InteractiveFly: GeneBrief

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

Projection neurons (PNs), located in the antennal lobe region of the insect brain, play a key role in processing olfactory information. To explore how activity is regulated at the level of single PNs within this central circuit recordings were made from these neurons in adult Drosophila melanogaster brains. A previous study demonstrated that PNs express voltage-gated calcium currents with a transient and sustained component. This study found that the sustained component is mediated by cac gene-encoded Ca_v2-type channels involved in regulating action potential-independent release of neurotransmitter at excitatory cholinergic synapses. The function of the transient calcium current and the gene encoding the underlying channels, however, were unknown. This study reports that the transient current blocked by prepulse inactivation is sensitive to amiloride, a vertebrate Ca_v3-type channel blocker. In addition PN-specific RNAi knockdown of α1T, the Drosophila Ca_v3-type gene, caused a dramatic reduction in the transient current without altering the sustained component. These data demonstrate that the α1T gene encodes voltage-gated calcium channels underlying the amiloride-sensitive transient current. Alterations in evoked firing and spontaneous burst firing in the α1T knockdowns demonstrate that the Cav3-type calcium channels are important in regulating excitability in adult PNs (Iniguez, 2013).

A wide variety of insect behaviors are driven or modulated by olfactory input and the ensemble of neurons involved in processing olfactory information is well defined. Olfactory perception begins when odorant molecules bind to receptors in olfactory receptor neurons (ORNs) located in the antennae and the maxillary palps. ORNs project to the antennal lobes, the insect equivalent of the vertebrate olfactory bulb, where they synapse onto the dendrites of projection neurons (PNs), the principal output cells that extend axons to higher order processing centers in the mushroom bodies and lateral horn. In Drosophila melanogaster, where genetic manipulations and behavioral assessment are routine, it is now feasible to record from identified neurons within this circuit in the adult brain. This has made it possible to explore the mechanisms that regulate activity in a circuit important in generating specific components of an adult behavior at the single cell level. Whole cell recordings from single PNs have demonstrated that olfactory processing begins in the antennal lobe where both intra- and interglomerular interactions influence activity of these cells (Iniguez, 2013).

To understand the molecular mechanisms underlying regulation of neuronal activity in individual PNs in the olfactory circuit requires identification of the ion channel subtypes that govern excitability and synaptic transmission in these cells. One important class of channels present in all neurons are voltage-gated calcium channels that mediate depolarization-induced calcium influx that influences a number of cellular processes including excitability and release of neurotransmitters at chemical synapses. The α1-subunit of these multimeric proteins forms the ion-conducting pore that defines many of the functional properties characteristic of the distinct calcium channel subtypes. There are three families of genes encoding α1 subunits and in the Drosophila genome there is one α1 subunit gene in each family: α1D (Ca_v1), cac (Ca_v2), and α1T (Ca_v3) (Zheng, 1995; Smith, 1996; Littleton, 2000; King 2007). A recent study found that voltage-gated calcium currents recorded from the cell bodies of PNs in the adult brain could be separated into two kinetically distinct components: a rapidly decaying transient current and a slowly decaying sustained current (Gu, 2009). Using a combination of pharmacological and molecular genetic strategies, this study has demonstrated that the Ca_v2-type cac gene encodes calcium channels that mediate PLTXII-sensitive sustained calcium currents. These studies show that the CAC channels regulate action potential-independent release of neurotransmitter at excitatory cholinergic synapses in the adult brain, a novel role not predicted from previous studies at peripheral synapses (Iniguez, 2013).

While a recent study suggests that Ca_v2-type CAC channels (aka Dmca1A; Cacophony) also contribute to the transient calcium currents in adult motor neurons (Ryglewski, 2012), the current results indicated that neither PLTXII nor mutations in the cac gene reduced the transient currents in PNs. This suggests that a distinct calcium channel subtype gives rise to the transient current in adult PNs. In vertebrates, previous studies have demonstrated that Ca_v3 genes encode channels underlying transient calcium currents (Iniguez, 2013).

