InteractiveFly: GeneBrief

ether a go-go: Biological Overview | References

BIOLOGICAL OVERVIEW

Drosophila Ether-a-go-go (Eag) is the founding member of a large family of voltage-gated K(+) channels, the KCNH family, which includes Kv10, 11 and 12. Concurrent binding of calcium/calmodulin (Ca(2+)/CaM) to N- and C-terminal sites inhibits mammalian EAG1 channels at sub-micromolar Ca(2+) concentrations, likely by causing pore constriction. Although the Drosophila EAG channel was believed to be Ca(2+)-insensitive, both the N- and C-terminal sites are conserved. This study shows that Drosophila EAG is inhibited by high Ca(2+) concentrations that are only present at plasma membrane Ca(2+) channel microdomains. To test the role of this regulation in vivo, mutations were engineered that block CaM-binding to the major C-terminal site of the endogenous eag locus, disrupting Ca(2+)-dependent inhibition. eag CaMBD mutants have reduced evoked release from larval motor neuron presynaptic terminals and show decreased Ca(2+) influx in stimulated adult projection neuron presynaptic terminals, consistent with an increase in K(+) conductance. These results are predicted by a conductance-based multi-compartment model of the presynaptic terminal in which some fraction of EAG is localized to the Ca(2+) channel microdomains that control neurotransmitter release. The reduction of release in the larval neuromuscular junction drives a compensatory increase in motor neuron somatic excitability. This misregulation of synaptic and somatic excitability has consequences for systems-level processes and leads to defects in associative memory formation in adults (Bronk, 2018).

Regulation of excitability is critical to tuning the nervous system for complex behaviors. This study demonstrates that the EAG family of voltage-gated potassium channels exhibits conserved gating by Ca2+/CaM. Disruption of this regulation in Drosophila results in a decreased evoked neurotransmitter release due to truncated Ca2+ influx in presynaptic terminals. In the larval motor system, the blunting of evoked responses triggers a homeostatic increase in somatic excitability, which would act to compensate for the decrease in muscle stimulation. In adults, disrupted Ca2+ dynamics lead to defects in memory formation. These data demonstrate that the biophysical details of channels can have widespread effects on behavior at the level of cellular compartments and circuits (Bronk, 2018).

The repolarization of neurons and muscle cells by K+ channels plays an important role in limiting the excitability of cells and returning them to a baseline membrane potential to be ready for the next action potential. K+ channels also play an indirect role in regulating Ca2+ levels inside the cell, preventing excitotoxicity and shaping the temporal profile of cellular responses to activity. Several families of Ca2+-activated K+ channels (e.g., small- and large-conductance, SK and BK channels) appear to act in a feedback mode, with internal Ca2+ activating the channels to hyperpolarize the cell and limit additional Ca2+ entry. However, there is one Ca2+-regulated K+ channel family that appears to act in a counterintuitive manner. The mammalian ether-à-go-go (EAG) K+ channel is completely inhibited at all membrane voltages in the presence of 100-300 nM Ca2+/calmodulin (Ca2+/CaM). This study sought to understand the role of this seemingly maladaptive regulation by disrupting the CaM interaction with EAG in Drosophila, where the presence of only one gene coding for EAG simplifies the analysis (Bronk, 2018).

The Drosophila ether-à-go-go (eag) gene encodes a voltage-gated delayed rectifier K+ channel subunit, EAG. The first eag mutant was discovered in Drosophila and was identified by its ether-induced leg shaking (Kaplan, 1969). Subsequently, Drosophila EAG was found to define a family of K+ channels with multiple homologs in mammals (Warmke, 1994). EAG is a member of the KCNH family of K+ channels and is also referred to as Kv10. Although the transmembrane domains of EAG are similar in structure to Shaker-type voltage-gated channels (Warmke, 1994), recent structural data have led to the hypothesis that EAG has an additional novel gating mechanism. This alternate gating mechanism allows cytoplasmic factors such as CaM to act on the pore region through the domain linking the COOH terminus to the S6 transmembrane domain (Whicher, 2016). Channel closure is achieved by the simultaneous binding of Ca2+/CaM to both an NH2-terminal and a COOH-terminal site. This requirement for binding at two sites means that disruption of a single CaM binding domain (CaMBD) should block Ca2+-dependent gating entirely (Bronk, 2018).

Diversity in the expression of K(+) channels among neurons allows a wide range of excitability, growth, and functional regulation. Ether-a-go-go (EAG), a voltage-gated K(+) channel, was first characterized in Drosophila mutants by spontaneous firing in nerve terminals and enhanced neurotransmitter release. Although diverse functions have been ascribed to this protein, its role within neurons remains poorly understood. The aim of this study was to characterize the function of EAG in situ in Drosophila larval motoneurons. Whole cell patch-clamp recordings performed from the somata revealed a decrease in I(Av) and I(Kv) K(+) currents in eag mutants and with targeted eag RNAi expression. Spontaneous spike-like events were observed in eag mutants but absent in wild-type motoneurons. Thus the results provide evidence that EAG represents a unique K(+) channel contributing to multiple K(+) currents in motoneurons helping to regulate excitability, consistent with previous observations in the Drosophila larval muscle (Bronk, 2018).

The biophysical properties of EAG have been determined in heterologous expression systems, and little is known about its function in the nervous system. Fly loss-of-function eag mutants, in which the conductance is completely missing, have learning deficits (Griffith, 1994) in addition to a robust hyperexcitability in larval motor neurons that also causes spontaneous neuronal firing. In cockroaches, knockdown of EAG revealed a role for Ca2+-dependent inhibition in the light response (Immonen, 2017). EAG1 knockout mice have normal learning and memory, sensorimotor function, social behavior, and anxiety and only display a mild hyperactivity (Ufartes, 2013). However, the existence of multiple EAG family members in mammals raises the possibility of compensation or redundancy and complicates in vivo analysis. Cellular physiology in EAG1 knockout mice has revealed enhanced synaptic facilitation during high-frequency stimulation (≥50 Hz) at the parallel fiber-Purkinje cell synapse in the cerebellum, accompanied by elevated presynaptic Ca2+ (Mortensen, 2015). In both insects and mammals, the dominant phenotype of loss of EAG channels appears to be increased presynaptic release (Bronk, 2018).

The role of gating by Ca2+/CaM in vivo is likely to be more complicated, and understanding the role of Ca2+ inhibition requires generating an EAG channel that is voltage gated but lacks Ca2+/CaM inhibition. Such a channel would be predicted to pass more current when Ca2+ concentration is high. This study shows that fly EAG is inhibited by Ca2+/CaM, and this study generate mutant alleles disrupting the CaM interaction with EAG to interrogate its function. Without Ca2+/CaM inhibition of EAG, evoked synaptic currents were found to be reduced, and presynaptic Ca2+ is reduced during high-frequency stimulation. These defects lead to changes in somatic excitability that go in the opposite direction, likely the result of homeostasis. The complex misregulation of excitability in these mutants disrupts higher level behavior, demonstrating that the biophysical details of channel regulation can have profound effects at the organismal level (Bronk, 2018).

An obvious role of protein localization is to limit the action of a molecule to a particular cell compartment; e.g., an anchored K+ channel will only affect the membrane potential of a small area surrounding it. For example, a scaffolded kinase will preferentially phosphorylate substrates in the same complex. Less obvious consequences of binding to a scaffold are alterations of the intrinsic activities of the complexed molecules and generation of novel complex-specific effects on neuronal processes. A well-known example of this is the fusion of vesicles carrying neurotransmitters. Cytosolic bulk Ca2+ in neurons is typically regulated in the 10 nM to 10 µM range, and only cytoplasmic Ca2+ microdomains close to a voltage-gated Ca2+ channel (VGCC) ever see Ca2+ at millimolar concentrations. The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) machinery has very low Ca2+ affinity and only functions when vesicles are docked to a complex with VGCCs. The inhibition of Drosophila EAG channels by Ca2+ is likely to be facilitated by a similar type of interaction. This study provides the first insights into its role by introducing mutations into the endogenous eag locus that block regulation by Ca2+ (Bronk, 2018).

The fly EAG channel had been thought to be an outlier with regard to regulation by Ca2+. This study shows that it is regulated in the same manner as mammalian EAG1. The major difference between fly and mammalian EAGs appears to be sensitivity. Inhibition of fly EAG is only see at high micromolar to millimolar Ca2+ levels in transfected cells. It is important to note, however, that this study has not measured the Ca2+ sensitivity in vivo. Although the most parsimonious expectation is that EAG Ca2+ sensitivity is low in Drosophila neurons, it remains possible that some fly-specific extrinsic factor (not present in mammalian or amphibian cells) could serve to increase it in the native context. Nonetheless, the data show a striking difference between the regulation of mammalian EAG1, which is fully inhibited at nanomolar levels of Ca2+, and the fly EAG, which retains activity even at millimolar Ca2+ levels. How the difference in sensitivity is achieved in the fly protein is unknown, but evidence is presented that the efficacy of regulation of EAG channels by Ca2+/CaM is likely controlled by the COOH-terminal CaMBDs, because alternative splicing in the fly gene at the site of the NH2-terminal CaMBD does not significantly modulate apparent affinity. This Ca2+-dependent inhibition of EAG channels is a robust and evolutionarily conserved feature of the channel family that has so far been without demonstrated function in central neurons (Bronk, 2018).

EAG channels are located in presynaptic terminals in both mammals and flies and characterization of knockouts suggest that EAG participates in repolarization after action potential invasion and controls action potential width. These loss-of-function studies make it clear that repolarizing K+ current is important for presynaptic function and that the channel has a similar overall role across species. The role of Ca2+-dependent inhibition of EAG currents is not as well understood. The finding that evoked release was decreased in the EAG CaMBD mutant in the face of normal spontaneous release dynamics and quantal size argued that presynaptic EAG in vivo is exposed to very high levels of Ca2+ during action potential-mediated neurotransmitter release, consistent with localization at active zones. The reduced neurotransmitter release suggests that the Ca2+-dependent inhibition of EAG is used to locally amplify the effects of depolarization at the active zone by decreasing the repolarizing current in that domain (Bronk, 2018).

In mammals it has been speculated that the inhibition of EAG1 by Ca2+/CaM serves as a feedback mechanism to reduce the electrochemical driving force for Ca2+ influx (Schönherr, 2000). The idea is that EAG channels, if colocalized with VGCCs, could control the local membrane potential after an action potential during the time that the Ca2+ channels are open. Inhibition of EAG by Ca2+ would allow the membrane potential to stay close the Ca2+ reversal potential and blunt the electrochemical drive for Ca2+ influx through the open VGCC. The current data would argue against this hypothesis for the presynaptic terminal at the larval NMJ, because loss of regulation by Ca2+ decreases presynaptic release and evoked Ca2+ influx. Whether this might be a viable mechanism for regulation of Ca2+ in dendrites will require further investigation (Bronk, 2018).

Another possible role for Ca2+-dependent inhibition might be maintaining neurotransmitter release during repetitive firing, essentially providing a way to turn down local repolarization to maintain the ability to catalyze vesicle fusion. The current data are consistent with this type of role. Future studies of the dynamics of EAG inhibition and its dependence of action potential pattern might provide clues to this aspect of EAG function (Bronk, 2018).

The fact that presynaptic release at the NMJ and Ca2+ responses in central axons are reduced is consistent with the gain-of-function nature of the CaMBD mutation, which allows the EAG K+ current to be maintained during neuronal activation. This suggests that there is little local homeostatic change in axon terminals when the ability of Ca2+ to inhibit EAG currents is lost. This lack of compensation is especially interesting in light of the ability of the NMJ to quickly compensate for toxin-mediated decreases in EJP amplitude in eag null mutants and to chronically compensate for EJP reductions, e.g., in glutamate receptor mutants or when muscle excitability is dampened. One possible interpretation of this is that EAG itself is part of the homeostatic machinery that would be engaged in this type of neuronal insult and that its regulation by Ca2+ is required for compensation. Perhaps consistent with this, a significant transcriptional induction of eag mRNA is seen in these mutants. Another possibility is that the increase in EAG current itself occludes the effects of the normal homeostatic compensation (Bronk, 2018).

