cacophony: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - cacophony

Synonyms - Dmca1A

Cytological map position - 10F8-11A1

Function - channel

Keywords - neuromuscular synapse, mating behavior, visual signal transduction

Symbol - cac

FlyBase ID: FBgn0005563

Genetic map position - chrX:11,931,464-11,985,240

Classification - voltage sensitive calcium channel

Cellular location - surface



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Saras, A. and Tanouye, M. A. (2016). Mutations of the calcium channel gene cacophony suppress seizures in Drosophila. PLoS Genet 12: e1005784. PubMed ID: 26771829
Summary:
Bang sensitive (BS) Drosophila mutants display characteristic seizure-like phenotypes resembling, in some aspects, those of human seizure disorders such as epilepsy. The BS mutant parabss1, caused by a gain-of-function mutation of the voltage-gated Na+ channel gene, is extremely seizure-sensitive with phenotypes that have proven difficult to ameliorate by anti-epileptic drug feeding or by seizure-suppressor mutation. It has been presented as a model for intractable human epilepsy. This study show that cacophony (cacTS2), a mutation of the Drosophila presynaptic Ca++ channel α1 subunit gene, is a particularly potent seizure-suppressor mutation, reverting seizure-like phenotypes for parebss1 and other BS mutants. Seizure-like phenotypes for parabss1 may be suppressed by as much as 90% in double mutant combinations with cacTS2. Unexpectedly, it was found that parebss1 also reciprocally suppresses cacTS2 seizure-like phenotypes. The cacTS2 mutant displays these seizure-like behaviors and spontaneous high-frequency action potential firing transiently after exposure to high temperature. This seizure-like behavior in cacTS2 is ameliorated by 85% in double mutant combinations with parebss1.
Lembke, K. M., Scudder, C. and Morton, D. B. (2017). Restoration of motor defects caused by loss of Drosophila TDP-43 by expression of the voltage-gated calcium channel, Cacophony, in central neurons. J Neurosci 37(39): 9486-9497. PubMed ID: 28847811
Summary:
Defects in the RNA-binding protein, TDP-43, are known to cause a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar dementia. A variety of experimental systems have shown that neurons are sensitive to TDP-43 expression levels, yet the specific functional defects resulting from TDP-43 dysregulation have not been well described. Using the Drosophila TDP-43 ortholog TBPH, it has been shown that TBPH-null animals display locomotion defects as third instar larvae. Furthermore, loss of TBPH caused a reduction in cacophony, a Type II voltage-gated calcium channel, expression and that genetically restoring cacophony in motor neurons in TBPH mutant animals was sufficient to rescue the locomotion defects. The present study examined the relative contributions of neuromuscular junction physiology and the motor program to the locomotion defects and identified subsets of neurons that require cacophony expression to rescue the defects. At the neuromuscular junction, it was shown that mEPP amplitudes and frequency require TBPH. Cacophony expression in motor neurons rescued mEPP frequency but not mEPP amplitude. It was also shown that TBPH mutants displayed reduced motor neuron bursting and coordination during crawling and restoring cacophony selectively in two pairs of cells located in the brain, the AVM001b/2b neurons, also rescued the locomotion and motor defects, but not the defects in neuromuscular junction physiology. These results suggest that the behavioral defects associated with loss of TBPH throughout the nervous system can be associated with defects in a small number of genes in a limited number of central neurons, rather than peripheral defects.
Lam, A., Karekar, P., Shah, K., Hariharan, G., Fleyshman, M., Kaur, H., Singh, H. and Gururaja Rao, S. (2018). Drosophila voltage-gated calcium channel alpha1-subunits regulate cardiac function in the aging heart. Sci Rep 8(1): 6910. PubMed ID: 29720608
Summary:
Ion channels maintain numerous physiological functions and regulate signaling pathways. They are the key targets for cellular reactive oxygen species (ROS), acting as signaling switches between ROS and ionic homeostasis. A paraquat (PQ) screen was carried out in Drosophila to identify ion channels regulating the ROS handling and survival in Drosophila melanogaster. The screen has revealed that α1-subunits (D-type, T-type, and cacophony) of voltage-gated calcium channels (VGCCs) handle PQ-mediated ROS stress differentially in a gender-based manner. Since ROS are also involved in determining the lifespan, it was discovered that the absence of T-type and cacophony decreased the lifespan while the absence of D-type maintained a similar lifespan to that of the wild-type strain. VGCCs are also responsible for electrical signaling in cardiac cells. The cardiac function of each mutant was evaluated through optical coherence tomography (OCT), which revealed that alpha1-subunits of VGCCs are essential in maintaining cardiac rhythmicity and cardiac function in an age-dependent manner. The results establish specific roles of alpha1-subunits of VGCCs in the functioning of the aging heart.
Lam, A., Karekar, P., Shah, K., Hariharan, G., Fleyshman, M., Kaur, H., Singh, H. and Gururaja Rao, S. (2018). Drosophila voltage-gated calcium channel alpha1-subunits regulate cardiac function in the aging heart. Sci Rep 8(1): 6910. PubMed ID: 29720608
Summary:
This study has carried out a paraquat (PQ) screen in Drosophila to identify ion channels regulating the ROS handling and survival in Drosophila melanogaster. The screen has revealed that alpha1-subunits (D-type, T-type, and cacophony) of voltage-gated calcium channels (VGCCs) handle PQ-mediated ROS stress differentially in a gender-based manner. Since ROS are also involved in determining the lifespan, this study discovered that the absence of T-type and cacophony decreased the lifespan while the absence of D-type maintained a similar lifespan to that of the wild-type strain. VGCCs are also responsible for electrical signaling in cardiac cells. The cardiac function of each mutant was evaluated through optical coherence tomography (OCT), which revealed that alpha1-subunits of VGCCs are essential in maintaining cardiac rhythmicity and cardiac function in an age-dependent manner. These results establish specific roles of alpha1-subunits of VGCCs in the functioning of the aging heart.
Brusich, D. J., Spring, A. M., James, T. D., Yeates, C. J., Helms, T. H. and Frank, C. A. (2018). Drosophila CaV2 channels harboring human migraine mutations cause synapse hyperexcitability that can be suppressed by inhibition of a Ca2+ store release pathway. PLoS Genet 14(8): e1007577. PubMed ID: 30080864
Summary:
Gain-of-function mutations in the human CaV2.1 gene CACNA1A cause familial hemiplegic migraine type 1 (FHM1). To characterize cellular problems potentially triggered by CaV2.1 gains of function, mutations encoding FHM1 amino-acid substitutions S218L (SL) and R192Q (RQ) were engineered into transgenes of Drosophila CaV2/cacophony. The transgenes were expressed pan-neuronally. Single mutant SL- and complex allele RQ,SL-expressing animals showed overt phenotypes, including sharply decreased viability. SL- and RQ,SL-expressing neuromuscular junctions (NMJs) exhibited enhanced evoked discharges, supernumerary discharges, and an increase in the amplitudes and frequencies of spontaneous events. Some spontaneous events were gigantic (10-40 mV), multi-quantal events. These were eliminated by application of TTX-or by lowered or chelated Ca2+-suggesting that gigantic events were elicited by spontaneous nerve firing. Some neuronal hyperexcitability phenotypes were reversed after knockdown of Drosophila phospholipase Cbeta (PLCbeta), IP3 receptor, or ryanodine receptor (RyR)-all factors known to mediate Ca2+ release from intracellular stores. Pharmacological inhibitors of intracellular Ca2+ store release produced similar effects. Interestingly, however, the decreased viability phenotype was not reversed by genetic impairment of intracellular Ca2+ release factors. On a cellular level, the data suggest inhibition of signaling that triggers intracellular Ca2+ release could counteract hyperexcitability induced by gains of CaV2.1 function.
BIOLOGICAL OVERVIEW