This study reports that amiloride, a vertebrate Ca_v3-type channel blocker, reduces the transient calcium current without significantly altering the sustained Ca_v2-type CAC channel-mediated current in adult PNs. In addition, RNAi mediated knockdown of the α1T gene, the Drosophila Ca_v3-type homolog, in PNs reduced the transient component significantly but the sustained component was not affected. Alterations in evoked and spontaneous firing were observed in the α1T knockdowns. These data demonstrate that α1T-encoded Ca_v3-type channels mediate transient calcium currents that are important in shaping the PN firing properties and, therefore, play an important role in regulating olfactory signal processing (Iniguez, 2013).

Projection neurons in the adult Drosophila antennal lobe process olfactory input from olfactory receptor neurons and relay this to neurons in the mushroom body and lateral horn. This well-defined neural pathway and access to these neurons in the whole brain preparation provide an excellent model system to explore how voltage-gated calcium channels regulate signaling in an identified neuronal subtype within the adult olfactory circuit. The results of this study demonstrate that the transient calcium current in the adult Drosophila antennal lobe PNs is mediated by amiloride-sensitive α1T-encoded channels. Furthermore, the α1T channels were found to modulate cell excitability, a novel role in the Drosophila central nervous system (Iniguez, 2013).

A recent analysis of adult Drosophila motor neurons reported three distinct calcium current phenotypes in an α1T null mutant: complete loss of all calcium currents (56%), reduction of both sustained and transient currents (28%), and no effect on calcium currents (16%) (Ryglewski, 2012). In contrast, whena PN-specific Gal4 driver was used in conjunction with two independently generated UAS-RNAi lines targeted to the α1T gene, this resulted in specific reductions in the transient calcium current. No significant change in the sustained calcium current was observed. Since the two RNAi lines were targeted to distinct regions of the α1T gene, this is compelling evidence that the α1T gene encodes channels underlying the transient current in PNs. The varied effects of an α1T null mutant of calcium currents previously reported in motor neurons may be associated with activation of homeostatic regulatory mechanisms caused by elimination of the channel in all cell types (Iniguez, 2013).

Similar to currents mediated by T-type channels in vertebrates, the transient calcium currents in adult PNs are inhibited by amiloride, a vertebrate Ca_v3 T-type calcium channel blocker. Amiloride has also been reported to block a portion of the calcium current that could be inactivated by depolarization in Drosophila larva body wall fibers and embryo motor neurons (Gielow, 1995; Baines, 1998). While the underlying gene encoding the current in muscle and motor neurons was not identified, the current data suggest the α1T gene may also encode the channels in these cells. In a recent study it appears that α1T channels also underlie an amiloride-sensitive transient current that represents a relatively small component of the total calcium current in adult motor neurons (Ryglewski, 2012). The majority of transient current in the motor neurons, however, appears to be mediated by Ca_v2-type CAC channels (Ryglewski, 2012). This suggests that, similar to finding in the mammalian central nervous system, stage and/or cell-specific splicing events give rise to calcium channels with distinct functional properties (Bell, 2004; Lipscombe, 2012; Iniguez, 2013 and references therein).

In contrast to T-type currents in vertebrates that typically activate at low voltages, the PN transient calcium currents are first activated at a membrane potential of between -50 and -40 mV. This activation profile is similar to that reported for transient calcium currents in neurons cultured from brains of late stage wild-type pupae, Drosophila embryonic motor neurons, Drosophila muscle fibers, and Drosophila larval motor neurons. In contrast, studies in cytokinesis-arrested neuroblasts, embryonic motor neurons, and adult motor neurons reported both low- and high-voltage-activated calcium currents. The discrepancies in the studies could arise from the differences in cell types and developmental stages. It is also possible that contributing to the differences noted is the use of barium vs. calcium as the charge carrier for studying calcium influx. Replacing calcium with barium substantially increases the current amplitude . Barium is also known to suppress Ca²⁺-dependent inactivation of calcium channels. In larval motor neurons when barium was used to replace calcium as the charge carrier, this caused a substantial increase in current amplitude and altered the kinetics of calcium current decay. All of the recordings in PNs in the present study were conducted in physiological concentrations of Ca²⁺, and therefore, calcium-dependent inactivation of the current could contribute to the transient nature of the current (Iniguez, 2013).