Whereas the phenotype of the nerve terminal appears relatively straightforward, somatic recordings demonstrated a surprising increase in excitability. This enhanced excitability is not directly reconcilable with the nature of the eag CaMBD mutation itself, because it would be expected to result in suppression of excitability due to enhanced K+ current. There are at least three possible scenarios that might explain this observation. One is that the loss of regulation of EAG triggers a compensation in the dendritic/somatic compartment, but this compensation 'overshoots' -- i.e., the expression of excitability-promoting channels/transporters is too high. A second possibility is that the motor neuron's somatic/dendritic increase in excitability is a homeostatic response to the decrease in release at the NMJ. Because the normal mechanisms of compensation (locally increased presynaptic release) are not engaged, the neuron may scale up its responses to inputs to allow more transmitter to be released via increased numbers of action potentials. This would imply that there are secondary tiers of compensation and that there can be interactions between homeostatic programs similar to what has been reported when neurons receive conflicting perturbations. These possibilities are speculative but in line with the robust homeostasis seen with other manipulations of this preparation. A third possibility is that at the action potential initiation zone, increased EAG current can act to enhance fast firing by, for example, blunting Na+ channel inactivation (Bronk, 2018).

The sequence conservation of both NH2- and COOH-terminal CaMBDs from insects to mammals is striking. Why would their apparent affinities be orders of magnitude apart? The difference in sensitivity might be driven by how subcellular complexes are constituted and the relative size of synaptic compartments in the two types of organisms. The Ca2+ sensitivity required to carry out a function depends on the localization of the effector with respect to the Ca2+ source and the nature of local Ca2+ buffering. Channels located in very close proximity to VGCCs do not require high Ca2+ sensitivity, because they are exposed to very high (mM) local Ca2+ concentrations. Conversely, EAG channels that are inhibited with high affinity could be affected by Ca2+ distant from the mouth of the VGCC. For fly neurons whose central processes are very small, high-affinity EAG inhibition might completely abolish all EAG current and render neurons hyperexcitable, much like the case for eag null animals. The Ca2+-dependent inhibition of EAG could therefore contribute to maintaining presynaptic activity with repetitive stimulation in both species but be tuned differently due to differences in the location of the channels, buffering capacity of the neurons, or the size of the cell and synapse. A better understanding of the localizations of VGCCs and EAG channels will be required to fully understand the role Ca2+-dependent inhibition of EAG family channels (Bronk, 2018).

In neurons, ion channel-containing signaling complexes are a key class of protein assemblage. The colocalization of proteins to the same complex can strongly influence the kinetics and even the energetics of their activities and produce unforeseen interactions. Most of a neuron's important biochemistry is not well predicted by simply considering the equilibrium state; Ca2+ microdomains are an excellent example of this principle. This study presents data demonstrating that blocking the regulation of EAG by Ca2+ can disrupt the ability to form memory. This argues that relatively subtle changes in the regulation of excitability can have profound effects. There are an increasing number of examples of proteins whose function requires their localization to a particular microdomain. To understand how neurons work, it is necessary to understand how protein complexes shape signaling, and the fly EAG channel will provide a useful model for this endeavor (Bronk, 2018).

Inter-relationships among physical dimensions, distal-proximal rank orders, and basal GCaMP fluorescence levels in Ca(2+) imaging of functionally distinct synaptic boutons at Drosophila neuromuscular junctions

GCaMP imaging is widely employed for investigating neuronal Ca(2+) dynamics. The Drosophila larval neuromuscular junction (NMJ) consists of three distinct types of motor terminals (type Ib, Is and II). This study investigated whether variability in synaptic bouton sizes and GCaMP expression levels confound interpretations of GCaMP readouts, in inferring the intrinsic Ca(2+) handling properties among these functionally distinct synapses. Analysis of large data sets accumulated over years established the wide ranges of bouton sizes and GCaMP baseline fluorescence, with large overlaps among synaptic categories. This study shows that bouton size and GCaMP baseline fluorescence were not confounding factors in determining the characteristic frequency responses among type Ib, Is and II synapses. More importantly, the drastic phenotypes that hyperexcitability mutations manifest preferentially in particular synaptic categories, were not obscured by bouton heterogeneity in physical size and GCaMP expression level. The data enabled an extensive analysis of the distal-proximal gradient of GCaMP responses upon genetic and pharmacological manipulations. The results illustrate the conditions that disrupt or enhance the distal-proximal gradients. For example, stimulus frequencies just above the threshold level produced the steepest gradient in low Ca(2+) (0.1 mM) saline, while supra-threshold stimulation flattened the gradient. Moreover, membrane hyperexcitability mutations (eag₁ Sh₁₂₀ and para_bss1) and mitochondrial inhibition by dinitrophenol (DNP) disrupted the gradient. However, a novel distal-proximal gradient of decay kinetics appeared after long-term DNP incubation. Focal recording was performed to assess the failure rates in transmission at low Ca(2+) levels, which yielded indications of a mild distal-proximal gradient in release probability (Xing, 2018b).

Regulation of presynaptic Ca2+ is critical for transmitter release as well as short-term and long-term synaptic plasticity. The Drosophila larval body-wall neuromuscular junction (NMJ) is an ideal system to contrast Ca2+ dynamics in synapses of different functional categories, as it contains in close proximity both tonic and phasic (type Ib and Is, respectively) glutamatergic synapses, as well as modulatory octopaminergic (type II) synapses, all of which can be imaged in the same microscopic field. A previous work has demonstrated characteristic frequency dependence of GCaMP signals for type Ib, Is and II synaptic boutons, indicating their distinct Ca2+ dynamics, i.e., type II, Is and Ib synapses being responsive to low, medium and high stimulus frequencies, respectively (Xing, 2018b).

Studies over the past years have accumulated a large body of single bouton records of GCaMP responses, along with quantifications of bouton sizes and GCaMP baseline fluorescence intensities. Despite the nomenclature implying size-related distinctions among these synapses (type Ib also known as 'I big', Is as 'I small'), significant heterogeneity is seen with overlapping bouton sizes between different synaptic categories. Furthermore, for each synaptic category, the level of GCaMP expression (as indicated by baseline fluorescence intensity F) varied significantly in the database. This study set out to determine the ranges of variation in these parameters and examined whether such high levels of heterogeneity confound interpretations from GCaMP measurements for salient physiological properties of the distinct synaptic bouton types (Xing, 2018b).

The database for this study also allowed a re-examination of the previously reported distal-to-proximal gradient in GCaMP response (ΔF/F). Analyses based on genetic and pharmacological manipulations further revealed the various conditions that can obscure or optimize the gradient. Lastly, simultaneous electrophysiological focal recording was carried out to map local synaptic transmission events to investigate the physiological significance of such distal-proximal GCaMP response gradient along the motor terminal branch (Xing, 2018b).

Among different cell types, neurons exhibit the most complex cellular morphology, with many specialized subcellular compartments, including presynaptic boutons and postsynaptic spines in different parts of soma and along axons and dendrites. Ca2+dynamics are crucial in the regulation of synaptic development, function, and plasticity. However, these structures of micrometer scale are highly variable and plastic in their size and location. Furthermore, synapses of different categories of neurons in the nervous systems, e.g. ionotropic and metabotropic, are distinct in morphology and distribution. It is therefore important to determine whether and how variation in geometric factors, such as physical size and location of synapses, sets constraints on local Ca2+ dynamics, and interferes with the readout of Ca2+ indicators in the investigation of intrinsic regulation mechanisms in functionally distinct synapses (Xing, 2018b).

Although a plethora of Drosophila GCaMP imaging studies has been published on CNS neuronal activity and peripheral synaptic function, the inter-relationships between synaptic parameters, such as bouton size, location, GCaMP baseline fluorescence F, and GCaMP signal amplitude ΔF/F, have not been fully established (Xing, 2018b).

The Drosophila NMJ provides a unique opportunity to contrast ionotropic synapses (type Ib and Is) with metabotropic synapses (type II) in close proximity for simultaneous imaging within the same microscopic field. Previous results show that type Ib, Is and II synapses manifest distinct frequency responses of GCaMP Ca2+ signals controlled by different ion channels and clearance mechanisms . In the present study, correlation analysis of large samples of boutons clarifies that neither bouton size nor GCaMP baseline fluorescence represents a confounding factor, when determining the intrinsic distinctions of frequency-dependent responses among type Ib, Is and II synapses (Xing, 2018b).

In principle, bouton size variation can be a contributing factor in determining the dynamics of GCaMP Ca2+ membrane properties, larger boutons should have a slower rate in accumulating cytosolic Ca2+ to produce detectable GCaMP signals. Thus, it can be argued that the size differences could be a possible explanation for the characteristic higher frequency response for type Ib bouton in contrast to the lower frequency ranges for type Is and even lower for type II synapses. In fact, the bouton size effect has been demonstrated in type Ib boutons to exist only for single-stimulus evoked Ca2+ transients, but not for plateau levels of Ca2+ signals evoked by trains of repetitive stimuli, using the fast indicator OGB-1. Theoretically, the rise of Ca2+ transients to a steady plateau level in response to a prolonged stimulus train is a process involving both Ca2+ influx and clearance, kinetically distinct from the measured amplitude of a single-stimulus evoked Ca2+ signal. In the current case, GCaMP signals are generally slow and do not resolve Ca2+ transients evoked by single action potentials. Instead, the plateaus of GCaMP signals report integration of Ca2+ influx and clearance over repetitive stimulus responses. In this study, the results demonstrate that the large overlaps of bouton sizes among the three synaptic types did not obscure their distinct frequency-dependent GCaMP signal characteristics. Therefore, physical dimension of synaptic boutons is not a practical predictor for the dynamics of GCaMP signals for boutons either within or between synaptic categories. Furthermore, it cannot account for the striking preferential effects of hyperexcitability mutations on different synaptic categories. Instead, intrinsic mechanisms such as membrane excitability and Ca2+ clearance capacity play far more important roles in the regulation of presynaptic Ca2+ dynamics (Xing, 2018b).

However, the data indeed confirm that GCaMP baseline fluorescence is significantly correlated with bouton size, as expected from their longer optical path in which GCaMP indicators interact with excitation light. Nonetheless, neither of these two absolute measures (in μm2 and mV) plays a significant role in determining GCaMP signal amplitudes, which are normalized, unit-less quantities (ΔF/F). A direct measurement of bouton width may be an alternative method to estimate bouton thickness and correlate with bouton fluorescence intensity, which can be investigated in further studies (Xing, 2018b).

Earlier Ca2+ imaging studies based on different Ca2+ indicators have led to somewhat different pictures on the distal-proximal gradient along synaptic terminals in the Drosophila NMJ. A relatively weak distal-proximal gradient of presynaptic Ca2+ dynamics has been detected by back-filling type Ib and Is synaptic terminals with the synthetic indicator Oregon Green BAPTA-1 (OGB-1) in muscles 4, 6 and 7 in high Ca2+ saline (1 mM). A different study using presynaptic expression of genetically encoded Ca2+ indicators (Cameleon2.3) has also demonstrated a presynaptic Ca2+ gradient of similar magnitude (Xing, 2018b).

Postsynaptic expression of Ca2+ indicators, including GCaMP and derivatives of Cameleon, has revealed a stronger distal-proximal gradient of nerve-evoked optical signal representing Ca2+ influx through postsynaptic glutamate receptors along type Ib synaptic terminals. However, a more recent study reports relative uniform distribution of evoked postsynaptic myrGCaMP5 signals. It is not known whether the differences in the indicator types and their cellular localization could account for the discrepancy between these observations (Xing, 2018b).