The cacophony (cac) locus was first identified in a screen for mutants exhibiting altered courtship song (von Schilcher, 1976, 1977) and was subsequently found to be allelic to the nightblind A (nbA) locus (Heisenberg, 1975; Smith, 1998b). A synaptic function for cac-encoded calcium channels was suggested by electroretinogram recordings from cac (nbA) mutants (Heisenberg, 1975; Smith, 1998b), by the sequence similarity between cac and mammalian a1 subunits implicated in synaptic transmission (Smith, 1996), and by the genetic interaction of cacTS2 with comatose (comt) (Dellinger, 2000). Functional analysis indeed demonstrates that cac encodes a primary a1 subunit (Smith, 1996) functioning in neurotransmitter release at the presynaptic terminus of the neuromuscular junction (Kawasaki, 2000). A more central function for the cac locus with respect to its role in courtship behavior is suggested by genetic mosaic studies reporting that the courtship song may be regulated by synaptic functions occurring locally within certain central nervous system ganglia; these are likely to be located in the ventral nerve cord, as inferred from song analyses of cacS//cac+ mosaics (Hall, 1990; cf. Schilcher, 1979). However, it is also possible that excitable-cell etiology of cac-induced song defects resides more peripherally, at neuromuscular junctions (Kawasaki, 2000).

