InteractiveFly: GeneBrief

Ca²⁺-channel protein α1 subunit D: Biological Overview | References

Neuronal network function critically depends on the tight control of synaptic vesicle (SV) release probability at chemical synapses over wide ranges of activity regimes. At the same time, synaptic gain remains adjustable to render network function flexible. To maintain synapse function over time, SV recycling rates must be matched to vastly different activity patterns and synaptic gains. While SV release and recycling as well as their plasticity-related adjustments all include Ca²⁺-dependent steps, they operate in parallel but on different time scales. A tight spatial and temporal coordination of presynaptic Ca²⁺ signals and their effectors is thus needed for both the induction of changes in synaptic strength and the maintenance of robust synapse function. However, the mechanisms that effectively separate Ca²⁺ signals in time and space (e.g., through different voltage-gated calcium channels [VGCCs]) to allocate these to different presynaptic functions are not well understood (Krick, 2021).

SV release probability depends on the sensitivity of the vesicular Ca²⁺ sensor and the positioning of VGCCs inside active zones (AZs). Various mechanisms that can tune release probability by modulating their precise localization or kinetic properties have been uncovered. Irrespective of such modulation, efficient Ca²⁺-triggered SV release through presynaptic VGCCs (mainly Ca_V2.1 and Ca_V2.2 in vertebrates) remains spatially restricted to a few hundred nanometers due to the limited abundance and brief opening of the channels and the presence of endogenous Ca²⁺ buffers. It is thus conceivable that Ca²⁺ signals originating within presynaptic terminals but outside AZs are engaged to tune SV recycling and plastic changes according to changes in activity (Krick, 2021).

Apart from the need for fast activating and inactivating Ca_V2 channels (Drosophila Cacophony) for SV release, other types of VGCCs have been implicated in presynaptic plasticity. In GABAergic synapses, pharmacological blockade of Ca_V1 channels does not affect AP-induced SV release but converts posttetanic potentiation into synaptic depression. In hippocampal CA3 mossy fiber boutons or in synapses of the lateral amygdala, Ca_V2.3 and Ca_V1.2 channels are required for presynaptic long-term plasticity but are unable to trigger SV release (Krick, 2021).

Differential functions of Ca_V2 and Ca_V1 channels in neurotransmitter release versus other Ca²⁺-dependent presynaptic processes can hardly be explained just by different coupling distances to SVs, since there are also situations where loose coupling is predominant. Moreover, compared with Ca_V2.1 and Ca_V2.2, Ca_V1 channels display higher conductances, suggesting that additional mechanisms are required to allocate Ca_V1-related Ca²⁺ signals to specific presynaptic functions while avoiding interference with SV release. SV recycling also includes regulation by presynaptic Ca²⁺ signals but operates mostly at different subsynaptic sites and at slower time scales than Ca²⁺-triggered SV release. It is hypothesized that activity-dependent regulation of SV recycling employs Ca_V1-dependent Ca²⁺ entry and that active mechanisms exist to regulate the relative contributions of Ca_V2 and Ca_V1 channels to SV release versus recycling. These hypotheses are addressed at the Drosophila larval neuromuscular junction (NMJ), an established model for glutamatergic synapse function (Krick, 2021).

Neuronal network function critically depends on the tight control of synaptic vesicle (SV) release probability at chemical synapses over wide ranges of activity regimes. At the same time, synaptic gain remains adjustable to render network function flexible. To maintain synapse function over time, SV recycling rates must be matched to vastly different activity patterns and synaptic gains. While SV release and recycling as well as their plasticity-related adjustments all include Ca²⁺-dependent steps, they operate in parallel but on different time scales. A tight spatial and temporal coordination of presynaptic Ca²⁺ signals and their effectors is thus needed for both the induction of changes in synaptic strength and the maintenance of robust synapse function. However, the mechanisms that effectively separate Ca²⁺ signals in time and space (e.g., through different voltage-gated calcium channels [VGCCs]) to allocate these to different presynaptic functions are not well understood (Krick, 2021).

SV release probability depends on the sensitivity of the vesicular Ca²⁺ sensor and the positioning of VGCCs inside active zones (AZs). Various mechanisms that can tune release probability by modulating their precise localization or kinetic properties have been uncovered. Irrespective of such modulation, efficient Ca²⁺-triggered SV release through presynaptic VGCCs (mainly Ca_V2.1 and Ca_V2.2 in vertebrates) remains spatially restricted to a few hundred nanometers due to the limited abundance and brief opening of the channels and the presence of endogenous Ca²⁺ buffers. It is thus conceivable that Ca²⁺ signals originating within presynaptic terminals but outside AZs are engaged to tune SV recycling and plastic changes according to changes in activity (Krick, 2021).

Apart from the need for fast activating and inactivating Ca_V2 channels for SV release, other types of VGCCs have been implicated in presynaptic plasticity. In GABAergic synapses, pharmacological blockade of Ca_V1 channels does not affect AP-induced SV release but converts posttetanic potentiation into synaptic depression. In hippocampal CA3 mossy fiber boutons or in synapses of the lateral amygdala, Ca_V2.3 and Ca_V^1.2 channels are required for presynaptic long-term plasticity but are unable to trigger SV release (Krick, 2021).

Differential functions of Ca_V2 and Ca_V1 channels in neurotransmitter release versus other Ca²⁺-dependent presynaptic processes can hardly be explained just by different coupling distances to SVs, since there are also situations where loose coupling is predominant. Moreover, compared with Ca_V2.1 and Ca_V2.2, Ca_V1 channels display higher conductances, suggesting that additional mechanisms are required to allocate Ca_V1-related Ca²⁺ signals to specific presynaptic functions while avoiding interference with SV release. SV recycling also includes regulation by presynaptic Ca²⁺ signals but operates mostly at different subsynaptic sites and at slower time scales than Ca²⁺-triggered SV release. It is hypothesized that activity-dependent regulation of SV recycling employs Ca_V1-dependent Ca²⁺ entry and that active mechanisms exist to regulate the relative contributions of Ca_V2 and Ca_V1 channels to SV release versus recycling. These hypotheses are addressed at the Drosophila larval neuromuscular junction (NMJ), an established model for glutamatergic synapse function (Krick, 2021).