The current work indicates that several factors and conditions may be manipulated to either enhance or obscure the detection of a gradient of GCaMP signals along the synaptic terminals. These include external Ca2+ concentration, stimulus frequency, and larval genotype. The initial choice to use low Ca2+ saline in earlier GCaMP imaging studies was to optimize the striking effects of hyperexcitable ion channel mutations and to avoid muscle contraction without relying on the usage of glutamate for postsynaptic receptor desensitization. Fortuitously, it was found that low Ca2+ saline could better reveal the distal-proximal gradients (a proximal/distal ratio about 30%), in contrast to the previously reported gradient (type Ib: in the range of 60%-80%; type Is: 80%-90%) at high Ca2+concentrations (1-1.5 mM). To sum up, the optimal detection of the distal-proximal gradient falls in the range just above the threshold of effective stimulus frequency (20 Hz for Is, 40-80 Hz for Ib) for GCaMP imaging experiments at 0.1 mM Ca2+ (Xing, 2018b).

For both type Is and type Ib, longitudinal ΔF/F gradients could be demonstrated corresponding to the bouton rank order, or their physical distance from the distal end of the terminals. It should be noted, however, that there were no corresponding longitudinal gradients in bouton sizes or GCaMP baseline fluorescence (Xing, 2018b).

This gradient was disrupted by hyperexcitable mutations, or DNP incubation. Interestingly, after prolonged DNP treatment, which saturated the ΔF/F response and obscured its gradient, a novel gradient in decay kinetics of the GCaMP signals was revealed in both type Ib and Is, i.e. slower half-decay time in distal boutons, suggesting a possible role of mitochondria in gradient formation (Xing, 2018b).

To investigate the functional significance of the GCaMP signal gradient, advantage was taken of the focal recording of efEJP events from distal and proximal boutons to correlate with the optical imaging results from type Ib boutons in muscle 4. With this approach, the failure rate of transmission could be readily quantified at low Ca2+ levels. There were indications for a relatively weak distal-proximal difference in release probability with repetitive 1-Hz stimulation. However, to assess the GCaMP response amplitude, a higher simulation frequency was applied (40 Hz), at which a clear distal-proximal gradient of GCaMP signals could be observed in all terminals that displayed ΔF/F above the noise level (0.1 mM Ca2+). In this case, it was found that GCaMP ΔF/F signals beyond the cut-off level (0.05) were correlated with drastically higher release probability. A clear trend of facilitation was observed during the 2 s stimulus train in both distal and proximal boutons. However, no striking distal-proximal difference of facilitation properties was observed under this condition (Xing, 2018b).

It should be stressed that GCaMP signals reflect cytosolic residual Ca2+ accumulation, which takes place in a time scale of hundreds of milliseconds to a few seconds, and involves both Ca2+ influx and clearance mechanisms. In contrast, Ca2+ entry and subsequent vesicle fusion and transmitter release occur in milliseconds. Therefore, depending on the stimulus protocols and local physiological conditions, the two measurements may yield rather different readouts that reflect the states of two steps in the chain of cellular Ca2+ dynamics, from influx, local actions, cytoplasmic accumulation and clearance. Thus, in type Ib synaptic boutons at low Ca2+condition, the distal-proximal gradient of GCaMP signal gradient does not necessarily imply a similar gradient in transmission strength (Xing, 2018b).

Since GCaMP signals reflect the dynamics of cytosolic residual Ca2+ accumulation, more investigations are needed to fully understand its relationship with short-term synaptic plasticity properties. Furthermore, a more definitive assessment of any gradient in transmitter release, and transmission efficacy of individual boutons along the synaptic terminal requires direct quantification of local synaptic currents at various Ca2+ levels under voltage-clamp conditions. This awaits future studies employing the focal loose-patch clamp, first pioneered in this preparation by Prof. Harold Atwood and associates (Xing, 2018b).

Unraveling synaptic GCaMP signals: differential excitability and clearance mechanisms underlying distinct Ca(2+) dynamics in tonic and phasic excitatory, and aminergic modulatory motor terminals in Drosophila

GCaMP is an optogenetic Ca(2+) sensor widely used for monitoring neuronal activities but the precise physiological implications of GCaMP signals remain to be further delineated among functionally distinct synapses. The Drosophila neuromuscular junction (NMJ), a powerful genetic system for studying synaptic function and plasticity, consists of tonic and phasic glutamatergic and modulatory aminergic motor terminals of distinct properties. This study reports a first simultaneous imaging and electric recording study to directly contrast the frequency characteristics of GCaMP signals of the three synapses for physiological implications. Distinct mutational and drug effects on GCaMP signals indicate differential roles of Na(+) and K(+) channels, encoded by genes including paralytic (para), Shaker (Sh), Shab, and ether-a-go-go (eag), in excitability control of different motor terminals. Moreover, the Ca(2+) handling properties reflected by the characteristic frequency dependence of the synaptic GCaMP signals were determined to a large extent by differential capacity of mitochondria-powered Ca(2+) clearance mechanisms. Simultaneous focal recordings of synaptic activities further revealed that GCaMPs were ineffective in tracking the rapid dynamics of Ca(2+) influx that triggers transmitter release, especially during low-frequency activities, but more adequately reflected cytosolic residual Ca(2+) accumulation, a major factor governing activity-dependent synaptic plasticity. These results highlight the vast range of GCaMP response patterns in functionally distinct synaptic types and provide relevant information for establishing basic guidelines for the physiological interpretations of presynaptic GCaMP signals from in situ imaging studies (Xing, 2018a).

Ca2+ influx on action potential arrival at synaptic terminals triggers neurotransmitter release, and residual Ca2+ accumulation following repetitive action potentials regulates activity-dependent synaptic plasticity. Na+ and K+ channels play fundamental roles in shaping the axonal action potential and its repetitive firing pattern and thus can profoundly influence the amplitudes and kinetics of synaptic Ca2+ elevation. Conversely, Ca2+ clearance mechanisms, including mitochondrial and endoplasmic reticulum (ER) sequestration and energy-dependent extrusion via plasma membrane Ca2+-ATPase (PMCA), are critical in the restoration of synaptic basal Ca2+ levels (Xing, 2018a).

GCaMPs are widely used genetically encoded Ca2+ indicators. Despite the frequent applications of GCaMPs in monitoring neuronal activities in nervous systems of various animal species, it is unclear how differences in membrane excitability and Ca2+ clearance mechanisms determine the amplitude and kinetics of GCaMP Ca2+ signals in functionally distinct categories of synapses (Xing, 2018a).

This analyzed GCaMP signals in the Drosophila larval neuromuscular junction (NMJ), in which both excitatory (glutamatergic tonic type Ib and phasic type Is) as well as modulatory (octopaminergic type II) synapses could be monitored simultaneously within the same optical microscopy field. The glutamatergic type I synapses have been extensively studied for their electrophysiological properties and striking phenotypes caused by ion channel mutations. Octopaminergic type II synaptic terminals are known to modulate the growth and transmission properties of type I synapses and to display remarkable excitability-dependent plasticity. However, differences in excitability control and Ca2+ handling properties among these three distinct synaptic types remain to be determined (Xing, 2018a).

This decade-long study, extended from earlier results (Ueda, 2006, 2009; Xing, 2014), employed different versions of GCaMPs, including GCaMPs 1, 5, and 6, to delineate the distinct frequency characteristics of GCaMP signals from type Ib, Is, and II synapses and their preferential sensitivities to different pharmacological or genetic perturbations. In particular, the results show that type II synapses were most strongly affected by manipulations of channels encoded by ether-a-go-go (eag, Eag, or KV10 ortholog), Shab (KV2 ortholog), and paralytic (para, NaV1) channels, whereas type Is synapses were most severely modified by manipulations of Shaker (Sh, KV1 ortholog). Strikingly, double insults through manipulating Sh together with either eag or Shab could generate extreme hyperexcitability in type Is synapses, leading to greatly enhanced GCaMP signals on individual nerve stimulation. In contrast, type Ib synapses remained largely intact in the above experimentations but could display similar extreme hyperexcitability following triple insults with combinations of mutations or blockers of K+ channels. Simultaneous focal electrical recordings of synaptic activities revealed that such extreme cases of enhanced GCaMP signals actually resulted from supernumerary high-frequency (>100 Hz) repetitive firing in the motor terminals following each single stimulus (Xing, 2018a).

Further kinetic analysis revealed different Ca2+ clearance capacity among three types of synaptic terminals. This study found that Na+ and K+ channel mutations or blockers influence mainly the rise kinetics of GCaMP signals, whereas inhibiting Ca2+ clearance mediated by PMCA (via high pH treatment) slowed the decay phase acutely. In addition, it was discovered that long-term inhibition of mitochondrial energy metabolism by incubation with either 2,4-dinitrophenol (DNP) or azideled to drastically lengthened decay time of the GCaMP signal and significantly altered its frequency responses to repetitive stimulation, over a time course of tens of minutes (Xing, 2018a).

Overall, this study demonstrates a wide range of GCaMP response patterns indicating differential membrane excitability and Ca2+ clearance mechanisms in functionally distinct types of synapses. Although the slow kinetics of GCaMP signals could not adequately resolve the rapid process of Ca2+ influx triggered by individual action potentials, they could nevertheless report cytosolic residual Ca2+ accumulation on repetitive synaptic activities. These data thus provide essential baseline information for refined interpretations of GCaMP signals when monitoring in vivo neural circuit activities that often result from interplay among different categories of synapses (Xing, 2018a).

Genetically encoded GCaMP indicators are widely used for detecting neuronal circuit activities in vivo. However, the analytic power of GCaMP signals has not been fully exploited to extract information regarding basic synaptic physiology. This study took advantage of the special anatomic features of the Drosophila larval NMJ to contrast properties of metabotropic aminergic (type II) and ionotropic glutamatergic (tonic type Ib and phasic type Is) synapses using several GCaMP Ca2+ indicators. Simultaneous monitoring of GCaMP signals from the three synapses within the same microscopic field demonstrates differential excitability control of Ca2+ influx by Na+ and K+ channels. Analyses of both kinetic and amplitude features of GCaMP signals reveal the extreme effects of particular Na+ and K+ channels on each of the three synaptic types, as well as the prominent roles of mitochondria-powered Ca2+ clearance mechanisms in shaping their distinct Ca2+ handling properties (Xing, 2018a).

A summary diagram is presented of how the various genetic and pharmacological manipulations influence Ca2+ influx and clearance, hence the amplitude and kinetics of GCaMP signals (Presynaptic cytosolic residual Ca2+ regulation in Drosophila NMJ synapses). Action potentials, generated and fine-tuned by Na+ and K+ channels, depolarize synaptic terminals and allows Ca2+ influx, which triggers synaptic transmission rapidly in milliseconds. The influx of Ca2+ ions are either actively extruded by PMCA locally, or sequestered by intracellular organelles such as mitochondria and ER, or buffered by Ca2+ binding proteins. The rise of GCaMP signals spans from hundreds of milliseconds up to seconds before peaking, depending on stimulation frequencies and external Ca2+ concentrations. Even with improved sensitivity, GCaMP6 signals are not faster compared to GCaMP1.3, taking at least 100 ms after a single stimulus to reach the peak of fluorescence at high external Ca2+ concentration. Thus, GCaMP signals are several orders slower than individual action potentials and the ensuing postsynaptic potentials. Further, unlike the synthetic Ca2+ indicators such as Oregon Green BAPTA (Hill coefficient 1.48), a GCaMP protein, with calmodulin as the Ca2+ sensor, typically binds four Ca2+ ions allosterically to produce enhanced fluorescence (Hill coefficients of GCaMP1 = 3.3). The magnitude of enhancement is thus limited especially at low levels of Ca2+ elevations evoked by single action potentials. Therefore, GCaMP signals better serve as the readout of a leaky integrator that registers cytosolic residual Ca2+, i.e., the net Ca2+ accumulation as determined by the process of influx and clearance over repetitive firing of action potentials, which can be induced either by trains of stimulation, or hyperexcitability (Xing, 2018a).