The increase in evoked firing frequency in PNs reported in the RNAi-1T knockdowns demonstrates that the α1T channels are important in regulating excitability in adult Drosophila neurons. The increased excitability caused by reducing expression of Ca_v3-type channels in the adult PNs is similar to the upregulation of firing frequency documented in larval motor neurons following genetic reduction of Ca_v1 or Ca_v2 type calcium channels (Worrell, 2008). In the wild-type motor neurons, the increase in firing frequency was mimicked by acute removal of calcium from the recording solution suggesting the change in excitability reflects reduced activation of Ca²⁺-activated K⁺ channels. However, Peng (2007) was not able to rule out the possibility that there was compensatory downregulation of other K⁺ channel genes in the Ca_v1 and Ca_v2 mutants that could have the same effect of increasing motor neuron-evoked firing frequency (Iniguez, 2013).

To avoid the possible contribution of developmental regulation of other channel types, it would be helpful to explore the role of α1T channels in regulating excitability by acutely blocking these channels in wild-type PNs. Unfortunately Drosophila express nonvoltage-gated sodium channels that are also sensitive to amiloride, and preliminary studies indicate these can affect membrane potential depolarizations directly. Therefore, additional experiments with double mutant combinations will be necessary to determine if reduced activation of Ca-activated K channels and/or reduced expression of other K channels contribute to the increase in firing frequency seen in adult PNs in α1T knockdowns (Iniguez, 2013).

Low-voltage-activated T-type Ca²⁺ channels in mammals have been shown to be important in pacemaking activity in sino-artrial node of the heart, and they are crucial for generation of rhythmic bursts of action potentials in thalamic relay neurons of the thalamus. In PNs there was no significant change in the burst firing frequency, but the burst duration in α1T knockdowns was significantly increased. This suggests that while T-type calcium channels do have a role in regulating spontaneous burst firing in Drosophila PNs, their relatively high activation voltage may limit their contribution to pacemaking type activities (Iniguez, 2013).

Is the third calcium channel subtype encoded by the DMCA1D gene also expressed in adult PNs? Preliminary studies revealed that calcium currents examined in PNs from hypomorphic α1D mutant AR66 and α1D-knockdowns (VDRC no.51491) had both transient and sustained currents that were not significantly different than wild-type. However, alteration in the firing properties of DMCA1D knockdown and mutant indicates that these channels are expressed in PNs. This suggests that the currents mediated by these channels are located in the axonal compartment, electrically distant from the soma. If calcium influx is initiated at some distance from the electrode, this current change will be substantially attenuated when it reaches the soma. Unfortunately, the soma of PNs is the only location in these cells that is accessible to the patch-clamp electrode (Iniguez, 2013).

In conclusion, these results demonstrate for the first time that α1T-encoded voltage-gated calcium channels are expressed in adult PNs where they are important in regulating excitability. Further studies on how these channels regulate olfactory related behavior will be an important contribution to understanding of how activity within specific neurons in a well-defined circuit guides behavior (Iniguez, 2013).

Ca-α1T, a fly T-type Ca²⁺ channel, negatively modulates sleep

Mammalian T-type Ca²⁺ channels are encoded by three separate genes (Cav3.1, 3.2, 3.3). These channels are reported to be sleep stabilizers important in the generation of the delta rhythms of deep sleep, but controversy remains. The identification of precise physiological functions for the T-type channels has been hindered, at least in part, by the potential for compensation between the products of these three genes and a lack of specific pharmacological inhibitors. Invertebrates have only one T-type channel gene, but its functions are even less well-studied. Ca-α1T, the only Cav3 channel gene in Drosophila melanogaster, was cloned and expressed in Xenopus oocytes and HEK-293 cells and was confirmed to pass typical T-type currents. Voltage-clamp analysis revealed the biophysical properties of Ca-alpha1T show mixed similarity, sometimes falling closer to Cav3.1, sometimes to Cav3.2, and sometimes to Cav3.3. Ca-α1T was found to be broadly expressed across the adult fly brain in a pattern vaguely reminiscent of mammalian T-type channels. In addition, flies lacking Ca-alpha1T show an abnormal increase in sleep duration most pronounced during subjective day under continuous dark conditions despite normal oscillations of the circadian clock. Thus, this study suggests invertebrate T-type Ca(2+) channels promote wakefulness rather than stabilizing sleep (Jeong, 2015).