The data show strict functional separation of AP-triggered neurotransmitter release by Ca_v2 and activity-dependent modulation of SV recycling and short-term plasticity by Ca_v1 VGCCs. Although task sharing and partial redundancy among Ca_v2 isoforms is known for mammalian synapses, and the dynamic regulation of their relative abundance within AZs can add to synaptic plasticity, insight into mechanisms that allow for the separate regulation of different aspects of presynaptic function by Ca_v2 and Ca_v1 channels is currently sparse (Krick, 2021).

Ultrastructural support for the coexistence of Ca_v2 and Ca_v1 channels has been obtained in rat hippocampal neurons, where Ca_v2 localizes to AZs and Ca_v1 outside AZs, largely as is found for Drosophila. Moreover, pharmacological data in mammals indicate that Ca_v1 and Ca_v2 VGCCs separately control SV release and synaptic plasticity. In synapses of the amygdala, Ca_v1 is not required for SV release but for presynaptic forms of LTP; in GABAergic basket cells, Ca_v1 is not required for evoked release but for posttetanic potentiation; and at mouse neuromuscular synapses, anatomical and physiological data indicate the presence of both presynaptic Ca_v1 and Ca_v2 channels, but again with little contribution of Ca_v1 to evoked SV release. Therefore, studies of different synapse types in various species support the idea that multiple fundamental aspects of presynaptic function are executed in parallel on the basis of spatially separated VGCCs with different kinetics and conductances. This study provides a mechanism for functional separation in the small space of the axon terminal (Krick, 2021).

The fast activation and inactivation kinetics of Ca_v2 channels in the AZ seem well suited for tight excitation-release coupling, and Ca_v2 activation mediates release mostly in an all or none fashion, though dynamic modulation of channel-SV coupling to adjust release probability is reported. By contrast, Ca_v1 channels typically have larger single-channel conductances and slower inactivation kinetics, suggesting that they are well suited to cope with the need for relatively high Ca²⁺ and the slow time course of endocytic vesicle retrieval (Krick, 2021).

Endocytosis regulation by activity-dependent Ca²⁺ influx is discussed for mammalian and invertebrate synapses. At the Drosophila NMJ, separate Ca²⁺ entry routes for differential exo- and endocytosis regulation have been postulated, and the SV-associated calcium channel Flower has been suggested to contribute to this function. This study identified Ca_v1 channels within the periphery of AZs as a distinct entry route for Ca²⁺-dependent augmentation of SV endocytosis. Although the precise underlying mechanisms remain to be investigated, an attractive hypothesis is that Ca_v1 may serve as an activity-dependent switch to direct recycling into different SV pools. In basket cells, Ca_v1 mediated Ca²⁺ influx has been speculated to mobilize vesicles into the releasable pool to maintain synaptic transmission during high-frequency bursting. Similarly, at the mouse NMJ, pharmacological blockade of L-type Ca_v1 channels decreases FM2-10 loading and quantal release upon high-frequency stimulation. This is in line with the findings of increased synaptic depression, reduced SV reacidification, decreased FM1-43 uptake, and reduced PSC recovery after RRP depletion upon reduction of presynaptic Ca_v1 function. However, the effects of Ca_v1-kd manifest within a few seconds. Unless recycling and SV reformation are ultrafast, this seems too fast for SV reuse. In cultured hippocampal neurons, for example, SVs are not reused during the first 200 APs, irrespective of stimulation frequency between 5 and 40 Hz. However, given that endocytic proteins can also function in release site clearance, reduced endocytosis in Ca_v1-kd may increase synaptic depression and decrease recovery from RRP depletion indirectly as a result of reduced release site clearance. Additional effects of Ca_v1 channels on other steps in the SV cycle, such as SV priming, can also not be excluded (Krick, 2021).

For the mouse NMJ, it has been inferred that Ca_v1 activity directs recycled SVs into a high-probability release pool. Ultrastructural analysis of Drosophila synapses has also revealed two different recycling modes, one that depends on external Ca²⁺ and directs recycled SVs to AZs and another one that does not depend on external Ca²⁺ and replenishes other SV pools. Taken together, peri-AZ localization of presynaptic Ca_v1 channels as found in hippocampus and at the Drosophila NMJ may provide a common control mechanism to direct SV recycling to different pools in an activity-dependent manner. Protection of AZs by the peri-AZ PMCA provides a mechanism to maintain mean quantal content, and thus coding reliability, in the face of Ca²⁺-mediated endocytosis regulation (Krick, 2021).

As in many mammalian neurons, in Drosophila motoneurons, Ca_v1 channels localize also to dendrites to boost excitatory synaptic input. Therefore, cooperative functions of Ca_v1 channels in different subneuronal compartments coordinate firing and SV recycling rates. Moreover, as in spinal motoneurons, Drosophila Ca_v1 channel function is modulated by biogenic amines, thus providing means for integrative regulation of motoneuron excitability and SV recycling rates in the context of internal state and behavioral demands (Krick, 2021).

This study shows that 1) axon terminal Ca_v1 segregates into the peri-AZ compartment to augment SV endocytosis, and 2) PMCA, rather than directly acting on Ca²⁺ entering through Ca_v2, actively controls Ca_v1-dependent Ca²⁺ changes, thereby enabling side-by-side Ca²⁺ domains with profiles that meet the different requirements for SV release and recycling. This is consistent with reports on spatially restricted expression and/or regulation of PMCA in small T lymphocytes as a means to steer Ca²⁺-dependent processes specifically within cellular microdomains. In consequence, this study proposes to expand the concept of controlling release probability by presynaptic Ca²⁺ buffering systems after nanodomain collapse, which has been scrutinized in many studies, with the idea of nanodomain protection from presynaptic Ca²⁺ signals originating outside the AZ (Krick, 2021).