A number of studies have implicated specific classes of voltage-gated calcium channels in neurotransmitter release (for review, see Wheeler, 1995; Stanley, 1997; Catterall, 1998). These channels are composed of multiple subunits, including a1, the primary structural subunit, as well as a2d, ß, and g subunits. a1 subunits of the A and B classes have been localized to synaptic terminals (Robitaille, 1990; Westenbroek, 1995), and heterologous expression shows that their pharmacology resembles that of calcium channels involved in neurotransmitter release. a1A (Cav2.1) and a1B (Cav2.2) encode P/Q- and N-type channels, respectively, and the pharmacology of these channels when expressed in heterologous systems is similar to that observed for neurotransmitter release. These a1 subunits contain a defined synaptic protein interaction (SYNPRINT) domain (Mochida, 1996; Rettig, 1997; Sheng, 1998) that interacts directly with the neurotransmitter release apparatus and may participate in coupling calcium influx to fast synaptic vesicle fusion. In contrast to the closely related a1 subunit genes encoding vertebrate presynaptic calcium channels, cacophony (cac) appears to be the only homologous gene in Drosophila (Smith, 1996; Littleton, 2000). Functional diversity of a1 subunits is generated by alternative splicing and A-to-I mRNA editing (see Drosophila Adar). Fly genes encoding L-type and T-type calcium channels, as well as each of the known accessory subunits, have also been identified (Kawasaki, 2000; Kawasaki, 2002 and references therein).

The gating of presynaptic calcium channels is regulated by several mechanisms, including direct a1 subunit interactions with G-proteins, calcium/calmodulin, and components of the neurotransmitter release apparatus. Inhibition of neurotransmitter release by G-protein-linked receptor agonists occurs through direct interactions between the calcium-channel a1 subunit and ßgamma subunits of heterotrimeric G-proteins (De Waard, 1997; Zamponi, 1997; Mirotznik, 2000; Colecraft, 2001). Regulation by G-proteins is antagonized by protein kinase C-mediated phosphorylation of the a1 subunit (Zamponi, 1997; Herlitze, 2001), which has also been reported to increase basal calcium current (Yang, 1993). Another regulatory mechanism involves direct binding of calcium/calmodulin to the IQ motif within the C-terminal cytoplasmic domain of the a1 subunit and is thought to mediate calcium-dependent channel gating, including facilitation and inactivation (Lee, 1999; DeMaria, 2001; Erickson, 2001). An EF hand calcium-binding motif within the same C-terminal region of the a1 subunit may also contribute to calcium-dependent inactivation (Peterson, 2000). Finally, interaction of presynaptic calcium-channel a1 subunits with syntaxin, a core protein of the neurotransmitter release apparatus, has been shown to regulate channel gating (Bezprozvanny, 1995, 2000; Degtiar, 2000) and also to promote regulation by G-proteins (Stanley, 1997; Jarvis, 2001; Lü, 2001; Kawasaki, 2002 and references therein).

What is the mechanism by which calcium influx is coupled to synaptic vesicle fusion in Drosophila? Binding of syntaxin and several other synaptic proteins to mammalian calcium channels led to identification of a synaptic-protein interaction (SYNPRINT) domain within the intracellular loop linking domains II and III of a1A (P/Q-type) and a1B (N-type) subunits (for review, see Sheng, 1998). This domain is proposed to mediate fast coupling of calcium influx to synaptic vesicle fusion by tethering calcium channels and the release apparatus and by participating in calcium-channel regulation (Mochida, 1996; Sheng, 1998; Wu, 1999; Zhong, 1999). Although cac-encoded calcium channels function in fast, calcium-triggered neurotransmitter release (Kawasaki, 2000), no sequence homologous to known calcium-channel synaptic-protein interaction domains is present in cac or elsewhere in the fly genome (Kawasaki, 2000; Littleton and Ganetzky, 2000). These findings suggest either a novel synaptic-protein interaction domain or an alternative mechanism for the fast coupling of calcium influx to synaptic vesicle fusion (Kawasaki, 2002 and references therein).