T-type Ca²⁺ channels are a subfamily of voltage-dependent Ca²⁺ channels (VDCCs) that produce low-voltage-activated (LVA) Ca²⁺ currents implicated in NREM sleep in mammals (Lee, 2004). Three different genes encode the pore-forming alpha subunits of mammalian T-type channels, Ca_v3.1, 3.2, and 3.3. Of these, Ca_v3.1 and 3.3 are highly expressed in the thalamus, where the oscillations required for NREM sleep are generated. Mice lacking Ca_v3.1 show reduced delta-wave activity and reduced sleep stability, suggesting that mammalian T-type currents have a sleep-promoting or stabilizing function (Jeong, 2015).

Unlike mammals, Drosophila melanogaster has only one T-type Ca²⁺ channel, Ca-α1T, which is also known as DmαG. A recent study found that motor neurons in flies lacking Ca-α1T show reduced LVA but also reduced high-voltage-activated (HVA) Ca2+ currents, suggesting that although Ca-α1T seems to be a genuine T-type channel, it may have interesting biophysical properties (Ryglewski, 2012). Therefore a single isoform of Ca-α1T was cloned, it was expressed in Xenopus oocytes or HEK-293 cells, and its biophysical properties were compared with those of the rat T-type channel Cav3.1. Several Ca-α1T mutant alleles were generated, and a defect was generated in their sleep/wake cycles. Contrary to results in mammals, the fly T-type Ca2+ channel destabilizes sleep. It is anticipated that these findings will help clarify species-dependent differences in the in vivo functions of T-type Ca2+ channels, particularly their role in sleep physiology (Jeong, 2015).

Ca-α1T is the largest T-type channel cloned to date, measuring 3205 amino acids. Electrophysiological characterization of Ca-α1T in Xenopus oocytes showed that Ca-α1T has the hallmark properties of a T-type channel: low-threshold activation at around -60 mV, a maximal current output at -20 mV, transient current kinetics elicited by a step-pulse protocol producing a “criss-crossing” pattern, and slow deactivation of tail currents. These biophysical properties are also consistent with previous studies that implicated Ca-α1T in low-voltage-activated (LVA) currents in both the central and peripheral nervous systems of the fly (Ryglewski, 2012; Iniguez, 2013; Jeong, 2015 and references therein).

Mammalian genomes contain three T-type Ca²⁺ channel genes (i.e., Ca_v3.1-3.3), while the fly genome contains only one. Therefore Ca-α1T was measured for some of the characteristics that distinguish the three mammalian channels. In terms of current kinetics, Ca-α1T is more similar to mammalian Ca_v3.1 and Ca_v3.2 than Ca_v3.3, which exhibits considerably slower kinetics. In terms of both its relative permeability to Ba²⁺ over Ca²⁺ and its sensitivity to nickel inhibition, Ca-α1T is most similar to Ca_v3.2 (Jeong, 2015).

The three mammalian T-type Ca²⁺ channels, each with their own distinct biophysical properties, are expressed in largely complementary patterns of neurons throughout the brain, conferring considerable functional diversity. Areas of particularly strong expression include those important for the gating and processing of sensory inputs, motor control, learning and memory, as well as reward circuits (Talley, 1999). Using a GFP-tagged knock-in allele, this study reports that Ca-α1T is expressed broadly across the adult fly brain in structures reminiscent of the mammalian T-type Ca²⁺ channels. These include sensory neuropils (i.e., the optic and antennal lobes, the antennal mechanosensory and motor centers, the anterior ventrolateral protocerebrum, and the subesophageal zone), motor-associated neuropils (i.e., the central complex), and those associated with learning, memory, and reward (i.e., the mushroom bodies). It is still unclear, however, whether the different isoforms predicted to originate from the Ca-α1T locus will have different biophysical properties or different distributions around the brain (Jeong, 2015).