PMCAs have high Ca²⁺ affinity and can accelerate Ca²⁺ clearance on millisecond timescales. While isolating AZs from Ca²⁺ influx through Ca_v1, PMCA otherwise does not affect the spatiotemporal properties of AZ Ca²⁺ nanodomains, because transmission amplitudes are not altered by PMCA-kd in the absence of Ca_v1 channels. Instead, it ensures stable release probability in the face of presynaptic Ca²⁺ signals that augment SV recycling, shape APs, and control synaptic plasticity. In contrast to soluble Ca²⁺ buffers and fixed ones in the AZ, the membrane-bound peri-AZ PMCA can be regulated on short time scales (e.g., by downstream effectors of Ca²⁺ and phospholipids). In addition, release from autoinhibition by binding of Ca²⁺/calmodulin, which is conserved across phyla, provides a molecular memory due to the slow time course of calmodulin release, allowing PMCA to persist in a preactivated state and to respond instantaneously to the next Ca²⁺ signal. Therefore, PMCA-mediated control of SV release probability is likely adjusted by the local activity at the synaptic terminal. The data show that changes in PMCA-dependent AZ protection largely impact SV release probability by allowing or preventing functional coupling of Ca_v1 channels with readily releasable SVs. It is proposed that the distant localization of Ca_v1 channels and PMCA in between AZs enables effective and versatile regulation of synaptic strength on a short time scale. In fact, theoretical considerations and recent studies on Ca_v2.1 dynamic coupling in hippocampal synapses and on differential spacing of Ca_v2 channels in cerebellar synapses suggest that modulation of SV release probability favors loose coupling of VGCCs to SV. Thus, regulation of presynaptic PMCA activity emerges as an effective means to dynamically regulate plasticity and SV recycling rates downstream of Ca_v1 (Krick, 2021).

Dstac Regulates Excitation-Contraction Coupling in Drosophila Body Wall Muscles

Stac3 regulates excitation-contraction coupling (EC coupling) in vertebrate skeletal muscles by regulating the L-type voltage-gated calcium channel (Cav channel). Recently a Stac-like gene, Dstac, was identified in Drosophila and found to be expressed by both a subset of neurons and muscles. This study shows that Dstac and Dmca1D, the Drosophila L-type Cav channel, are necessary for normal locomotion by larvae. Immunolabeling with specific antibodies against Dstac and Dmca1D found that Dstac and Dmca1D are expressed by larval body-wall muscles. Furthermore, Ca(2+) imaging of muscles of Dstac and Dmca1D deficient larvae found that Dstac and Dmca1D are required for excitation-contraction coupling. Finally, Dstac appears to be required for normal expression levels of Dmca1D in body-wall muscles. These results suggest that Dstac regulates Dmca1D during EC coupling and thus muscle contraction (Hsu, 2020).

Dstac is required for normal circadian activity rhythms in Drosophila

The genetic, molecular and neuronal mechanism underlying circadian activity rhythms is well characterized in the brain of Drosophila. The small ventrolateral neurons (s-LNVs) and pigment dispersing (PDF) expressed bfactory them are especially important for regulating circadian locomotion. This study describes a novel gene, Dstac, which is similar to the stac genes found in vertebrates that encode adaptor proteins which bind and regulate L-type voltage-gated Ca(2+) channels (CaChs). Dstac is coexpressed with PDF by the s-LNVs and regulates circadian activity. Furthermore, the L-type CaCh, Dmca1D, appears to be expressed by the s-LNVs. Since vertebrate Stac3 regulates an L-type CaCh it is hypothesized that Dstac regulates Dmca1D in s-LNVs and circadian activity (Hsu, 2018).

Tyramine action on motoneuron excitability and adaptable tyramine/octopamine ratios adjust Drosophila locomotion to nutritional state

Adrenergic signaling profoundly modulates animal behavior. For example, the invertebrate counterpart of norepinephrine, octopamine, and its biological precursor and functional antagonist, tyramine, adjust motor behavior to different nutritional states. In Drosophila larvae, food deprivation increases locomotor speed via octopamine-mediated structural plasticity of neuromuscular synapses, whereas tyramine reduces locomotor speed, but the underlying cellular and molecular mechanisms remain unknown. This study shows that tyramine is released into the CNS to reduce motoneuron intrinsic excitability and responses to excitatory cholinergic input, both by tyramine(honoka) receptor activation and by downstream decrease of L-type calcium current. This central effect of tyramine on motoneurons is required for the adaptive reduction of locomotor activity after feeding. Similarly, peripheral octopamine action on motoneurons has been reported to be required for increasing locomotion upon starvation. It was further shown that the level of tyramine-beta-hydroxylase (TBH), the enzyme that converts tyramine into octopamine in aminergic neurons, is increased by food deprivation, thus selecting between antagonistic amine actions on motoneurons. Therefore, octopamine and tyramine provide global but distinctly different mechanisms to regulate motoneuron excitability and behavioral plasticity, and their antagonistic actions are balanced within a dynamic range by nutritional effects on TBH (Schutzler, 2019).

Although intermediate steps remain to be investigated, this study identified MN L-type Ca2+ current through Dmca1D channels as an essential downstream target. Following targeted RNAi knockdown of Dmca1D in MNs tyramine has no effect on MN excitability. While this does not exclude the possibility of additional ion channels as downstream targets of honoka, it shows that a reduction of L-type Ca2+ current is required for the observed TA effects. TA-mediated decreases of MN L-type Ca2+ current may effectively decrease locomotor activity because Ca2+ influx through axonal and dendritic Dmca1D channels cooperatively increases MN intrinsic excitability and MN responses to excitatory cholinergic synaptic input (Kadas, 2017). In sum, this study identified MNs as a cellular target and L-type Ca2+ channels as a molecular target of behaviorally relevant TA action. Given the importance of biogenic amines in the control of motor, mood, social, and cognitive behaviors, the identification of cellular and molecular amine targets is of broad significance (Schutzler, 2019).