Electrophysiological recording of postsynaptic EJCs or EJPs generally detects the ensemble effects of type Ib, Is, and II synapses. Unlike type Ib and Is synapses, electrophysiological characterization of aminergic type II synapses is more technically challenging because they do not generate readily detectable postsynaptic electrical responses. In contrast, GCaMP signals offer the necessary spatial resolution, and thus enabled demonstration for the first time that mutations or blockers of specific ion channels lead to drastically different effects on type II, as well as type Ib and Is, axonal terminals (Xing, 2018a).

These results demonstrated that type Ib synapses were most enriched in the reserve of repolarizing capacity pooled from different K+ channel subtypes and could sustain multiple insults of K+ channel elimination or blockage before exhibiting the 'hallmark' of extreme hyperexcitability (single pulse-evoked giant GCaMP signals at 0.1 mM Ca2+). In comparison, type II synapses had the smallest repertoire of K+ channels and simply knocking down either Shab or eag could induce the hallmark hyperexcitability effect. In type Is synapses, Sh appeared to be the central player for repolarization and perturbing the Sh channel together with either Eag or Shab channels induced the hallmark ceiling effect of extreme hyperexcitability. This finding also resolved type Is but not Ib motor axons as the major source of the striking electrophysiological phenotype, i.e., axonal high-frequency repetitive firing (Xing, 2018a).

Alleles of para also have differential effects on type Ib, Is, and II synapses, possibly reflecting differential expression of the Para product, e.g., different splice isoforms, or posttranslational modifications (Xing, 2018a).

Type II synapses were more prone to conduction failure on high-frequency stimulation, as indicated by GCaMP signals that frequently became intermittent, or even totally missing during 10- to 40-Hz stimulation. This reflects the well-known axonal passive cable properties; thinner axons have proportionally higher longitudinal internal resistance relative to trans-membrane resistance, resulting in a more limited safety margin of axonal conduction and a longer refractory period for action potentials. Therefore, type II terminals are more prone to K+ and Na+ channels modifications (Xing, 2018a).

Morphometric analysis confirms that the differential excitability and distinct Ca2+ dynamics found in this study reflect intrinsic properties of type Ib, Is, and II synapses. The GCaMP responses characteristic of each synaptic type were independent of different sizes of boutons along individual axonal synaptic terminals, implying that differences in the physical dimensions among the three synaptic bouton types do not contribute to the distinct properties of type Ib, Is, and II synapses reported in this study (Xing, 2018a).

Obviously, besides Na+ and K+ channels, other channels may contribute to excitability-controlled Ca2+ influx. In particular, different types of Ca2+ channels await further study. Notably, previous anatomic studies have shown differences in presynaptic density area among different types of boutons. Ca2+ channels are known to be closely associated with active zones embedded within presynaptic density areas. It has been shown that type Is has higher density of active zones than type Ib synapses (He, 2009; Lu, 2016; Xing, 2018a).

It should be noted that differences in Ca2+ clearance capacity correlate well with the distinct frequency responses in the Ca2+ dynamics of these synaptic categories. Type II synapses apparently have the slowest rate of Ca2+ clearance, as evidenced by its slowest decay of GCaMP signals after secession of stimulation. The faster Ca2+ clearance in type Ib when compared to type Is synapses (He, 2009) appears to parallel its higher firing frequency (40-60 Hz in Ib vs 10-20 Hz in type Is) during natural bursting activities in semi-intact larval preparations, whereas presynaptic cytosolic Ca2+ elevation during repetitive firing stimulate mitochondrial oxidative phosphorylation so as to meet temporary burst energy needs. It is conceivable that type Ib synapses thus require a more efficient Ca2+ clearance system to avoid intracellular Ca2+ build-up. Interestingly, earlier electron microscopy studies have shown that tonic (type Ib) synapses contain more mitochondria than phasic (type Is) synapses in both Drosophila larval and crayfish NMJs. The current observation using mitochondrial staining confirmed this conclusion and also revealed a far lower density of mitochondria in type II synapses (Xing, 2018a).

This study showed the importance of mitochondria-powered Ca2+ clearance in shaping the distinct dynamics of cytosolic residual Ca2+ build-up in type Ib, Is, and II synapses. Inhibiting mitochondrial function with two different means, incubation with either DNP, a proton ionophore that dissipates mitochondrial proton gradient, or azide, an electron-transport chain inhibitor (complex IV), consistently resulted in slower GCaMP signal decay time course and shifted the frequency dependence in type II, Is, and Ib over a period of tens of minutes (Xing, 2018a).

In contrast to the slow effect of mitochondrial inhibition, high-pH inhibition of PMCA clearly impedes the GCaMP signal decay time course acutely. Ca2+ extrusion via PMCA, a Ca2+-ATPase, has been characterized in the Drosophila NMJ, as well as goldfish retina. Although under in vitro conditions, the fluorescence intensity of GCaMP protein can be affected by pH change, intracellularly expressed GCaMP protein is less likely to be affected by extracellular pH manipulation. This notion was supported by lack of change in presynaptic GCaMP baseline fluorescence intensity on external pH changes. Therefore, impaired ATP production from mitochondria can lead to PMCA-mediated Ca2+ extrusion shut-down, which could account for the striking effect of long-term DNP incubation (Xing, 2018a).

Notably, DNP treatment significantly impeded the GCaMP signal decay time course only after long-term incubation (beyond 20 min). Previous studies employing other proton ionophores such as carbonyl cyanide m-chlorophenyl hydrazine (CCCP) has demonstrated that inhibition of mitochondrial proton gradient does not significantly alter overall cytosolic Ca2+ dynamics acutely (Xing, 2018a).

Besides mitochondria, ER may also actively sequestrate Ca2+ via sarco/ER Ca2+ ATPase (SERCA) in synapses. This study inhibited SERCA with thapsigargin (1-2 µM, 1-h treatment) and found no obviously detectable effects on GCaMP signals comparable to the effect of DNP on any of the three types of synapses (in 4 larvae). Previous publications with a higher thapsigargin concentration (10 µM;) or more sensitive Ca2+ indicator (Oregon Green BAPTA) have demonstrated only mildly increased Ca2+ signal amplitude and slower time course in type Ib synapses. Therefore, the contributions of mitochondrial and ER Ca2+ sequestration may be masked by other high-capacity ATP-dependent Ca2+ clearance mechanisms, such as PMCA. Nevertheless, when ATP production by mitochondria is inhibited, these active Ca2+ clearance mechanisms could be diminished on gradual depletion of ATP reserve (Xing, 2018a).

In this study, synapses remain viable, and Ca2+ clearance system remains functioning for at least tens of minutes, despite mitochondrial inhibition by DNP or azide. Nonmitochondrial sources of ATP such as glycolysis or ATP binding proteins might sustain for some time, until the first sign of depletion, i.e., the appearance of slower GCaMP signal decay kinetics (Xing, 2018a).

In fact, some vertebrate central nervous system synapses are known to operate without local presynaptic mitochondria. Similarly, Drosophila mutant drp1 and dMiro larval NMJs, with greatly reduced numbers of synaptic mitochondria, remain viable and display essentially normal Ca2+ dynamics and buffer capacity unless challenged by prolonged stimulation beyond minutes. In these studies, type II synapses had a lower abundance in mitochondria and thus more limited ATP reserve and in consequence were most vulnerable to DNP treatment. They were the first to show lengthened decay and to become completely nonresponsive subsequently during DNP incubation (Xing, 2018a).

Overall, this study indicates that analysis of GCaMP signals can be extended to extract information about specific synaptic physiologic properties. GCaMP signals offer higher spatial resolution and can complement electrophysiology data to pinpoint critical differences in channel expression and excitability properties among neighboring synaptic terminals (Xing, 2018a).

Systematic kinetic analysis of GCaMP signals revealed the predominant effects of hyperexcitability on the rise kinetics and Ca2+ clearance capacity on the decay kinetics. In conjunction with focal electrophysiological recording, genetic and pharmacological analyses indicate a close relationship between GCaMP signals and cytosolic residual Ca2+ accumulation rather than the rapid process of Ca2+ influx that triggers transmitter release. This approach also revealed the striking hyperexcitable effects caused by insults to multiple K+ channels, leading to the hallmark giant GCaMP signals evoked by single stimuli that generated high-frequency supernumerary firing of nerve action potentials. Thus, GCaMP signals may be further exploited to shed new light on activity-dependent plasticity in synapses of distinct properties. This work may help to establish guidelines for refined interpretations of GCaMP signals beyond the first-order, qualitative indications for gross neuronal activities in neural circuits (Xing, 2018a).

Regulation of synaptic architecture and synaptic vesicle pools by Nervous wreck at Drosophila Type 1b glutamatergic synapses

Nervous wreck (Nwk), a protein that is present at Type 1 glutamatergic synapses that contains an SH3 domain and an FCH motif, is a Drosophila homolog of the human srGAP3/MEGAP protein, which is associated with mental retardation. Confocal microscopy revealed that circles in Nwk reticulum enclosed T-shaped active zones (T-AZs) and partially colocalized with synaptic vesicle (SV) markers and both exocytosis and endocytosis components. Results from an electron microscopic (EM) analysis showed that Nwk proteins localized at synaptic edges and in SV pools. Both the synaptic areas and the number of SVs in the readily releasable (RRPs) and reserve (RPs) SV pools in nwk² were significantly reduced. Synergistic, morphological phenotypes observed from eag¹;;nwk² neuromuscular junctions suggested that Nwk may regulate synaptic plasticity differently from activity-dependent Hebbian plasticity. Although the synaptic areas in eag¹;;nwk² boutons were not significantly different from those of nwk², the number of SVs in the RRPs was similar to those of Canton-S. In addition, three-dimensional, high-voltage EM tomographic analysis demonstrated that significantly fewer enlarged SVs were present in nwk² RRPs. Furthermore, Nwk formed protein complexes with Drosophila Synapsin and Synaptotagmin 1 (DSypt1). Taken together, these findings suggest that Nwk is able to maintain synaptic architecture and both SV size and distribution at T-AZs by interacting with Synapsin and DSypt1 (Hur, 2018).

The interaction between the Drosophila EAG potassium channel and the protein kinase CaMKII involves an extensive interface at the active site of the kinase

The Drosophila EAG (dEAG) potassium channel is the founding member of the superfamily of KNCH channels, which are involved in cardiac repolarization, neuronal excitability and cellular proliferation. In flies, dEAG is involved in regulation of neuron firing and assembles with CaMKII to form a complex implicated in memory formation. This study has characterized the interaction between the kinase domain of CaMKII and a 53-residue fragment of the dEAG channel that includes a canonical CaMKII recognition sequence. Crystal structures together with biochemical/biophysical analysis show a substrate-kinase complex with an unusually tight and extensive interface that appears to be strengthened by phosphorylation of the channel fragment. Electrophysiological recordings show that catalytically active CaMKII is required to observe active dEAG channels. A previously identified phosphorylation site in the recognition sequence is not the substrate for this crucial kinase activity, but rather contributes importantly to the tight interaction of the kinase with the channel. The available data suggest that the dEAG channel is a docking platform for the kinase and that phosphorylation of the channel's kinase recognition sequence modulates the strength of the interaction between the channel and the kinase (Castro-Rodrigues, 2018).

The KCNH voltage-gated potassium channels (EAG, ERG and ELK) are involved in important physiological processes like cardiac repolarization, neuronal excitability and cellular proliferation. KCNH potassium channels have a typical tetrameric assembly, where each subunit has six transmembrane helices harboring a voltage sensor and a K+ selectivity filter. These channels also include unique long N- and C-terminal cytoplasmic regions, which are thought to function as interfaces with cellular signaling cascades including kinases. The Drosophila EAG (dEAG) potassium channel is the founding member of the KCNH superfamily. Interestingly, it has been shown that this channel interacts with the Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Castro-Rodrigues, 2018).

CaMKII is a Ser/Thr protein kinase with a central role in the mechanism of long-term potentiation and synaptic plasticity. CaMKII is a dodecamer or tetradecamer; each subunit includes a protein kinase domain at the N terminus and a hub or multimerization domain at the C terminus. These two domains are separated by a regulatory segment (RS) containing a Ca2+/CaM-binding motif and a variable linker. Binding of Ca2+/CaM to the RS activates the kinase by releasing the inhibitory interaction between RS and the kinase domain (Castro-Rodrigues, 2018).