Considering their broad expression, T-type knockout mice appear healthy and subtle mutant phenotypes emerge only upon close inspection. Sleep, in particular, has become a focal point in the search for a physiological function for the T-type channels. Mammalian T-type Ca²⁺ channels may act as sleep stabilizers and may help generate the burst firing necessary for the sleep oscillations of deep NREM sleep. Unfortunately, the three separate mammalian T-type genes all undergo alternative splicing to produce various channel isoforms that each have specific biophysical properties, neuroanatomical and subcellular localizations, and varying abilities to interact with other ion channels. All these variables and more combine to make it difficult if not impossible to define a precise physiological role in sleep for T-type channels as a group. Although Ca_v3.1 knockout mice lack the delta oscillations characteristic of deep sleep and show reduced total sleep (Lee, 2004), when the knockout is limited to the rostral midline thalamus, sleep is still reduced, but delta waves are mildly increased (Anderson, 2005). Another more recent study showed that treatment with the T-type-specific channel blocker TTA-A2 enhances sleep and delta rhythms in wild type mice but not Ca_v3.1/Ca_v3.3 double knockout mice (Kraus, 2010). In other words, manipulation of T-type channels can both enhance and reduce total sleep and deep delta-wave sleep depending on the experimental context (Jeong, 2015).

Although perhaps an underestimate of the actual complexity of the situation, the subtlety of the phenotypes of the homozygous viable Ca_v3 mutant mice are often ascribed to functional compensation among the various Ca_v3.1-3 isoforms. It was therefore expected that a behavioral investigation of the sole fly T-type channel, Ca-α1T, would uncover less subtle sleep phenotypes. It was thus surprising to find, that despite its broad and relatively strong expression across adult fly brains, Ca-α1T-null mutants, like the Ca_v3.1-null mice, are homozygous viable and lack any overt phenotypes. Upon closer examination, however, it was observed that Ca-α1T-null mutants sleep more than controls, especially in constant darkness (Jeong, 2015).

The reason for this relative specificity in the sleep phenotype caused by Ca-α1T loss-of-function to constant darkness is still unclear. Flies exhibit a burst of activity upon exposure to the early morning light but then sleep through most of the rest of the day. Since control flies show less sleep during subjective daytime under continuous darkness than under the light phase of light-dark conditions, it is clear that light exposure can also have sleep-promoting effects. Through a series of imaging experiments, Shang (2011) reported that although dopamine (DA) is potently wake-promoting, light exposure can suppress this action of DA at least partly by causing the up-regulation of the inhibitory DA receptor D2R in PDF neurons, which are themselves wake-promoting (Hendricks, 2003). This modulation of the wake-promoting PDF neurons by light may help explain why the Ca-α1T loss-of-function phenotype is biased toward continuous dark conditions if Ca-α1T functions downstream of the PDF neurons. It would mean the responsible Ca-α1T-positive neurons are also modulated by light (Jeong, 2015).

It was possibe to replicate the increased sleep phenotype of Ca-α1T-null mutants via pan-neuronal knock-down of Ca-α1T, but it was not possible to further narrow the cause of this phenotype to a more specific neuronal subpopulation. This was in spite of numerous attempts with neuronal Gal4 driver lines ranging from broadly expressed enhancer traps and neurotransmitter Gal4 drivers to much more narrowly expressed neuropeptide drivers. This difficulty suggests Ca-α1T may function in novel sleep circuits (Jeong, 2015).

In addition to their sleep phenotype, Ca-α1T-null mutants also have a circadian phenotype: an elongated circadian period and a reduction in rhythmic power. It is difficult to say, however, whether these altered circadian parameters are independent of or secondary to the sleep phenotype. Rhythmic power is proportional to the magnitude of the changes in activity level and the regularity with which they occur. Since the increased sleep observed in the Ca-α1T-null mutants does reduce the change in overall activity level between subjective day and subjective night, the increased sleep must also cause a reduction in rhythmic power (Jeong, 2015).