Conservation of cardiac L-type Ca(2+) channels and their regulation in Drosophila: A novel genetically-pliable channelopathic model

Dysregulation of L-type Ca(2+) channels (LTCCs) underlies numerous cardiac pathologies. Understanding their modulation with high fidelity relies on investigating LTCCs in their native environment with intact interacting proteins. Such studies benefit from genetic manipulation of endogenous channels in cardiomyocytes, which often proves cumbersome in mammalian models. Drosophila melanogaster, however, offers a potentially efficient alternative as it possesses a relatively simple heart, is genetically pliable, and expresses well-conserved genes. Fluorescence in situ hybridization confirmed an abundance of Ca-alpha1D and Ca-alpha1T mRNA in fly myocardium, which encode subunits that specify hetero-oligomeric channels homologous to mammalian LTCCs and T-type Ca(2+) channels, respectively. Cardiac-specific knockdown of Ca-alpha1D via interfering RNA abolished cardiac contraction, suggesting Ca-alpha1D (i.e. A1D) represents the primary functioning Ca(2+) channel in Drosophila hearts. Moreover, viable single cardiomyocytes were successfully isolated and Ca(2+) currents were recorded via patch clamping, a feat never before accomplished with the fly model. The profile of Ca(2+) currents recorded in individual cells when Ca(2+) channels were hypomorphic, absent, or under selective LTCC blockage by nifedipine, additionally confirmed the predominance of A1D current across all activation voltages. T-type current, activated at more negative voltages, was also detected. Lastly, A1D channels displayed Ca(2+)-dependent inactivation, a critical negative feedback mechanism of LTCCs, and the current through them was augmented by forskolin, an activator of the protein kinase A pathway. In sum, the Drosophila heart possesses a conserved compendium of Ca(2+) channels, suggesting that the fly may serve as a robust and effective platform for studying cardiac channelopathies (Limpitikul, 2018).

In Vivo Calcium Signaling during Synaptic Refinement at the Drosophila Neuromuscular Junction

Neural activity plays a key role in pruning aberrant synapses in various neural systems, including the mammalian cortex, where low frequency (0.01 Hz) calcium oscillations refine topographic maps. However, the activity-dependent molecular mechanisms remain incompletely understood. Activity-dependent pruning also occurs at embryonic Drosophila neuromuscular junctions (NMJs), where low frequency Ca2+ oscillations are required for synaptic refinement and the response to the muscle-derived chemorepellant Sema2a. This study examined embryonic growth-cone filopodia in vivo to directly observe their exploration and to analyze the episodic Ca2+ oscillations involved in refinement. Motoneuron filopodia repeatedly contacted off-target muscle fibers over several hours during late embryogenesis, with episodic Ca2+ signals present in both motile filopodia as well as in later-stabilized synaptic boutons. The Ca2+ transients matured over several hours into regular low frequency (0.03Hz) oscillations. In vivo imaging of intact embryos of both sexes revealed that the formation of ectopic filopodia is increased in Sema2a heterozygotes. Genetic evidence is provided suggesting a complex presynaptic Ca2+-dependent signaling network underlying refinement that involves the phosphatases Calcineurin and PP1, as well the serine/threonine kinases CaMKII and PKA. Significantly, this network influenced the neuron's response to the muscle's Sema2a chemorepellant, critical for the removal of off-target contacts (Vonhoff, 2017).

As Ca2+ signals were detected at both ectopic and native motoneuron terminals during the critical period for synaptic refinement, the effect of knocking-down Ca2+ channels on synaptic connectivity and development was tested. In separate experiments, RNAi constructs were expressed pan-neurally during embryonic and larval development to target each of the known genes that encode α subunits of voltage-gated Ca2+ channels (VGCCs): the Ca(v)1 channel gene Dmca1D, the Ca(v)2.1 gene cacophony (also known as Dmca1A), and Ca(v)3 gene Dmca1G. Compared with control larvae, animals expressing RNAi-knockdowns of either cacophony or Dmca1G had an elevated frequency of ectopic contacts. By contrast, RNAi-knockdown of Dmca1D did not have a miswiring phenotype, although Dmca1D-RNAi has been shown to reduce Ca2+ currents in Drosophila larval motoneurons. An elevated ectopic frequency was also observed in larvae expressing the RNAi construct for the auxiliary Ca2+ channel β subunit that is required for channel function (Vonhoff, 2017).

Dendritic and axonal L-type calcium channels cooperate to enhance motoneuron firing output during Drosophila larval locomotion

Behaviorally adequate neuronal firing patterns are critically dependent on the specific types of ion channel expressed and on their subcellular localization. This study combines in situ electrophysiology with genetic and pharmacological intervention in larval Drosophila melanogaster of both sexes to address localization and function of L-type like calcium channels in motoneurons. Dmca1D (Cav1 homolog) L-type like calcium channels localize to both the somatodendritic and the axonal compartment of larval crawling motoneurons. In situ patch-clamp recordings in genetic mosaics reveal that Dmca1D channels increase burst duration and maximum intraburst firing frequencies during crawling-like motor patterns in semi-intact animals. Genetic and acute pharmacological manipulations suggest that prolonged burst durations are caused by dendritically localized Dmca1D channels, which activate upon cholinergic synaptic input and amplify EPSPs, thus indicating a conserved function of dendritic L-type channels from Drosophila to vertebrates. By contrast, maximum intraburst firing rates require axonal calcium influx through Dmca1D channels, likely to enhance sodium channel de-inactivation via a fast afterhyperpolarization through BK channel activation. Therefore, in unmyelinated Drosophila motoneurons different functions of axonal and dendritic L-type like calcium channels likely operate synergistically to maximize firing output during locomotion (Kadas, 2017).

The Dmca1D channel mediates Ca inward currents in Drosophila embryonic muscles

The channel mechanisms were studied that underly membrane excitation in the Drosophila embryonic body wall muscle, whose biophysical properties have been poorly characterized. The inward current underlying the action potential was solely mediated by a high-threshold class of voltage-gated Ca2+ channels, which exhibited slow inactivation, Ca2+-permeability with saturation at high [Ca2+]OUT and sensitivity to a Ca2+ channel blocker, Cd2+. The Ca2+ current in the embryonic muscle was completely eliminated in Dmca1D mutants, indicating that the Dmca1D-encoded Ca2+-channel is the major mediator of inward currents in the body wall muscles throughout the embryonic and larval stages (Hara, 2015).