It has been proposed that phosphorylation of the dEAG potassium channel by CaMKII modulates channel activity and reciprocally that the functional properties of CaMKII are altered upon interaction with the channel. CaMKII phosphorylates dEAG at residue T787, within a CaMKII recognition sequence present in the channel's C-terminal cytoplasmic region. A similar sequence in the GluN2B subunit of the NMDA receptors has long been recognized as an interaction and phosphorylation site for CaMKII. Other KCNH channels such as the human EAG and ERG (hERG) channels do not have this recognition sequence; in these channels, the region corresponds to a section of the C terminus that in the recent cryo-EM structures was truncated as it is thought to be unstructured. In the fly, dEAG and CaMKII can be found at neuron synapses as part of a signaling complex. Importantly, the two proteins can be co-purified and it has been proposed that the kinase recognition sequence is the major region of interaction (Castro-Rodrigues, 2018).

As in many other cases of regulation of potassium channel activity by kinases and phosphatases, the molecular details of the partnering between CaMKII and dEAG are not completely understood. A biochemical, structural and functional characterization was performed of determinants of the interaction between this potassium channel and the kinase. The complex formed between a channel fragment, spanning the CaMKII recognition sequence in dEAG, and the kinase domain of CaMKII displays the expected features of a kinase-substrate complex but is unusually stable for a typical kinase-substrate pair, which have KDs around 200 µM. The tight interaction involves residues previously defined for an ideal CaMKII substrate together with main-chain interactions and water-mediated contacts. Functional data show that interaction between the channel fragment and the kinase domain alters the activity of the kinase and that the presence of active CaMKII is crucial for a fully functional channel. However, phosphorylation at the kinase recognition sequence in the channel does not alter the functional properties of the channel. Instead, the recognition sequence contributes to the stability of the channel/kinase complex with phosphorylation of the recognition site likely modulating complex stability. Overall, this analysis gives a detailed view of the interaction established between CaMKII and dEAG, providing molecular insights into the relationship established by CaMKII and other ion channels like the NMDA receptor and voltage-dependent calcium channels (Castro-Rodrigues, 2018).

This study has characterized the interaction between the kinase domain of CaMKII and a C-terminal fragment of the dEAG channel, which spans the kinase recognition sequence. In particular, this interaction was found to display the general features found in a kinase-substrate complex, with the channel fragment interacting with the active site of the kinase. The interaction is unusually tight for a kinase and its substrate, with a long interface between the two protein components, determined not only from specific contacts established by channel side chains but also from contacts established by channel main-chain atoms and by water-mediated interactions. Catalytically active CaMKII was found to be required to observe active dEAG channels on the oocyte membrane but, interestingly, this does not involve phosphorylation of T787, a residue in the kinase recognition sequence. Instead, the results indicate that phosphorylation at T787, previously implicated in dEAG function, enhances the interaction between the channel fragment and kinase (Castro-Rodrigues, 2018).

A question arising from this study is, what makes the CaMKII kinase domain-dEAG fragment an unusual kinase-substrate complex? Although there are many structures of protein kinases, structures of Ser/Thr protein kinases in complex with their cognate peptide substrates are not abundant probably because this interaction is typically of low affinity (KD ~ 200 μM). The following kinase structures bound to high-affinity substrates or high-affinity inhibitory peptides were studied: phosphorylase kinase (PHK) with a 7-residue optimal substrate (2PHK), protein kinase B in complex with a 10-residue substrate peptide derived from glycogen synthetase kinase 3β (GSK3β) (1O6K, 1O6L), protein kinase A with a 20-residue protein kinase inhibitor (PKI) (1ATP), and protein kinase A with a PKI-derived substrate peptide (SP20) (1JBP, 1JLU). Structures were also analyzed of CaMKII kinase bound in trans to the RS from another CaMKII subunit (3KK8, 2WEL) or in complex with the inhibitor CaMKII-Ntide (3KL8). In addition, this analysis includeds an example of a 'normal affinity' peptide substrate, the structure of cyclin-dependent kinase 2 (CDK2)-cyclin A3 (1QMZ); this complex was obtained from co-crystallization of the kinase with large concentrations of a low-affinity peptide substrate. Comparison of these structures reveals that many of the side-chain interactions described for the CaMKII kinase domain bound the dEAG fragment are specific to this kinase-substrate pair and not present in other kinase-substrate pairs. In contrast, the buried surface area for some of the high-affinity kinase-peptide complexes, around 1000 Å 2, is comparable to that found in CaMKII-dEAG (~ 945 Å 2). In a few high-affinity structures, 2PHK and 1O6K, where the substrates are shorter (7 and 10 residues long, respectively), the buried surface area is smaller, ~ 600-700 Å2. However, for the low-affinity complex CDK2-cyclin A3-peptide substrate (7 residues long), the buried surface area is even smaller (~ 470 Å2). In addition, the high-affinity substrate structures display a main-chain hydrogen bond network; this network is absent from the low-affinity substrate structure. A general feature of the structures analyzed with high enough resolution is the presence of water molecules in the kinase-substrate interface and mediating interactions between the pair of molecules. This characteristic is independent of the kinase-substrate affinity. It is concluded that the combination of structural features described for the dEAG-CaMKII are also present in other high-affinity kinase-substrate complexes, probably underlying the unusually tight interaction as they are absent from the 'more common' low affinity complex structure (Castro-Rodrigues, 2018).

What are the functional consequences of the tight interaction between the kinase domain and the channel? First, from the point of view of the kinase, it has been demonstrated that the interaction with the channel fragment changes the properties of full-length CaMKII, giving rise to a sustained Ca2+/calmodulin- and autophosphorylation-independent activation of the kinase. This study now showns that the interaction also reduces the rate of ATP hydrolysis in a single kinase domain. This observation can be explained by a slowing down of the catalytic turnover due to the tight binding of the channel fragment to the substrate-binding site in the kinase and the stabilization of the closed active site. The two outcomes of the tight interaction between the CaMKII and the dEAG channel fragment, sustained activation and reduction in catalytic activity, are compatible. Interaction between a channel kinase-recognition sequence with a kinase domain in the dodecameric CaMKII reduces catalytic activity of that particular domain while competing the kinase RS. This is likely to cause a rearrangement of the dodecamer, destabilize the inhibited state of other kinase domains in CaMKII and result in Ca2+-calmodulin- and autophosphorylation-independent activity (Castro-Rodrigues, 2018).

Second, from the point of view of the channel, this study has shown that a catalytically active kinase is required for the display of channel activity on the membrane. The basis for this effect is not yet clear, and it might result from impact of the kinase on the functional properties of the channel, on the levels of channel protein in the membrane or on the folding/processing of channel in the membrane or from a combination of these. Further studies are needed to dissect this effect. This finding contrasts with previous findings of the Griffith laboratory, where they found that T787A modestly increased the rate of channel inactivation and decreased channel current. At present, there is no explanation for the discrepancy (Castro-Rodrigues, 2018).

What then is the role of the interaction between the kinase domain of CaMKII and its recognition sequence in the dEAG channel? Other proteins that harbor amino acid sequences similar to the CaMKII-binding region of dEAG provide clues about this question. Such a sequence is present in the C-terminus of β1 and β2 subunits of human voltage-dependent calcium channel and has been shown to play a role in the formation of stable complexes between CaMKII and the calcium channel. This interaction, together with β2a phosphorylation, is required for the facilitation of L-type Ca2+channels. CaMKII was described as a scaffold for proteasome recruitment to dendritic spines and was found to phosphorylate Ser120 (at the minimal R-x-x-S motif) of the Rpt6 regulatory subunit and concurrently enhance proteasome activity. A segment resembling the recognition sequence in dEAG but with a non-phosphorylatable residue (Arg instead of Thr/Ser, as in the CaMKII-Ntide inhibitor) can be found in the 26S proteasome p45/Rpt6 subunit. This protein segment is distal from the phosphorylation site and appears occluded in the structure of the fully assembled 26S proteasome particle. Nevertheless, it is tempting to envisage the identified segment playing a role in the interaction of CaMKII with the proteosome in some specific conformation (Castro-Rodrigues, 2018).

Crucially, there is the well-described interaction between CaMKII and the NMDA receptor through a C-terminal sequence that is very similar to the one found in this study. The interaction between the NMDA receptor subunit GluN2B and CaMKII is an important element of the long-term potentiation mechanism at the synapse. There is a strong similarity in the properties of the CaMKII/dEAG channel complex and the CaMKII/NMDA receptor. Both channels contain CaMKII recognition sequences in their cytoplasmic C-terminal regions, forming very stable complexes with CaMKII, which give rise to Ca2+/calmodulin- and autophosphorylation-independent activity of the kinase. The interaction between the two channels and CaMKII has a role in learning and memory formation. In flies, the dEAG channel and CaMKII are localized to the synaptic compartment, and both proteins are involved in a pathway that regulates neuronal plasticity and memory formation. In mammals, long-term potentiation depends on the establishment and long-term stability of the interaction between CaMKII and the NMDA receptor at the post-synaptic density, where this interaction is a defining molecular event of synaptic plasticity that leads to enhanced synaptic strength (Castro-Rodrigues, 2018).

These parallels strongly suggest that the interaction between the two ion channels and CaMKII follows similar rules and has related biochemical purposes. To explain the functional relationship between CaMKII and the NMDA receptor, it has been proposed that the channel acts as a docking/recruiting platform for the kinase. Together with the demonstration that CaMKII and dEAG form a complex in Drosophila, this leads to a proposal that the dEAG channel is a docking or recruiting platform for CaMKII, where the contacts mediating the channel/kinase complex include the interaction between the kinase domain and the recognition sequence seen in the structures. Moreover, the data suggest that the role of phosphorylation at T787 is to modulate the strength of the interaction between the kinase and the channel. An apparent increased stability of the kinase/fragment complex upon phosphorylation of T787 and a competitor peptide has an enhanced functional effect on the mutant T787A channel relative to the wild-type channel. Importantly, this study reveals structural data that explain the stabilization effect of T787 phosphorylation by the formation of extra interactions between the phosphorylated channel fragment and the kinase. Overall, these results are consistent with a scenario where phosphorylation of the kinase recognition sequence in the protein does not alter the function of the channel but instead alters the action of the kinase by stabilizing its location (Castro-Rodrigues, 2018).

The functional results also suggest that the interaction between CaMKII and the full-length channel involves other contacts besides those characterized in this study. It is speculated that the other interacting regions include stretches in the long disordered C-terminal region of the dEAG channel since these are the amino acid regions that differ the most between dEAG and either the rat EAG or hERG channels, both of which are not known to interact with CaMKII. Overall, to fully understand the functional and structural impact of CaMKII in the dEAG channel, further work will be required (Castro-Rodrigues, 2018).

The amino acid sequence similarity identified in the C-terminal stretches of dEAG and GluN2B subunit of the NMDA receptor involved in the interaction with CaMKII also strongly suggests that this region of GluN2B binds to the kinase domain of CaMKII in a similar manner to that observed in the structures described in this study, with the GluN2B region interacting as a substrate. In addition, the similarities also suggest that phosphorylation of the recognition sequence in GluN2B will lead to a tightening of the interaction with the kinase as observed for dEAG. In fact, phosphorylation of the GluN2B recognition sequence has been shown to alter its interaction with CaMKII, but these results have been interpreted as indicating a destabilization of the complex upon phosphorylation. However, these experiments were performed differently from the current and do not directly explore the role of phosphorylation on the stability of the kinase/fragment complex formed at the end of the catalytic reaction, with ADP bound in the active site (Castro-Rodrigues, 2018).