The length of time animals spend sleeping is controlled by both the circadian clock and by a homeostatic drive to sleep that is proportional to time spent awake. Thus, most 'sleep mutants' described so far have had defects in one or the other—they are either circadian sleep mutants or homeostatic sleep mutants. After 24 hours of mechanically-induced sleep deprivation, it was observed that Ca-α1T-null mutants regain slightly more of their lost sleep than control flies, although the increase was not statistically significant. This suggests that, in addition to their circadian phenotype, Ca-α1T-null mutants may also have a slightly stronger homeostatic drive to sleep than controls. Although neither the circadian phenotype nor the homeostatic phenotype are particularly strong, together they produce a robust increase in sleep (Jeong, 2015).

The 'three channel' compensation hypothesis in mice may yet turn out to be correct, but the current results in flies suggest that other factors -- isoform-specific differences, differences related to protein-protein interactions, or even something completely unforeseen -- may allow mice and flies lacking these broadly expressed and highly conserved ion channels to still function remarkably well. It will be interesting to see whether future studies focused on the technically demanding study of isoform-specific expression patterns and isoform-specific rescues in both mice and flies will clarify how T-type channels can at various times and in various contexts both enhance and reduce sleep (Jeong, 2015).

Ca_v2 channels mediate low and high voltage-activated calcium currents in Drosophila motoneurons

Different blends of membrane currents underlie distinct functions of neurons in the brain. A major step towards understanding neuronal function, therefore, is to identify the genes that encode different ionic currents. This study combined in situ patch clamp recordings of somatodendritic calcium currents in an identified adult Drosophila motoneuron with targeted genetic manipulation. Voltage clamp recordings revealed transient low voltage-activated (LVA) currents with activation between -60 mV and -70 mV as well as high voltage-activated (HVA) current with an activation voltage around -30 mV. LVA could be fully inactivated by prepulses to -50 mV and was partially amiloride sensitive. Recordings from newly generated mutant flies demonstrates that DmαG (Ca-α1T, the subject of this Interactive Fly page), the Ca_v3 homolog, encodes the amiloride-sensitive portion of the transient LVA calcium current. This study further demonstrated that the Ca_v2 homolog, Dmca1A (Cacophony), mediated the amiloride-insensitive component of LVA current. This novel role of Ca_v2 channels was substantiated by patch clamp recordings from conditional mutants, RNAi knock-downs, and following Dmca1A overexpression. In addition, it was shown that Dmca1A underlies the HVA somatodendritic calcium currents in vivo. Therefore, the Drosophila Ca_v2 homolog, Dmca1A (Cacophony), underlies HVA and LVA somatodendritic calcium currents in the same neuron. Interestingly, DmαG (Ca-α1T, the subject of this Interactive Fly page) is required for regulating LVA and HVA derived from Dmca1A in vivo. In summary, each vertebrate gene family for voltage-gated calcium channels is represented by a single gene in Drosophila, namely Dmca1D (Ca_v1), Dmca1A (Ca_v2) and DmalphaG (Ca_v3), but the commonly held view that LVA calcium currents are usually mediated by Ca_v3 rather than Ca_v2 channels may require reconsideration (Ryglewski, 2012).

Transient LVA calcium currents have previously been recorded in Drosophila muscle, and indirect evidence for neuronal LVA currents came from the embryonic CNS. In other insects neuronal LVA, although not transient, has been demonstrated, but the underlying gene(s) were not determined. This study demonstrates that DmαG encodes a transient LVA calcium current in Drosophila neurons that activates between -70 mV and -60 mV in vivo. Small-amplitude LVA current was absent in DmαG mutants and selectively blocked by the vertebrate T-type channel blocker amiloride, thus confirming genome sequence predictions that revealed strong similarity between DmαG and vertebrate Ca_v3 channels. Therefore, the three vertebrate VGCC families (Ca_v1, Ca_v2 and Ca_v3) each have one Drosophila homolog, namely Dmca1D, Dmca1A and DmαG (Ryglewski, 2012).