Dmca1D was identified as the channel that mediates the Ca2+ inward current in the Drosophila embryonic muscle. Dmca1D has been shown to be the primary Ca2+ channel involved in the generation of voltage-dependent inward current in the larval abdominal muscles (Ren, 1998). This means that Drosophila skeletal muscles express the same α-subunit throughout the embryonic and larval stages. The Dmca1D channel current in the larval muscle as carried by Ba2+ was detected at - 20 mV and more positive potential, with the peak current attained at 0 mV (Gielow, 1995; Ren, 1998). In the present study, Ca2+ currents appeared at - 20 mV and peaked at + 20 mV with normal [Ca2+]OUT. A similar shift in the voltage at which the inward current reaches the peak was observed when Dmca1D was heterologously expressed in Xenopus oocytes (Ren, 1998). It might be that the internal milieu of the cell from which currents were recorded had an impact on the voltage dependence of channel activation. The Ba2+ current in the larval muscle slowly declined during a command step of 500 ms in the voltage range between - 20 and 0 mV (Gielow, 1995; Ren, 1998), indicating that its inactivation is slow and least voltage dependent, similar to the Ca2+ current in the embryonic muscle. The Ca2+ inward current recorded in this study exhibited ultraslow inactivation kinetics with time constants of ∼1000 ms, which is on the same order of magnitude as that for a slow inactivation component of L-type Ca2+ currents in dorsal horn neurons (∼2000 ms1). A study of mammalian Ca2+ channels expressed in Xenopus oocytes demonstrated that substitution of a β1b-subunit with a β2a-subunit in the α1A+α2-δ Ca2+ channel complex converts Ca2+ currents from inactivating into non-inactivating. This finding raises the intriguing possibility that the ultraslow inactivation observed in the current experiments was dependent on β-subunit subtypes recruited to the Ca2+ channel complex in Drosophila embryonic muscles. Another important question to be addressed by a future study is to determine whether or not the Ca2+ entry contributed to the inactivation of Ca2+ current, as was the case in the skeletal muscle fibers of the stick insect. The fact that the inactivation became evident only at the prepulse potentials beyond which the Ca2+ inward current was activated (i.e., ~- 20 mV) opens the possibility that the observed inactivation was, at least in part, dependent on Ca2+ influx. This possibility can be tested using a prepulse to the reversal potential of Ca2+ current so that the Ca2+ entry during the prepulse is minimal. Since the biophysical properties of the Dmca1D Ca2+ channel in the larval muscle have not been fully described, more detailed comparisons of Dmca1D Ca2+ channels in the embryonic and larval muscles are difficult. The larval abdominal muscles have an additional Ca2+ current component, which is sensitive to amiloride and similar to the vertebrate T-type current in kinetics (Gielow, 1995). In fact, some of the Ca2+ current traces in the current experiment had a rapidly decaying component. It remains to be determined whether the amiloride-sensitive transient Ca2+ current is present or not in the embryonic muscle (Hara, 2015).

This study found that the Ca2+ conductance saturates as the [Ca2+]OUT increases. Compared with the saturating Ca2+ conductance reported in other insect muscles, the saturation of the Ca2+ current in Drosophila embryonic muscle becomes evident at much lower [Ca2+]OUT, giving rise to a smaller dissociation constant for a presumptive binding site within the channel pore. This suggests that the interaction of Ca2+ with the channel wall during permeation is several folds stronger in the Dmca1D channel analyzed in this study. In a future study, it would be of interest to examine how other permeable and impermeable cations interact with the Dmca1D channel in penetrating or blocking it (Hara, 2015).

The body wall muscles formed in the embryo are used across the developmental stages. These muscles serve primarily for hatching in embryos and for crawling during the larval stage. Despite this change in their function, the current results and published works together suggest that Dmca1D remains the primary α-subunit type of Ca2+ channels in the body wall muscles across the developmental stages. It is envisaged that evolution preferentially regulated muscle excitability by means other than alteration of the Ca2+ channel subunits during development. In fact, Drosophila muscles have a variety of K+ channels with different compositions of subunits each encoded by different genes, including Shaker, Hyperkinetic, ether-á-gogo, Shab, slowpoke, and others, which confer distinct kinetics, and permeability properties and sensitivities to voltage, Ca2+, and/or cyclic nucleotides on the K+ channels thus formed. It remains to be determined how these K+ channels conform muscle membrane excitability to the requirement for different functions of body wall muscles in different developmental stages and how they cooperate with Dmca1D in modulating muscle contractile properties according to such functional requirements (Hara, 2015).

Distinct roles of Drosophila cacophony and Dmca1D Ca(2+) channels in synaptic homeostasis: genetic interactions with slowpoke Ca(2+) -activated BK channels in presynaptic excitability and postsynaptic response

Ca(2+) influx through voltage-activated Ca(2+) channels and its feedback regulation by Ca(2+) -activated K(+) (BK) channels is critical in Ca(2+) -dependent cellular processes, including synaptic transmission, growth and homeostasis. This study reports differential roles of cacophony (CaV 2) and Dmca1D (CaV 1) Ca(2+) channels in synaptic transmission and in synaptic homeostatic regulations induced by slowpoke (slo) BK channel mutations. At the Drosophila larval neuromuscular junctions (NMJs), a well-established homeostatic mechanism of transmitter release enhancement is triggered by experimentally suppressing postsynaptic receptor response. In contrast, a distinct homeostatic adjustment is induced by slo mutations. To compensate for the loss of BK channel control presynaptic Sh K(+) current is upregulated to suppress transmitter release, coupled with a reduction in quantal size. This study demonstrates contrasting effects of cac and Dmca1D channels in decreasing transmitter release and muscle excitability, respectively, consistent with their predominant pre- vs. postsynaptic localization. Antibody staining indicated reduced postsynaptic GluRII receptor subunit density and altered ratio of GluRII A and B subunits in slo NMJs, leading to quantal size reduction. Such slo-triggered modifications were suppressed in cac;;slo larvae, correlated with a quantal size reversion to normal in double mutants, indicating a role of cac Ca(2+) channels in slo-triggered homeostatic processes. In Dmca1D;slo double mutants, the quantal size and quantal content were not drastically different from those of slo, although Dmca1D suppressed the slo-induced satellite bouton overgrowth. Taken together, cac and Dmca1D Ca(2+) channels differentially contribute to functional and structural aspects of slo-induced synaptic modifications (Lee, 2014).

Synapse formation is tightly associated with neuronal excitability. This study found striking synaptic overgrowth caused by Drosophila K(+)-channel mutations of the seizure and slowpoke genes, encoding Erg and Ca(2+)-activated large-conductance (BK) channels, respectively. These mutants display two distinct patterns of "satellite" budding from larval motor terminus synaptic boutons. Double-mutant analysis indicates that BK and Erg K(+) channels interact with separate sets of synaptic proteins to affect distinct growth steps. Post-synaptic L-type Ca(2+) channels, Dmca1D, and PSD-95-like scaffold protein, Discs large, are required for satellite budding induced by slowpoke and seizure mutations. Pre-synaptic cacophony Ca(2+) channels and the NCAM-like adhesion molecule, Fasciclin II, take part in a maturation step that is partially arrested by seizure mutations. Importantly, slowpoke and seizure satellites were both suppressed by rutabaga mutations that disrupt Ca(2+)/CaM-dependent adenylyl cyclase, demonstrating a convergence of K(+) channels of different functional categories in regulation of excitability-dependent Ca(2+) influx for triggering cAMP-mediated growth plasticity (Lee, 2014).