Faced with the multiple occurrences of CaMKII protein complexes where the kinase catalytic site appears to be an important determinant of the interaction, it is worthwhile considering that CaMKII, like other protein kinases, may also have non-catalytic functions. In the particular case of the dEAG channel, a simple consideration of the oligomeric structures of the channel and CaMKII together with the well-established dynamic nature of the structure of CaMKII raises two intriguing possibilities. First, the interaction between these two proteins may involve avidity and clustering effects. The EAG potassium channel is a homotetramer with four CaMKII recognition sites per channel, and CaMKII is a dodecamer (and sometimes a tetradecamer), and therefore, the interaction of four kinase domains with the four channel subunits will result in increased stability of the complex through an avidity effect. Second, the dynamic architecture of CaMKII, where the 12 kinase domains project from the hub domain and display a 'wing span' that can vary from 200 to 320 Å, naturally suggests the possibility that activated kinase can bind multiple channels simultaneously and promote clustering of EAG channels. Future studies will be required to assess the role of this interaction on the organization of the synaptic membrane (Castro-Rodrigues, 2018).

Drosophila CRY entrains clocks in body tissues to light and maintains passive membrane properties in a non-clock body tissue independent of light

Circadian clocks regulate daily rhythms in physiology, metabolism, and behavior via cell-autonomous transcriptional feedback loops. In Drosophila, the blue-light photoreceptor Cryptochrome (Cry) synchronizes these feedback loops to light:dark cycles by binding to and degrading Timeless (Tim) protein. CRY also acts independently of TIM in Drosophila to alter potassium channel conductance in arousal neurons after light exposure, and in many animals CRY acts independently of light to repress rhythmic transcription. CRY expression has been characterized in the Drosophila brain and eyes, but not in peripheral clock and non-clock tissues in the body. To investigate CRY expression and function in body tissues, a GFP-tagged-cry transgene was generated that rescues light-induced behavioral phase resetting in cry⁰³ mutant flies and sensitively reports GFP-CRY expression. In bodies, CRY is detected in clock-containing tissues including Malpighian tubules, where it mediates both light-dependent TIM degradation and clock function. In larval salivary glands, which lack clock function but are amenable to electrophysiological recording, CRY prevents membrane input resistance from falling to low levels in a light-independent manner. The ability of CRY to maintain high input resistance in these non-excitable cells also requires the K+ channel subunits Hyperkinetic, Shaker, and Ether-a-go-go. These findings for the first time define CRY expression in Drosophila peripheral tissues and reveal that CRY acts together with K+ channels to maintain passive membrane properties in a non-clock-containing peripheral tissue independent of light (Agrawal, 2017).

CRYPTOCHROME-mediated phototransduction by modulation of the potassium ion channel β-subunit redox sensor

Blue light activation of the photoreceptor Cryptochrome (Cry) evokes rapid depolarization and increased action potential firing in a subset of circadian and arousal neurons in Drosophila melanogaster. This study shows that acute arousal behavioral responses to blue light significantly differ in mutants lacking Cry, as well as mutants with disrupted opsin-based phototransduction. Light-activated Cry couples to membrane depolarization via a well conserved redox sensor of the voltage-gated potassium (K+) channel β-subunit (Kvβ) Hyperkinetic (Hk). The neuronal light response is almost completely absent in hk-/- mutants, but is functionally rescued by genetically targeted neuronal expression of WT Hk, but not by Hk point mutations that disable Hk redox sensor function. Multiple K+ channel α-subunits that coassemble with Hk, including Shaker, Ether-a-go-go, and Ether-a-go-go-related gene, are ion conducting channels for Cry/Hk-coupled light response. Light activation of Cry is transduced to membrane depolarization, increased firing rate, and acute behavioral responses by the Kvβ subunit redox sensor (Fogle, 2015).

Acute behavioral arousal to blue light is significantly attenuated in CRY mutants. This study identified a redox signaling couple between blue light-activated CRY and rapid membrane depolarization via the redox sensor of Kvβ channel subunits coassembled with Kvα channel subunits. Additional unknown factors may act as intermediates between CRY and Hk. This finding provides in vivo validation of a very longstanding hypothesis that the highly conserved redox sensor of Kvβ subunit functionally senses cellular redox events to physiological changes in membrane electrical potential. Genetic loss of any single component functionally disrupts the CRY-mediated blue light response, which is functionally rescued by LNv restricted expression of their WT genes in the null backgrounds. Although little is known about the structural contacts between Kvβ and EAG subunits, Kvβ subunits make extensive physical contacts in a fourfold symmetric fashion in 1:1 stoichiometry with the T1 assembly domain of other coassembled tetrameric Kvα subunits that form the complete functional channel (Fogle, 2015).

Application of Kvβ redox chemical substrate modulates voltage-evoked channel peak current, steady-state current, and inactivation in heterologously expressed α-β channels, which are reversed by fresh NADPH. These results indicate that measurements of channel biophysical properties can reflect the redox enzymatic cycle of Kvβ as these channel modulatory effects are absent in preparations that lack the expression of WT Kvβ subunits or express redox sensor mutant Kvβ subunits. Whether direct chemical redox reactions occur between CRY and Hk is unclear. For CRY, light or chemical reduction induces one-electron reduction of the FAD cofactor of CRY, whereas the reductive catalytic mechanism of AKRs (such as Hk) requires a hydride ion transferred from NADPH to a substrate carbonyl, then a solvent-donated proton reduces the substrate carbonyl to an alcohol. These differences in redox chemistry between CRY and Hk suggest that other intermediates, such as oxygen, are possibly required for redox coupling (Fogle, 2015).

Spectroscopic analysis of animal and plant CRYs suggest that light activation causes reduction of the FAD oxidized base state. Light activation of Drosophila CRY also evokes conformational changes in the C terminus of CRY that clearly promotes CRY C-terminal access to proteolytic degradation and subsequent interactions with the Timeless clock protein, thus signaling degradation and circadian entrainment. However, all existing evidence suggests that light activated CRY-mediated circadian entrainment and membrane electrical phototransduction operate under different mechanisms, including their different activation thresholds and relative dependence on the C terminus of CRY. Further distinguishing the distinct mechanisms of the downstream effects of light-activated CRY, the light-induced conformational changes that couple CRY to ubiquitin ligase binding (thus causing circadian entrainment) occur in oxidized and reduced states of CRY and are unaffected in CRY tryptophan mutants that presumably are responsible for intraprotein electron transfer reactions following light-evoked reduction of the FAD cofactor. Another recent study shows that light- or chemical-evoked reduction of Drosophila CRY FAD is coupled to conformational changes of the CRY C terminus, along with reporting a surprising negative result that DPI has no effect on the reoxidation of the reduced anionic semiquinone of purified Drosophila CRY. DPI could hypothetically influence the electrophysiological light response by blocking the pentose phosphate pathway, which produces the Hk redox cofactor NADPH, but this does not explain the light dependence for DPI blocking the electrophysiological light response herein. The available evidence indicates that CRY-mediated light evoked membrane depolarization occurs independently of conformational changes in the CRY C-terminal domain but depends on redox changes in CRY, whereas CRY-mediated light evoked circadian entrainment depends on conformational changes in the CRY C-terminal domain and may or may not depend on CRY redox state (Fogle, 2015).

Light-activated CRY evokes rapid membrane depolarization through the redox sensor of the Kvβ subunit Hk. A general role for circadian regulation of redox state coupled to membrane excitability has been described recently in mammalian suprachiasmatic neurons. Redox modulation of circadian neural excitability may be a well-conserved feature (Fogle, 2015).

RNA editing in eag potassium channels: biophysical consequences of editing a conserved S6 residue

RNA editing at four sites in eag, a Drosophila voltage-gated potassium channel, results in the substitution of amino acids into the final protein product that are not encoded by the genome. These sites and the editing alterations introduced are K467R (Site 1, top of the S6 segment), Y548C, N567D and K699R (sites 2-4, within the cytoplasmic C-terminal domain). These residues were individually mutated, and the channels were expressed in Xenopus oocytes. A fully edited construct (all four sites) has the slowest activation kinetics and a paucity of inactivation, whereas the fully unedited channel exhibits the fastest activation and most dramatic inactivation. Editing Site 1 inhibits steady-state inactivation. Mutating Site 1 to the neutral residues resulted in intermediate inactivation phenotypes and a leftward shift of the peak current-voltage relationship. Activation kinetics display a Cole-Moore shift that is enhanced by RNA editing. Normalized open probability relationships for 467Q, 467R and 467K are superimposable, indicating little effect of the mutations on steady-state activation. 467Q and 467R enhance instantaneous inward rectification, indicating a role of this residue in ion permeation. Intracellular tetrabutylammonium blocks 467K significantly better than 467R. Block by intracellular, but not extracellular, tetraethylammonium interferes with inactivation. The fraction of inactivated current is reduced at higher extracellular Mg(+2) concentrations, and channels edited at Site 1 are more sensitive to changes in extracellular Mg(+2) than unedited channels. These results show that even a minor change in amino acid side-chain chemistry and size can have a dramatic impact on channel biophysics, and that RNA editing is important for fine-tuning the channel's function (Ryan, 2012).

Contribution of EAG to excitability and potassium currents in Drosophila larval motoneurons

Diversity in the expression of K(+) channels among neurons allows a wide range of excitability, growth, and functional regulation. Ether-a-go-go (EAG), a voltage-gated K(+) channel, was first characterized in Drosophila mutants by spontaneous firing in nerve terminals and enhanced neurotransmitter release. Although diverse functions have been ascribed to this protein, its role within neurons remains poorly understood. The aim of this study was to characterize the function of EAG in situ in Drosophila larval motoneurons. Whole cell patch-clamp recordings performed from the somata revealed a decrease in I(Av) and I(Kv) K(+) currents in eag mutants and with targeted eag RNAi expression. Spontaneous spike-like events were observed in eag mutants but absent in wild-type motoneurons. Thus these results provide evidence that EAG represents a unique K(+) channel contributing to multiple K(+) currents in motoneurons helping to regulate excitability, consistent with previous observations in the Drosophila larval muscle (Srinivasan, 2012).

Intracellular regions of the Eag potassium channel play a critical role in generation of voltage-dependent currents

Folding, assembly, and trafficking of ion channels are tightly controlled processes and are important for biological functions relevant to health and disease. This study reports that functional expression of the Eag channel is temperature-sensitive by a mechanism that is independent of trafficking or surface targeting of the channel protein. Eag channels in cells grown at 37 degrees C exhibit voltage-evoked gating charge movements but fail to conduct K(+) ions. By mutagenesis and chimeric channel studies, this study shows that the N- and C-terminal regions are involved in controlling a step after movement of the voltage sensor, as well as in regulating biophysical properties of the Eag channel. Synthesis and assembly of Eag at high temperature disrupt the ability of these domains to carry out their function. These results suggest an important role of the intracellular regions in the generation of Eag currents (Li, 2011).

Intracellular linkers are involved in Mg2+-dependent modulation of the Eag potassium channel

Modulation of activation kinetics by divalent ions is one of the characteristic features of Eag channels. TWhis study reports that Mg(2+)-dependent deceleration of Eag channel activation is significantly attenuated by a G297E mutation, which exhibits a gain-of-function phenotype in Drosophila by suppressing the effect of shaker mutation on behavior and neuronal excitability. The G297 residue is located in the intracellular linker of transmembrane segments S2 and S3, and is thus not involved in direct binding of Mg(2+) ions. Moreover, mutation of the only positively charged residue in the other intracellular linker between S4 and S5 also results in a dramatic reduction of Mg(2+)-dependent modulation of Eag activation kinetics. Collectively, the two mutations in eag eliminate or even paradoxically reverse the effect of Mg(2+) on channel activation and inactivation kinetics. Together, these results suggest an important role of the intracellular linker regions in gating processes of Eag channels (Liu, 2010).