Three lines of evidence show that Dmca1A (Cacophony) encodes somatodendritic HVA calcium currents. First, RNAi knock-down of Dmca1A abolished all HVA calcium current in MN5. Second, acute conditional knock-down of Dmca1A also abolished all HVA current, excluding the possibility that there were indirect developmental effects of Dmca1A knock-down on adult calcium currents. Third, application of the Dmca1A channel blocker PLTXII blocked all HVA current. Similarly, in cultured embryonic Drosophila cytokinesis-arrested neuroblasts Dmca1A mediates sustained HVA calcium current. HVA current in MN5 has a fast onset and does not fully inactivate over the duration of the voltage step (200 ms), and the shape of the current suggests two different components. Although electrical separation with depolarizing pre-pulses into a transient and a sustained HVA component was not possible, and the genetic and pharmacological manipulations employed in this study affected all HVA current and not only a portion of it, the possibility cannot be excluded that different splice variants of Dmca1A may underlie fast and slow components of HVA in MN5. Future studies will test this by expression of different splice variants in a Dmca1A null mutant background (Ryglewski, 2012).

However, the data demonstrate that Dmca1A encodes both HVA and LVA calcium current in vivo. Three lines of evidence proved that a large portion of MN5 LVA current was mediated by Dmca1A. First, RNAi knock-down of Dmca1A in MN5 abolished 70-80% of the LVA current. Second, acute genetic knock-down in temperature-sensitive mutants as well as acute pharmacological block of Dmca1A by PLTXII abolished about 60-70% of the LVA current in MN5. Third, the remaining PLTXII-insensitive LVA current is blocked by amiloride. This strongly indicates that Dmca1A encodes a fast, transient, PLTXII-sensitive LVA current in MN5 that activates between -70 mV and -60 mV. Therefore, Dmca1A encodes both HVA and LVA somatodendritic calcium current in the same neuron in vivo. This was further confirmed by targeted expression of a Dmca1A transgene in MN5 (along with endogenous Dmca1A) in a DmαG mutant background, which reliably produced LVA and HVA calcium currents that were blocked by PLTXII (Ryglewski, 2012).

This study is the first report of a Ca_v2-like channel mediating LVA calcium current with activation voltages between -70 mV and -60 mV. At present it remains unclear whether vertebrate Ca_v2 channels can mediate currents with similarly negative activation voltages, because sequence differences exist between Dmca1A and Ca_v2 (see below), and both may assemble with different accessory subunits. However, sustained HVA calcium current and fast inactivating calcium current with hyperpolarized activation voltages of -40 mV, both based on Dmca1A, have previously been reported in cultured Drosophila neuroblasts. Furthermore, some Ca_v1.3 channels activate in the LVA range, as alternative splicing can shift their activation voltages towards more hyperpolarized potentials (Ryglewski, 2012).

In contrast to voltage-gated potassium and sodium channels voltage-gated calcium channels are assembled from four homologous repeats (I-IV) each containing six transmembrane domains (TMD, S1-S6), all read from a single gene. Therefore, heterotetramerization as reported to cause different current properties in potassium channels is not an option. However, Dmca1A contains 34 exons, at least four of which undergo alternative splicing. An attractive hypothesis is that different activation voltages of Dmca1A channels may result from alternative splicing of mutually exclusive exons coding for part of the S4/S5 extracellular loop and the voltage sensor in the S4 TMD of the first of the four homologous repeats of the gene. Other known alternative splice sites in Dmca1A affect interactions with accessory subunits as well as a small exon coding for a part of repeat IV. Additional experiments are needed to determine whether different splice variants of Dmca1A underlie LVA and HVA somatodendritic neuronal calcium current (Ryglewski, 2012).

Dmca1A also contains at least ten known sites for A to I RNA editing which theoretically yields more than 1000 possible channel isoforms. Although genetic suppression of RNA editing causes disruption of coordinated motor behaviour including courtship, at present it remains speculative whether RNA editing may cause different activation voltages of Dmca1A channels. The Drosophila system offers genetic tools to directly address this possibility in future (Ryglewski, 2012).