The central importance of presynaptic calcium channels has motivated genetic analysis to investigate the in vivo functions of specific calcium-channel proteins at native synapses (Schafer, 1995; Dove, 1998; Lorenzon, 1998; Saegusa, 2000; Ino, 2001). This presents several challenges, including the long-term compensatory changes that may occur in null or hypomorphic mutant animals (Jun, 1999; Saegusa, 2000; Ino, 2001). Therefore, temperature-sensitive (TS) paralytic mutants of Drosophila provide an important and complementary tool allowing acute perturbation of specific gene products for analysis of the molecular mechanisms underlying physiological processes (Kawasaki, 2002 and references therein)

Evidence that cac functions in neurotransmitter release arises from its genetic interactions with comatose (comt), coding for a homolog of the N-ethylmaleimide-sensitive fusion protein that functions in priming synaptic vesicles for fast, calcium-triggered fusion. comt mutants exhibit rapid temperature sensitive paralysis. These mutants typically develop and function normally at permissive temperature and can be shifted to restrictive temperature to examine the acute functional consequences of perturbing a specific gene product. To broaden the analysis of the biology of neurotransmitter release to other gene products functioning in synaptic vesicle trafficking, a genetic screen was conducted to identify mutations exhibiting functional interactions with comt. One enhancer of comt was determined to be a TS allele of cac and has been designated cacTS2. Electrophysiological analysis at neuromuscular synapses has revealed that neurotransmitter release in cacTS2 is markedly reduced at elevated temperatures, indicating that cac functions in synaptic transmission (Kawasaki, 2000). Notably, rescue of rapid, calcium-triggered neurotransmitter release can be achieved in comatose mutants by neural expression of a single cDNA containing a subset of alternative exons and lacking any conserved synaptic-protein interaction sequence (Kawasaki, 2002).

How does Cacophony regulate separate and complex biological functions? The cacS mutant, which exhibits defects in the patterning of courtship lovesong and a newly revealed but subtle abnormality in visual physiology, is mutated such that a highly conserved phenylalanine (in one of the quasi-homologous intrapolypeptide regions called IIIS6) is replaced by isoleucine. The cacH18 mutant exhibits defects in visual physiology (including complete unresponsiveness to light in certain genetic combinations) and visually mediated behaviors; this mutant (originally nbAH18) has a stop codon in an alternative exon (within the cac ORF), which is differentially expressed in the eye. Analysis of the various courtship and visual phenotypes associated with this array of cac mutants demonstrates that Cacophony-type calcium channels mediate multiple, separable biological functions; these correlate in part with transcript diversity generated via alternative splicing (Smith, 2002).

Transsynaptic control of presynaptic Ca(2)(+) influx achieves homeostatic potentiation of neurotransmitter release

Given the complexity of the nervous system and its capacity for change, it is remarkable that robust, reproducible neural function and animal behavior can be achieved. It is now apparent that homeostatic signaling systems have evolved to stabilize neural function. At the neuromuscular junction (NMJ) of organisms ranging from Drosophila to human, inhibition of postsynaptic neurotransmitter receptor function causes a homeostatic increase in presynaptic release that precisely restores postsynaptic excitation. This study addresses what occurs within the presynaptic terminal to achieve homeostatic potentiation of release at the Drosophila NMJ. By imaging presynaptic Ca(2+) transients evoked by single action potentials, a retrograde, transsynaptic modulation of presynaptic Ca(2+) influx was revealed that is sufficient to account for the rapid induction and sustained expression of the homeostatic change in vesicle release. The homeostatic increase in Ca(2+) influx and release is blocked by a point mutation in the presynaptic CaV2.1 channel, demonstrating that the modulation of presynaptic Ca(2+) influx through this channel is causally required for homeostatic potentiation of release. Together with additional analyses, this study establishes that retrograde, transsynaptic modulation of presynaptic Ca(2+) influx through CaV2.1 channels is a key factor underlying the homeostatic regulation of neurotransmitter release (Muller, 2012a).