Alternative splicing of the eag potassium channel gene in Drosophila generates a novel signal transduction scaffolding protein

The Drosophila eag gene has been shown to regulate neuronal excitability, olfaction, associative learning and larval locomotion. Not all of the roles of this gene in these processes can be explained by its function as a voltage-gated potassium channel. This study shows that the eag gene is spliced in a PKA- and PKC-regulated manner to produce a protein lacking channel domains. This protein, in the context of activated PKA, can engage cellular signaling pathways that alter cell structure. Nuclear localization is necessary for C-terminal-mediated effects, which also require MAPK. The requirement for PKA/PKC activation in the synthesis and function of this novel protein suggests that it may couple membrane events to nuclear signaling to regulate neuronal function on long time scales (Sun, 2009).

A voltage-driven switch for ion-independent signaling by ether-a-go-go K+ channels

Voltage-gated channels maintain cellular resting potentials and generate neuronal action potentials by regulating ion flux. This study shows that Ether-a-go-go (EAG) K+ channels also regulate intracellular signaling pathways by a mechanism that is independent of ion flux and depends on the position of the voltage sensor. Regulation of intracellular signaling was initially inferred from changes in proliferation. Specifically, transfection of NIH 3T3 fibroblasts or C2C12 myoblasts with either wild-type or nonconducting (F456A) eag resulted in dramatic increases in cell density and BrdUrd incorporation over vector- and Shaker-transfected controls. The effect of EAG was independent of serum and unaffected by changes in extracellular calcium. Inhibitors of p38 mitogen-activated protein (MAP) kinases, but not p44/42 MAP kinases (extracellular signal-regulated kinases), blocked the proliferation induced by nonconducting EAG in serum-free media, and EAG increased p38 MAP kinase activity. Importantly, mutations that increased the proportion of channels in the open state inhibited EAG-induced proliferation, and this effect could not be explained by changes in the surface expression of EAG. These results indicate that channel conformation is a switch for the signaling activity of EAG and suggest an alternative mechanism for linking channel activity to the activity of intracellular messengers, a role that previously has been ascribed only to channels that regulate calcium influx (Hegle, 2006).

Genetic modifications of seizure susceptibility and expression by altered excitability in Drosophila Na₊ and K₊ channel mutants

A seizure-paralysis repertoire characteristic of Drosophila 'bang-sensitive' mutants can be evoked electroconvulsively in tethered flies, in which behavioral episodes are associated with synchronized spike discharges in different body parts. Flight muscle DLMs (dorsal longitudinal muscles) display a stereotypic sequence of initial and delayed bouts of discharges (ID and DD), interposed with giant fiber (GF) pathway failure and followed by a refractory period. This study examined how seizure susceptibility and discharge patterns are modified in various K⁺ and Na⁺ channel mutants. Decreased numbers of Na⁺ channels in nap^ts flies (Drosophila mutant with a temperature-sensitive block in nerve conduction) drastically reduced susceptibility to seizure induction, eliminated ID, and depressed DD spike generation. Mutations of different K⁺ channels led to differential modifications of the various components in the repertoire. Altered transient K⁺ currents in Sh¹³³ and Hyperkinetic (Hk) mutants promoted ID induction. However, only Sh¹³³ but not Hk mutations increased DD seizure and GF pathway failure durations. Surprisingly, modifications in sustained K⁺ currents in eag and Shab mutants increased thresholds for DD induction and GF pathway failure. Nevertheless, both eag and Shab, like Sh¹³³, increased DD spike generation and recovery time from GF pathway failure. Interactions between channel mutations with the bang-sensitive mutation Shaker bss demonstrated the role of membrane excitability in stress-induced seizure-paralysis behavior. Seizure induction and discharges were suppressed by nap^ts in bss nap double mutants, whereas Sh heightened seizure susceptibility in bss Sh¹³³ and bss Sh^M double mutants. The results suggest that individual seizure repertoire components reflect different neural network activities that could be differentially altered by mutations of specific ion channel subunits (Lee, 2006: Full text of article).

Camguk/CASK enhances Ether-a-go-go potassium current by a phosphorylation-dependent mechanism

Signaling complexes are essential for the modulation of excitability within restricted neuronal compartments. Adaptor proteins are the scaffold around which signaling complexes are organized. This study demonstrates that the Camguk (CMG)/CASK adaptor protein functionally modulates Drosophila Ether-a-go-go (EAG) potassium channels. Coexpression of CMG with EAG in Xenopus oocytes results in a more than twofold average increase in EAG whole-cell conductance. This effect depends on EAG-T787, the residue phosphorylated by calcium- and calmodulin-dependent protein kinase II. CMG coimmunoprecipitates with wild-type and EAG-T787A channels, indicating that T787, although necessary for the effect of CMG on EAG current, is not required for the formation of the EAG-CMG complex. Both CMG and phosphorylation of T787 increase the surface expression of EAG channels, and in COS-7 cells, EAG recruits CMG to the plasma membrane. The interaction of EAG with CMG requires a noncanonical Src homology 3-binding site beginning at position R1037 of the EAG sequence. Mutation of basic residues, but not neighboring prolines, prevents binding and prevents the increase in EAG conductance. These findings demonstrate that membrane-associated guanylate kinase adaptor proteins can modulate ion channel function; in the case of CMG, this occurs via an increase in the surface expression and phosphorylation of the EAG channel (Marble, 2005; full text of article).

Amnesiac controls perineural glial growth as part of interacting neurotransmitter-mediated signaling pathways

Drosophila peripheral nerves, similar structurally to the peripheral nerves of mammals, comprise a layer of axons and inner glia, surrounded by an outer perineurial glial layer. Although it is well established that intercellular communication occurs among cells within peripheral nerves, the signaling pathways used and the effects of this signaling on nerve structure and function remain incompletely understood. The Drosophila peripheral nerve is a favorable system for the study of intercellular signaling. Growth of the perineurial glia is controlled by interactions among five genes: inebriated (ine), which encodes a member of the Na⁺/Cl^--dependent neurotransmitter transporter family; ether a go-go (eag), which encodes a potassium channel; pushover (push), which encodes a large, Zn2+-finger-containing protein; amnesiac, which encodes a putative neuropeptide related to the pituitary adenylate cyclase activator peptide, and NF1, the Drosophila ortholog of the human gene responsible for type 1 neurofibromatosis. In other Drosophila systems, push and NF1 are required for signaling pathways mediated by Amn or the pituitary adenylate cyclase activator peptide. These results support a model in which the Amn neuropeptide, acting through Push and NF1, inhibits perineurial glial growth, whereas the substrate neurotransmitter of Ine promotes perineurial glial growth. Defective intercellular signaling within peripheral nerves might underlie the formation of neurofibromas, the hallmark of neurofibromatosis (Yager, 2001).

Mutations in two genes that affect neuronal excitability also affect the structure of the peripheral nerve: double mutants defective in ine, and push exhibit an extremely thickened nerve, which is a phenotype that is clearly visible with the dissecting microscope. To understand the cellular basis for this phenotype, transmission electron microscopy was performed on cross-sections of peripheral nerves. This analysis demonstrated that the push¹ and ine¹;push¹ double mutants exhibit a normal axon and peripheral glial layer, but a thickened perineurial glial layer. This increased perineurial thickness is expressed only moderately in push¹ but very strongly in the ine¹;push¹ double mutant. This increase in thickness is accompanied by an increase in the number of mitochondria within perineurial glial thin sections, suggesting that an increase in cell material accompanies this increased thickness. The ine¹;push¹ phenotype is significantly rescued in transgenic larvae expressing the 943-aa Ine isoform, called Ine-P1, under the transcriptional control of the heat-shock promoter. In particular, perineurial glial thickness in ine¹ push¹; hs-ine-P1 larvae, even in the absence of heat shock, was reduced to 2.0 ± 0.2 µm from 3.1 ± 0.3 in ine¹;push¹. The observed synergistic interaction between ine and push mutations suggests that each gene controls perineurial glial growth through partially redundant pathways (Yager, 2001).

In certain respects, mutations in ine confer phenotypes similar to mutations in the K⁺ channel structural gene eag. In particular, both eag and ine mutations interact synergistically with mutations in the K⁺ channel encoded by Shaker to cause a characteristic 'indented thorax and down-turned wings' phenotype, which is not exhibited by any of the single mutants. Because of this phenotypic similarity, the possibility that eag mutations might also affect perineurial glial thickness was tested. eag¹ resembles ine¹ in the control of perineurial glial growth: eag¹;push¹ double mutants, but not the eag¹ single mutant, exhibit strongly potentiated perineurial glial growth. This increased growth is similar to, but less extreme than, what is observed in ine¹;push¹. Double mutants for eag¹; push² also exhibit a thickened perineurial glial layer. In contrast, eag and ine mutations fail to display a comparable synergistic interaction (Yager, 2001).

Mutations in push were identified independently on the basis of defective segregation of nonrecombinant chromosomes in the female meiosis. push was implicated in this process as an intermediate in a signaling pathway mediated by the PACAP-like neuropeptide encoded by amn (S. Hawley, personal communication to Yager, 2001). This observation raised the possibility that push likewise affects perineurial glial growth by acting as an intermediate from an Amn signal. Consistent with this hypothesis, the amn^X8 deletion mutation increases perineurial glial thickness, and this increase is significantly rescued in transgenic flies expressing amn⁺ (Yager, 2001).

A second signaling pathway mediated by a PACAP-like neuropeptide has been identified in Drosophila. In this pathway, the larval muscle responds to application of PACAP by activating a voltage-gated potassium channel. This activation requires NF1, the ortholog of the human gene responsible for type 1 neurofibromatosis. The possibility was tested that NF1 might affect perineurial glial growth. The NF1^P2-null mutant exhibits strong potentiation of perineurial glial thickness in combination with ine¹. This thickness is much greater than the thickness observed in ine¹ mutants carrying K33, the NF1⁺ parent chromosome of NF1^P2. The increased glial thickness of ine¹; NF1^P2 is fully rescued by heat-shock-induced expression of the NF1⁺ transgene. However, unlike push, the phenotype of NF1^P2 is potentiated only moderately by the eag¹ mutation. In contrast, perineurial glial thickness in the push¹; NF1^P2 double mutant was 2.1 ± 0.15 µm, which is significantly thicker than either push¹ or NF1^P2, but not significantly different from amn^X8. These results are consistent with the possibility that push and NF1 mediate the amn signal through parallel partially redundant pathways (Yager, 2001).

These results are consistent with a model in which two neurotransmitter-mediated signaling pathways exert opposing effects on perineurial glial growth. One pathway, mediated by the Amn neuropeptide, inhibits perineurial glial growth. This pathway requires NF1 and Push activity. The second pathway, mediated by the substrate neurotransmitter of Ine (which will be called NT here), promotes perineurial glial growth. In this pathway, mutations in ine or eag each increase signaling by NT: ine mutations increase NT signaling by eliminating the NT reuptake transporter thus increasing NT persistence, whereas eag mutations increase NT signaling by increasing NT release as a consequence of increased excitability. These pathways interact such that the most extreme effects on perineurial glial growth are observed when the NT pathway is overstimulated and the Amn pathway is disrupted simultaneously. The genetic interactions that form the basis for this interpretation require that the mutations under investigation be null. Although the eag¹ mutation tested has not been characterized molecularly, the mutations in each of the other four genes analyzed are known to be or are strongly suspected to be null. Direct neuron-perineurial glia signaling is unlikely because the peripheral glia, which form the blood-brain barrier, are expected to be an impervious barrier to intercellular traffic. Two alternative mechanisms could underlie this signaling. In the first mechanism (direct peripheral glia-perineurial glia signaling), the peripheral glia release each neurotransmitter, and the perineurial glia respond. In the second mechanism (indirect signaling), each neurotransmitter is released by neurons, and the peripheral glia respond by regulating the release of a trophic factor that acts on perineurial glia (Yager, 2001).