Distinct satellite patterns induced by sloand sei mutations support the notion that the two K+ channels act on separate growth steps in concert with localized molecular partners. Double-mutant analysis leads to a minimal model involving functional interactions of Slo and Sei K+ channels with distinct assemblies of pre- and post-synaptic regulators in the sequential steps of synaptic growth and differentiation. Expression of slomutant phenotypes depends on scaffold protein, Dlg, and post-synaptic Dmca1D Ca2+ channels, both of which appear to be important for initial budding of satellites. Double-mutant analysis reveals a tight association between Sei, but not Slo, K+ channels and adhesion molecule, FasII, and pre-synaptic Cac Ca2+ and Para Na+ channels in initial satellite formation as well as the ensuing process. In the same vein, manipulations of pre-synaptic cAMP affect only sei-induced satellite formation, whereas slo satellites are more susceptible to modulations in post-synaptic cAMP signaling (Lee, 2014).

Whereas these pre- and post-synaptic molecules can contribute to the initial growth of satellites in sloand sei mutants, they may also be important for further differentiation and stabilization of such intermediate structures. The stabilized satellites could accumulate over time and would facilitate their capture in fixed preparations. Immunohistochemical and electron-microscopic analyses has indicated that the majority of sloand sei satellites are well differentiated in molecular composition and ultrastructure. As live imaging studies have demonstrated, differentiation of early 'ghost boutons' occurs at a slow rate, taking hours to days. Consistently, preliminary live imaging indicates type B and M satellites abundant in mutants as stable structures with no active morphological changes over the observation period up to 1 h, during which new satellites were sighted budding from primary boutons after high K+ stimulation. Thus, the synaptic differentiation process involving Slo or Sei K+ channels and their interacting partners may occur at a slower time scale (Lee, 2014).

The results demonstrate a more profound influence of post-synaptic molecules on initial induction of satellite formation and major pre-synaptic contribution in subsequent steps. This picture is in line with potential retrograde signaling during the sequential growth process. Recent studies at Drosophila larval NMJs have revealed significant contributions of retrograde factors, such as bone morphogenetic protein, to synaptic development and function. It will be important to examine whether and how these factors take part in particular steps of the proposed sequential growth process (Lee, 2014).

There has been emerging evidence for colocalization of post-synaptic BK channels with L-type Ca2+ channels and with PSD-95 scaffold protein at vertebrate synapses. This genetic analysis thus demonstrates the functional significance of the homologous post-synaptic macromolecular association (Slo BK, Dmca1D/L-type Ca2+ channels, and Dlg/PSD-95) in synaptic growth at the Drosophila NMJ. Whether interactions among these players are also important for regulation of synaptic transmission awaits further investigations (Lee, 2014).

It has been shown that sei^ts1 mutants display increased spontaneous activities in the giant-fiber neuron and enhanced synaptic growth at larval NMJs when exposed to high temperature. However, this study observed synaptic overgrowth even at room temperature in sei^ts2 and, to a lesser extent, sei^ts2/sei^ts1. DNA sequencing predicts truncated versus full-length polypeptides in the sei^ts1 and sei^ts2 alleles, respectively, which could explain the observed allele-dependent differences. Notably, altered pre-synaptic Ca2+ and cAMP regulation drastically suppressed sei phenotypes, but was ineffective on slo-induced overgrowth, suggesting significant interactions between Erg K+ channels and these pre-synaptic components, although pre-synaptic interaction of the Sei K+ channel with the Ca2+/cAMP pathway has not been well established in Drosophila. DNA sequence analysis suggests a putative cyclic nucleotide-binding domain in Sei K+ channels, similar to Eag that belongs to the same K+-channel family. Whether cAMP-dependent modification of sei phenotypes is related to the action of this putative domain should be further investigated in future studies (Lee, 2014).

Multimeric assembly of K+ channels, including Sei Erg and Slo BK, has been implicated in regulating the channel properties. Indeed, sei^ts2/+ and slo/+ larvae, presumably containing a mixture of mutated and WT subunits in their Erg and BK channels, display dominant mutational effects on satellite formation and associated synaptic growth. Importantly, pre-synaptic expression of a mutated sei transgene (UAS-sei^ts2) in WT led to a similar, but less extreme, phenotype, confirming the pre-synaptic action of sei^ts2 and its dominant effects in multimeric Sei channels (Lee, 2014).

It is interesting to ask whether simply reducing the amount of Sei and Slo channel proteins may produce phenotypes similar to heterozygous sei^ts2/+ and slo/+ animals. The RNA interference (RNAi) technique was used to test this possibility, using multiple combinations of GAL4 drivers and UAS-slo/sei-RNAi constructs, with Dicer-2 to facilitate RNA interference in some combinations. However, none of these combinations caused characteristic behavioral and physiological abnormalities of sei and slo. Only marginal and inconsistent synaptic growth phenotypes was observed among these combinations. For instance, the expression of sloand sei RNAi in motoneurons with the driver C164-GAL4 led to a slightly elevated satellite frequency, but the pan-neuronal driver C155-GAL4 produced even less overgrowth. Bouton formation was enhanced in these GAL4-UAS-RNAi combinations but not significantly above the elevated levels intrinsic to individual GAL4 and RNAi lines (Lee, 2014).

The results suggest that dysfunctions induced by RNAi knockdown may not reproduce all aspects of mutant phenotypes. A match in protein levels or altered protein properties may be required to produce the phenotype of interest. At this time, the efficiency of these RNAi lines has not been documented. Since it was not possible to measure the levels of Slo and Sei proteins because of a lack of appropriate antibodies, it is not possible to determine the levels of each RNAi knockdown. The sloand sei mutations induced by a chemical mutagen, ethyl methanesulfonate, may affect the properties and/or the amount of the gene product. For example, sei^ts2 mutants carry a point mutation near the pore domain of the channels, and thus may act as neomorphs that confer dominant effects in the heterozygote, a property difficult to be mimicked by RNAi knockdown (Lee, 2014).