Clearly, the Ca_v2 homolog Dmca1A underlies all HVA and 60 to 70% of adult MN5 LVA current. By contrast, DmαG makes up only for about 35% of all adult LVA current. What might be the function of DmαG channels if Dmca1A channels underlie most LVA current? One possibility is that DmαG channels might be localized differently from Dmca1A channels. In Purkinje and in mitral cells, Ca_v3 channels localize postsynaptically and interact with mGluRs (Hildebrand, 2009; Isope, 2010; Johnston, 2010). Alternatively, DmαG channels may contribute to the normal development of LVA and HVA currents through Dmca1A channels, as indicated by the absence or reduction of adult calcium currents in the majority of all DmαG mutant animals. DmαG is not absolutely necessary for Dmca1A transcription, post-transcriptional processing or targeting of channels, because all DmαG excision mutant animals with over-expression of Dmca1A channels had both LVA and HVA calcium currents. However, this does not exclude the possibility that expression of DmαG might facilitate any of these processes, particularly transcription of the endogenous Dmca1A gene. The observed variability of calcium current amplitudes in DmαG mutants suggests that there may be activity-dependent plasticity of Dmca1A expression. The current working model is that calcium influx through dendritic LVA DmαG channels, as induced by early synaptic activity during pupal development, will facilitate Dmca1A expression. MN5 receives excitatory cholinergic synaptic drive through Dα7 nAChRs. A postsynaptic localization of Ca_v3 channels in dendrites has been reported in several types of vertebrate neurons (Hildebrand, 2009; Isope, 2010). Therefore, it seems likely that synaptic activity will cause calcium influx through DmαG LVA channels, which in turn may facilitate Dmca1A channel expression. Since insect nAChRs conduct sodium and calcium, strong synaptic activity may be sufficient to signal Dmca1A expression without activation of DmαG channels, as was observed in 16% of all DmαG mutants. Since there are many potential ways by which activity-dependent calcium influx may regulate Dmca1A channels, ranging from transcriptional regulation to alternative splicing, targeting, translational regulation etc., the nature of the mechanisms has yet to be unraveled. Functional interactions of Ca_v3 and Ca_v2 channel expression have been reported in absence epilepsy models in mouse cortical neurons, but the underlying mechanisms remain unknown. The genetic tools available in Drosophila may facilitate future investigation of the mechanisms by which DmαG regulates Ca_v2 channel expression (Ryglewski, 2012).

Inhibitory peptidergic modulation of C. elegans serotonin neurons is gated by T-type calcium channels

Serotonin is an evolutionarily ancient molecule that functions in generating and modulating many behavioral states. Although much is known about how serotonin acts on its cellular targets, how serotonin release is regulated in vivo remains poorly understood. In the nematode C. elegans, serotonin neurons (see Drosophila serotonergic system) that drive female reproductive behavior are directly modulated by inhibitory neuropeptides. This study reports the isolation of mutants in which inhibitory neuropeptides fail to properly modulate serotonin neurons and the behavior they mediate. The corresponding mutations affect the T-type calcium channel CCA-1 (see Drosophila Ca-α1T) and symmetrically re-tune its voltage-dependencies of activation and inactivation towards more hyperpolarized potentials. This shift in voltage dependency strongly and specifically bypasses the behavioral and cell physiological effects of peptidergic inhibition on serotonin neurons. These results indicate that T-type calcium channels are critical regulators of a C. elegans serotonergic circuit and demonstrate a mechanism in which T-type channels functionally gate inhibitory modulation in vivo (Zang, 2017).

Search PubMed for articles about Drosophila Ca-α1T

Anderson, M. P., Mochizuki, T., Xie, J., Fischler, W., Manger, J. P., Talley, E. M., Scammell, T. E. and Tonegawa, S. (2005). Thalamic Cav3.1 T-type Ca2+ channel plays a crucial role in stabilizing sleep. Proc Natl Acad Sci U S A 102: 1743-1748. PubMed ID: 15677322

Baines, R. A. and Bate, M. (1998). Electrophysiological development of central neurons in the Drosophila embryo. J Neurosci 18: 4673-4683. PubMed ID: 9614242

Bell, T. J., Thaler, C., Castiglioni, A. J., Helton, T. D. and Lipscombe, D. (2004). Cell-specific alternative splicing increases calcium channel current density in the pain pathway. Neuron 41: 127-138. PubMed ID: 14715140

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date revised: 15 January 2016

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