Although direct signaling seems to be the simplest possibility, indirect signaling is most consistent with previous studies. As described above, both invertebrate and mammalian motor neurons can release small molecule and peptide neurotransmitters that affect properties of Schwann cells. A similar motor nerve terminal-peripheral glia communication could occur in Drosophila, because first boutons at the larval neuromuscular junction are covered by peripheral glia. This observation raises the possibility that Drosophila peripheral glia might respond to Amn and NT released from motor nerve terminals, and propagate these signals along the length of the nerve via gap junctions. However, the alternative possibility of NT release from along the length of axons, as has been suggested in other systems, cannot be ruled out. In addition, mammalian Schwann cells release trophic factors such as Desert hedgehog (Dhh) to induce growth of the surrounding perineurium, and astrocytes can respond to glutamate application by releasing a substance that affects blood vessels. This model predicts that peripheral glia release a trophic factor that behaves similarly to Dhh. The prediction that Drosophila NF1 acts within peripheral glia is consistent with the likelihood that mammalian NF1 acts within Schwann cells as well (Yager, 2001).

The possible effects of the thickened perineurial glia on motor neuron function are unclear. Mutations in four of the genes that affect perineurial glial thickness (eag, NF1, ine, and push) were each shown in previous studies to increase either neuronal or muscle membrane excitability, which raises the possibility of a correlation between excitability and perineurial glial growth. However, no increases in neuronal excitability have been detected in the amn mutant or the ine; NF1 double mutant (greater than that conferred by the ine mutation alone), despite the presence of greatly thickened perineurial glia in these genotypes. It is possible that the effects on neuronal excitability of these genotypes might be subtler than the assays can detect, or that the participation of these genes in both perineurial glial growth and excitability is coincidental (Yager, 2001).

These results are consistent with the previous observations that push and NF1 act downstream of the Amn/PACAP receptor. However, the precise nature of the interactions among these proteins is unknown. Thus, it is possible that the interactions are direct, and that Push, the NF1-encoded protein Neurofibromin, and the Amn receptor bind to each other in a macromolecular complex. Alternatively, it is possible that Push and Neurofibromin mediate the effects of Amn only indirectly. In either case, the observation that the push¹; NF1^P2 double mutant exhibits a perineurial glial thickness much greater than push¹ or NF1^P2 alone is consistent with the possibility that Push and Neurofibromin mediate the Amn signal through parallel partially redundant pathways (Yager, 2001).

The indirect signaling model could explain the partial cell-nonautonomy of NF1 in neurofibroma formation. Neurofibromas most likely initiate in individuals heterozygous for NF1 mutations by loss of the NF1⁺ allele in Schwann cells. However, neurofibromas contain, in addition to Schwann cells, cells derived from fibroblasts, perineurial cells, and neurons, which are thought to remain phenotypically NF1⁺. It is suggested that NF1 mutant Schwann cells cause the overproliferation of their wild-type neighbors by oversecreting trophic factors, and that this oversecretion might ultimately occur as a consequence of defective receipt of a neurotransmitter signal from neurons (Yager, 2001).

Interaction of the K channel β subunit, Hyperkinetic, with Eag family members

Assembly of K channel α subunits of the Shaker (Sh) family occurs in a subfamily specific manner. It has been suggested that subfamily specificity also applies in the association of β subunits with Sh channels. This study shows that the Drosophila β subunit homologue Hyperkinetic (Hk) associates with members of the Ether go-go (Eag), as well as Sh, families. Anti-EAG antibody coprecipitates EAG and HK indicating a physical association between proteins. Heterologously expressed Hk dramatically increases the amplitudes of Eag currents and also affects gating and modulation by progesterone. Through their ability to interact with a range of α subunits, the β subunits of voltage-gated K channels are likely to have a much broader impact on the signaling properties of neurons and muscle fibers than previously suggested (Wilson, 1998).

Genetic dissection of functional contributions of specific potassium channel subunits in habituation of an escape circuit in Drosophila

Potassium channels have been implicated in central roles in activity-dependent neural plasticity. The giant fiber escape pathway of Drosophila has been established as a model for analyzing habituation and its modification by memory mutations in an identified circuit. Several genes in Drosophila encoding K+ channel subunits have been characterized, permitting examination of the contributions of specific channel subunits to simple conditioning in an identified circuit that is amenable to genetic analysis. The results show that mutations altering each of four K+ channel subunits (Sh, slo, eag, and Hk) have distinct effects on habituation at least as strong as those of dunce and rutabaga, memory mutants with defective cAMP metabolism. Habituation, spontaneous recovery, and dishabituation of the electrically stimulated long-latency giant fiber pathway response were shown in each mutant type. Mutations of Sh (voltage-gated) and slo (Ca2+-gated) subunits enhanced and slowed habituation, respectively. However, mutations of eag and Hk subunits, which confer K+-current modulation, had even more extreme phenotypes, again enhancing and slowing habituation, respectively. In double mutants, Sh mutations moderated the strong phenotypes of eag and Hk, suggesting that their modulatory functions are best expressed in the presence of intact Sh subunits. Nonactivity-dependent responses (refractory period and latency) at two stages of the circuit were altered only in some mutants and do not account for modifications of habituation. Furthermore, failures of the long-latency response during habituation, which normally occur in labile connections in the brain, could be induced in the thoracic circuit stage in Hk mutants. This work indicates that different K+ channel subunits play distinct roles in activity-dependent neural plasticity and thus can be incorporated along with second messenger 'memory' loci to enrich the genetic analysis of learning and memory (Engel, 1998).

Calcium/calmodulin-dependent protein kinase II and potassium channel subunit eag similarly affect plasticity in Drosophila

Similar defects in both synaptic transmission and associative learning are produced in Drosophila melanogaster by inhibition of calcium/calmodulin-dependent protein kinase II and mutations in the potassium channel subunit gene eag. These behavioral and synaptic defects are not simply additive in animals carrying both an eag mutation and a transgene for a protein kinase inhibitor, raising the possibility that the phenotypes share a common pathway. At the molecular level, a portion of the putative cytoplasmic domain of Eag is a substrate of calcium/calmodulin-dependent protein kinase II. These similarities in behavior and synaptic physiology, the genetic interaction, and the in vitro biochemical interaction of the two molecules suggest that an important component of neural and behavioral plasticity may be mediated by modulation of Eag function by calcium/calmodulin-dependent protein kinase II (Griffith, 1994).

Search PubMed for articles about Drosophila

Agrawal, P., Houl, J. H., Gunawardhana, K. L., Liu, T., Zhou, J., Zoran, M. J. and Hardin, P. E. (2017). Drosophila CRY entrains clocks in body tissues to light and maintains passive membrane properties in a non-clock body tissue independent of light. Curr Biol 27(16):2431-2441. PubMed ID: 28781048

Bronk, P., Kuklin, E. A., Gorur-Shandilya, S., Liu, C., Wiggin, T. D., Reed, M. L., Marder, E. and Griffith, L. C. (2018). Regulation of Eag by calcium/calmodulin controls presynaptic excitability in Drosophila. J Neurophysiol [Epub ahead of print]. PubMed ID: 29364071

Castro-Rodrigues, A. F., Zhao, Y., Fonseca, F., Gabant, G., Cadene, M., Robertson, G. A. and Morais-Cabral, J. H. (2018). The interaction between the Drosophila EAG potassium channel and the protein kinase CaMKII involves an extensive interface at the active site of the kinase. J Mol Biol. PubMed ID: 30381148

Engel, J. E. and Wu, C. F. (1998). Genetic dissection of functional contributions of specific potassium channel subunits in habituation of an escape circuit in Drosophila. J Neurosci 18: 2254-2267. PubMed ID: 9482810

Fogle, K. J., Baik, L. S., Houl, J. H., Tran, T. T., Roberts, L., Dahm, N. A., Cao, Y., Zhou, M. and Holmes, T. C. (2015). CRYPTOCHROME-mediated phototransduction by modulation of the potassium ion channel β-subunit redox sensor. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 25646452

Griffith, L. C., Wang, J., Zhong, Y., Wu, C. F. and Greenspan, R. J. (1994). Calcium/calmodulin-dependent protein kinase II and potassium channel subunit eag similarly affect plasticity in Drosophila. Proc Natl Acad Sci U S A 91(21): 10044-10048. PubMed ID: 7937834

He, T., Singh, V., Rumpal, N. and Lnenicka, G. A. (2009). Differences in Ca2+ regulation for high-output Is and low-output Ib motor terminals in Drosophila larvae. Neuroscience 159(4): 1283-1291. PubMed ID: 19409207

Hegle, A. P., Marble, D. D. and Wilson, G. F. (2006). A voltage-driven switch for ion-independent signaling by ether-a-go-go K+ channels. Proc Natl Acad Sci U S A 103(8): 2886-2891. PubMed ID: 16477030

Hur, J. H., Lee, S. H., Kim, A. Y. and Koh, Y. H. (2018). Regulation of synaptic architecture and synaptic vesicle pools by Nervous wreck at Drosophila Type 1b glutamatergic synapses. Exp Mol Med 50(3): e462. PubMed ID: 29568072

Immonen, E. V., French, A. S., Torkkeli, P. H., Liu, H., Vahasoyrinki, M. and Frolov, R. V. (2017). EAG channels expressed in microvillar photoreceptors are unsuited to diurnal vision. J Physiol 595(16): 5465-5479. PubMed ID: 28087896

Kaplan, W. D. and Trout, W. E., 3rd (1969). The behavior of four neurological mutants of Drosophila. Genetics 61(2): 399-409. PubMed ID: 5807804

Lee, and Wu, C.-F. (2006). Genetic modifications of seizure susceptibility and expression by altered excitability in Drosophila Na₊ and K₊ channel mutants. J Neurophysiol. 96: 2465-2478. PubMed ID: 17041230

Li, Y., Liu, X., Wu, Y., Xu, Z., Li, H., Griffith, L. C. and Zhou, Y. (2011). Intracellular regions of the Eag potassium channel play a critical role in generation of voltage-dependent currents. J Biol Chem 286(2): 1389-1399. PubMed ID: 21059657

Liu, X., Wu, Y. and Zhou, Y. (2010). Intracellular linkers are involved in Mg2+-dependent modulation of the Eag potassium channel. Channels (Austin) 4(4): 311-318. PubMed ID: 20855938

Lu, Z., Chouhan, A. K., Borycz, J. A., Lu, Z., Rossano, A. J., Brain, K. L., Zhou, Y., Meinertzhagen, I. A. and Macleod, G. T. (2016). High-probability neurotransmitter release sites represent an energy-efficient design. Curr Biol 26(19): 2562-2571. PubMed ID: 27593375

Marble, D. D., et al. (2005). Camguk/CASK enhances Ether-a-go-go potassium current by a phosphorylation-dependent mechanism. J. Neurosci. 25(20): 4898-907. Medline abstract: 15901771

Mortensen, L. S., Schmidt, H., Farsi, Z., Barrantes-Freer, A., Rubio, M. E., Ufartes, R., Eilers, J., Sakaba, T., Stuhmer, W. and Pardo, L. A. (2015). KV 10.1 opposes activity-dependent increase in Ca(2)(+) influx into the presynaptic terminal of the parallel fibre-Purkinje cell synapse. J Physiol 593(1): 181-196. PubMed ID: 25556795

Ryan, M. Y., Maloney, R., Fineberg, J. D., Reenan, R. A. and Horn, R. (2012). RNA editing in eag potassium channels: biophysical consequences of editing a conserved S6 residue. Channels (Austin) 6(6): 443-452. PubMed ID: 23064203

Schönherr, R., Lober, K. and Heinemann, S. H. (2000). Inhibition of human ether a go-go potassium channels by Ca(2+)/calmodulin. EMBO J 19(13): 3263-3271. PubMed ID: 10880439

Srinivasan, S., Lance, K. and Levine, R. B. (2012). Contribution of EAG to excitability and potassium currents in Drosophila larval motoneurons. J Neurophysiol 107(10): 2660-2671. PubMed ID: 22323637

Sun, X. X., Bostrom, S. L. and Griffith, L. C. (2009). Alternative splicing of the eag potassium channel gene in Drosophila generates a novel signal transduction scaffolding protein. Mol Cell Neurosci 40(3): 338-343. PubMed ID: 19130887

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date revised: 25 July 2019

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