These results point out the critical role of cAMP signaling in the expression of both sloand sei mutant phenotypes and further highlight the profound functional consequences of altered excitability in neuronal plasticity. Activation of rut AC by activity-dependent accumulation of intracellular Ca2+ is pivotal in several forms of synaptic plasticity. For instance, in the Aplysia siphon-gill withdrawal reflex model, sensitizing stimuli increase cAMP levels and subsequently enhance transmission efficacy at sensorimotor synapses, and repeated conditioning induces sensory varicosity growth. Similarly, cAMP-dependent activation of protein kinase A in hippocampal slices is required for late-phase LTP that involves formation of new dendritic spines (Lee, 2014).

At Drosophila larval NMJs, altered cAMP metabolism in rut and dnc mutants impairs synaptic transmission stability and post-tetanic potentiation. In addition, fewer docked vesicles and retarded reserve pool mobilization have been documented in these mutants, indicating vesicle targeting and cycling defects. Thus, it will be interesting to examine the possibility that suppression of sloand sei satellites by rut is associated with alterations in membrane recycling. Such studies can be facilitated by relevant mutations, such as shibire defective in Dynamin, which is responsible for vesicle pinch-off, or drp1 (Dynamin-related protein 1) defective in reserve pool mobilization (Lee, 2014).

In summary, these observations reveal distinct patterns of satellite formation induced by sei and slomutations affecting two separate categories of K+ channels, which are apparently regulated by pre- and post-synaptic Ca2+/cAMP signaling, respectively. Together with previous studies, convergence on the Ca2+/CaM-activated cAMP synthesis by rut AC in the regulation of synaptic growth induced by a variety of K+ channel mutations further establishes a central role of rut AC in activity-dependent plasticity of synaptic function and growth (Lee, 2014).

Characterization of voltage-dependent Ca2+ currents in identified Drosophila motoneurons in situ

Voltage-dependent Ca2+ channels contribute to neurotransmitter release, integration of synaptic information, and gene regulation within neurons. Thus understanding where diverse Ca2+ channels are expressed is an important step toward understanding neuronal function within a network. Drosophila provides a useful model for exploring the function of voltage-dependent Ca2+ channels in an intact system, but Ca2+ currents within the central processes of Drosophila neurons in situ have not been well described. The aim of this study was to characterize voltage-dependent Ca2+ currents in situ from identified larval motoneurons. Whole cell recordings from the somata of identified motoneurons revealed a significant influence of extracellular Ca2+ on spike shape and firing rate. Using whole cell voltage clamp, along with blockers of Na+ and K+ channels, a Ca2+-dependent inward current was isolated. The Drosophila genome contains three genes with homology to vertebrate voltage-dependent Ca2+ channels: Dmca1A, Dmca1D, and Dmalpha1G. This study used mutants of Dmca1A and Dmca1D as well as targeted expression of an RNAi transgene to Dmca1D to determine the genes responsible for the voltage-dependent Ca2+ current recorded from two identified motoneurons. The results implicate Dmca1D as the major contributor to the voltage-dependent Ca2+ current recorded from the somatodendritic processes of motoneurons, whereas Dmca1A has previously been localized to the presynaptic terminal where it is essential for neurotransmitter release. Altered firing properties in cells from both Dmca1D and Dmca1A mutants indicate a role for both genes in shaping firing properties (Worrell, 2008).

Genetic and developmental characterization of Dmca1D, a calcium channel alpha1 subunit gene in Drosophila melanogaster

To begin unraveling the functional significance of calcium channel diversity, this study identified mutations in Dmca1D, a Drosophila calcium channel alpha1 subunit cDNA that was recently cloned. These mutations constitute the l(2)35Fa lethal locus, which was rename Dmca1D. A severe allele, Dmca1D^X10, truncates the channel after the IV-S4 transmembrane domain. These mutants die as late embryos because they lack vigorous hatching movements. In the weaker allele, Dmca1D^AR66, a cysteine in transmembrane domain I-S1 is changed to tyrosine. Dmca1D^AR66 embryos hatch but pharate adults have difficulty eclosing. Those that do eclose have difficulty in fluid-filling of the wings. These studies show that this member of the calcium channel alpha1 subunit gene family plays a nonredundant, vital role in larvae and adults (Eberl, 1998).

A mutation affecting dihydropyridine-sensitive current levels and activation kinetics in Drosophila muscle and mammalian heart calcium channels

The Dmca1D gene encodes a Drosophila calcium channel alpha1 subunit. This study describes the first functional characterization of a mutation in this gene. This alpha1 subunit mediates the dihydropyridine-sensitive calcium channel current in larval muscle but does not contribute to the amiloride-sensitive current in that tissue. A mutation, which changes a highly conserved Cys to Tyr in transmembrane domain IS1, identifies a residue important for channel function not only in Drosophila muscle but also in mammalian cardiac channels. In both cases, mutations in this Cys residue slow channel activation and reduce expressed currents. Amino acid substitutions at this Cys position in the cardiac alpha1 subunit show that the size of the side chain, rather than its ability to form disulfide bonds, affects channel activation (Ren, 1998).

Cloning and characterization of a calcium channel alpha 1 subunit from Drosophila melanogaster with similarity to the rat brain type D isoform

Thia study reports the complete sequence of a calcium channel alpha 1 subunit cDNA cloned from a Drosophila head cDNA library. This cDNA encodes a deduced protein containing 2516 amino acids with a predicted molecular weight of 276,493. The deduced protein shares many features with vertebrate homologs, including four repeat structures, each containing six transmembrane domains, a conserved ion selectivity filter region between transmembrane domains 5 and 6, and an EF hand in the carboxy tail. The Drosophila subunit has unusually long initial amino and terminal carboxy tails. The region corresponding to the last transmembrane domain (IVS6) and the adjacent cytoplasmic domain has been postulated to form a phenylalkylamine-binding site in vertebrate calcium channels. This region is conserved in the Drosophila sequence, while domains thought to be involved in dihydropyridine binding show numerous changes. The Drosophila subunit exhibits 78.3% sequence similarity to the rat brain type D calcium channel alpha 1 subunit, and so has been designated as a Drosophila melanogaster calcium channel alpha 1 type D subunit (Dmca1D). In situ hybridization shows that Dmca1D is highly expressed in the embryonic nervous system. Northern analysis shows that Dmca1D cDNA hybridizes to three size classes of mRNA (9.5, 10.2, and 12.5 kb) in heads, but only two classes (9.5 and 12.5 kb) in bodies and legs. PCR analysis suggests that the Dmca1D message undergoes alternative splicing with more heterogeneity appearing in head and embryonic extracts than in bodies and legs (Zheng, 1995).

