4,5)P₃-Ca²⁺ signalling pathway mediates ventral signals. One possible target of Ins(1,4,5)P₃-Ca²⁺ signalling is the Ca²⁺/calmodulin (CaM)-dependent protein phosphatase, calcineurin, and the transcription factor -- positioned further downstream -- NF-AT. XNF-AT might receive inputs from tyrosine kinase signalling pathways, therefore the activity of XNF-AT in the wild-type embryo may reflect the activity of Wnt pathway. A proposed model for dorsoventral axis formation and the interaction between the Wnt/Ca²⁺ and the canonical Wnt pathways suggests that XNF-AT is a direct target of the Ins(1,4,5)P₃-Ca²⁺ signal downstream of the Wnt/Ca²⁺ pathway, and that XNF-AT mediates ventralizing signal by suppression of canonical Wnt activity during axis formation of the Xenopus embryo (Saneyoshi, 2002).

REFERENCES

Search PubMed for articles about Drosophila Inositol 1,4,5,-tris-phosphate receptor

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Bosanac, I., et al. (2002). Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand. Nature 420: 696-700. 12442173

Brind, S., Swann, K. and Carroll, J. (2000). Inositol 1,4,5-trisphosphate receptors are downregulated in mouse oocytes in response to sperm or adenophostin A but not to increases in intracellular Ca2+ or egg activation. Dev. Biol. 223: 251-265. PubMed ID: 10882514

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Chen, X. and Dickman, D. (2017). Development of a tissue-specific ribosome profiling approach in Drosophila enables genome-wide evaluation of translational adaptations. PLoS Genet 13(12): e1007117. PubMed ID: 29194454

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