Search PubMed for articles about Drosophila Ca-alpha1D

Gielow, M. L., Gu, G. G. and Singh, S. (1995). Resolution and pharmacological analysis of the voltage-dependent calcium channels of Drosophila larval muscles. J Neurosci 15(9): 6085-6093. PubMed ID: 7666192

Eberl, D. F., Ren, D., Feng, G., Lorenz, L. J., Van Vactor, D. and Hall, L. M. (1998). Genetic and developmental characterization of Dmca1D, a calcium channel alpha1 subunit gene in Drosophila melanogaster. Genetics 148(3): 1159-1169. PubMed ID: 9539432

Hara, Y., Koganezawa, M. and Yamamoto, D. (2015). The Dmca1D channel mediates Ca inward currents in Drosophila embryonic muscles. J Neurogenet: 1-28. PubMed ID: 26004544

Hsu, I. U., Linsley, J. W., Varineau, J. E., Shafer, O. T. and Kuwada, J. Y. (2018). Dstac is required for normal circadian activity rhythms in Drosophila. Chronobiol Int: 1-11. PubMed ID: 29621409

Hsu, I. U., Linsley, J. W., Reid, L. E., Hume, R. I., Leflein, A. and Kuwada, J. Y. (2020). Dstac Regulates Excitation-Contraction Coupling in Drosophila Body Wall Muscles. Front Physiol 11: 573723. PubMed ID: 33123029

Kadas, D., Klein, A., Krick, N., Worrell, J. W., Ryglewski, S. and Duch, C. (2017). Dendritic and axonal L-type calcium channels cooperate to enhance motoneuron firing output during Drosophila larval locomotion. J Neurosci 37(45): 10971-10982. PubMed ID: 28986465

Krick, N., Ryglewski, S., Pichler, A., Bikbaev, A., Gotz, T., Kobler, O., Heine, M., Thomas, U. and Duch, C. (2021). Separation of presynaptic Ca_V2 and Ca_V1 channel function in synaptic vesicle exo- and endocytosis by the membrane anchored Ca²⁺ pump PMCA. Proc Natl Acad Sci U S A 118(28). PubMed ID: 34244444

Lee, J., Ueda, A. and Wu, C. F. (2014). Distinct roles of Drosophila cacophony and Dmca1D Ca(2+) channels in synaptic homeostasis: genetic interactions with slowpoke Ca(2+) -activated BK channels in presynaptic excitability and postsynaptic response. Dev Neurobiol 74(1): 1-15. PubMed ID: 23959639

Lee, S. M., Yeh, P. W. L. and Yeh, H. H. (2022). L-Type Calcium Channels Contribute to Ethanol-Induced Aberrant Tangential Migration of Primordial Cortical GABAergic Interneurons in the Embryonic Medial Prefrontal Cortex. eNeuro 9(1). PubMed ID: 34930830

Limpitikul, W. B., Viswanathan, M. C., O'Rourke, B., Yue, D. T. and Cammarato, A. (2018). Conservation of cardiac L-type Ca(2+) channels and their regulation in Drosophila: A novel genetically-pliable channelopathic model. J Mol Cell Cardiol 119: 64-74. PubMed ID: 29684406

Nakagawa-Tamagawa, N., Kirino, E., Sugao, K., Nagata, H. and Tagawa, Y. (2021). Involvement of Calcium-Dependent Pathway and beta Subunit-Interaction in Neuronal Migration and Callosal Projection Deficits Caused by the Cav1.2 I1166T Mutation in Developing Mouse Neocortex. Front Neurosci 15: 747951. PubMed ID: 34955712

Ren, D., Xu, H., Eberl, D. F., Chopra, M. and Hall, L. M. (1998). A mutation affecting dihydropyridine-sensitive current levels and activation kinetics in Drosophila muscle and mammalian heart calcium channels. J Neurosci 18(7): 2335-2341. PubMed ID: 9502794

Schutzler, N., Girwert, C., Hugli, I., Mohana, G., Roignant, J. Y., Ryglewski, S. and Duch, C. (2019). Tyramine action on motoneuron excitability and adaptable tyramine/octopamine ratios adjust Drosophila locomotion to nutritional state. Proc Natl Acad Sci U S A 116(9): 3805-3810. PubMed ID: 30808766

Vierra, N. C., O'Dwyer, S. C., Matsumoto, C., Santana, L. F. and Trimmer, J. S. (2021). Regulation of neuronal excitation-transcription coupling by Kv2.1-induced clustering of somatic L-type Ca(2+) channels at ER-PM junctions. Proc Natl Acad Sci U S A 118(46). PubMed ID: 34750263

Vonhoff, F. and Keshishian, H. (2017). In Vivo Calcium Signaling during Synaptic Refinement at the Drosophila Neuromuscular Junction. J Neurosci 37(22): 5511-5526. PubMed ID: 28476946

Wang, X., Nawaz, M., DuPont, C., Myers, J. H., Burke, S. R., Bannister, R. A., Foy, B. D., Voss, A. A. and Rich, M. M. (2022). The role of action potential changes in depolarization-induced failure of excitation contraction coupling in mouse skeletal muscle. Elife 11. PubMed ID: 34985413

Worrell, J. W. and Levine, R. B. (2008). Characterization of voltage-dependent Ca2+ currents in identified Drosophila motoneurons in situ. J Neurophysiol 100(2): 868-878. PubMed ID: 18550721

Zheng, W., Feng, G., Ren, D., Eberl, D. F., Hannan, F., Dubald, M. and Hall, L. M. (1995). Cloning and characterization of a calcium channel alpha 1 subunit from Drosophila melanogaster with similarity to the rat brain type D isoform. J Neurosci 15(2): 1132-1143. PubMed ID: 7869089

date revised: 5 February 2022

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