The homeostatic modulation of presynaptic neurotransmitter release has been observed in organisms ranging from Drosophila to human, at both central and neuromuscular synapses. However, the molecular mechanisms underlying this form of synaptic plasticity are poorly understood. The Drosophila neuromuscular synapse has emerged as a powerful model system to dissect the cellular and molecular basis of this phenomenon. Forward genetic screens at this synapse have begun to identify loss-of-function mutations that prevent this form of neural plasticity. Among the loss-of-function mutations that have been shown to block this process is a mutation in the presynaptic CaV2.1 Ca2+ channel (Frank, 2006). However, these prior genetic data do not inform us regarding whether this calcium channel normally participates in homeostatic plasticity or how it might do so. It remains to be shown that a change in presynaptic Ca2+ influx through the CaV2.1 Ca2+ channel occurs during homeostatic plasticity. It is equally likely that a genetic disruption of the CaV2.1 Ca2+ channel simply occludes this form of plasticity by generally impairing calcium influx or synaptic transmission. Furthermore, if a change in Ca2+ influx occurs during homeostatic plasticity, can it be shown that this change is causally required for the observed homeostatic change in presynaptic release? Finally, if a change in presynaptic Ca2+ influx occurs, can it account for both the rapid induction of homeostatic plasticity as well as the long-term maintenance of homeostatic plasticity, which has been observed to persist for several days (Muller, 2012a)?

To address these outstanding questions, this study probed Ca2+ influx during homeostatic plasticity by imaging presynaptic Ca2+ transients at the Drosophila neuromuscular junction (NMJ). This was done by comparing wild-type controls with animals harboring a mutation in the glutamate receptor subunit IIA (GluRIIA) of the muscle-specific ionotropic GluR at the fly NMJ (GluRIIASP16). The GluRIIASP16 mutation causes a reduction in miniature excitatory postsynaptic potential (mEPSP) amplitude, and induces a homeostatic increase in presynaptic release that precisely offsets the postsynaptic perturbation thereby restoring EPSP amplitudes toward wildtype levels. The GluRIIASP16 mutation is present throughout the life of the animal, and therefore this assay reports the sustained expression of homeostatic plasticity (Muller, 2012a).

If the homeostatic enhancement of release is solely due to a change in presynaptic Ca2+ influx without concomitant changes in the number of releasable vesicles, then repetitive stimulation of homeostatically challenged synapses is expected to result in more pronounced vesicle depletion. Indeed, there is evidence for increased synaptic depression in GluRIIA mutants and following PhTX application. However, in agreement with a recent study, this study observed that homeostatic potentiation was paralleled by an increased number of release-ready vesicles, as assayed by the method of back extrapolation of cumulative excitatory postsynaptic current amplitudes during stimulus trains. The increase in the number of release-ready vesicles detected by this assay could be a direct or an indirect consequence of the homeostatic change in presynaptic Ca2+ influx and might help the potentiated synapse to sustain release during ongoing activity (Muller, 2012a).

Work from several laboratories has provided evidence that chronic manipulation of neural activity in cultured mammalian neurons is associated with a compensatory change in presynaptic neurotransmitter release and a change in presynaptic Ca2+ influx. However, it remains unknown whether these homeostatic changes in release are caused by altered pre- versus postsynaptic activity, and it is unclear whether a change in presynaptic calcium influx is essential for this form of homeostatic plasticity in mammalian central neurons. Ultimately, it will be exciting to determine whether the molecular mechanisms identified in Drosophila, such as those described in this study, will translate to mammalian central synapses (Muller, 2012a).

An auxiliary subunit of the presynaptic calcium channel, α2δ-3, is required for rapid transsynaptic homeostatic signaling

The homeostatic modulation of neurotransmitter release, termed presynaptic homeostatic potentiation (PHP), is a fundamental type of neuromodulation, conserved from Drosophila to humans, that stabilizes information transfer at synaptic connections throughout the nervous system. This study demonstrates that α2δ-3 (straitjacket), an auxiliary subunit of the presynaptic calcium channel, Cacophony, is required for PHP. The α2δ gene family has been linked to chronic pain, epilepsy, autism, and the action of two psychiatric drugs: gabapentin and pregabalin. Loss of α2δ-3 blocks both the rapid induction and sustained expression of PHP due to a failure to potentiate presynaptic calcium influx and the RIM-dependent readily releasable vesicle pool. These deficits are independent of α2δ-3-mediated regulation of baseline calcium influx and presynaptic action potential waveform. α2δ proteins reside at the extracellular face of presynaptic release sites throughout the nervous system, a site ideal for mediating rapid, transsynaptic homeostatic signaling in health and disease (Wang, 2016).

Presynaptic homeostatic potentiation (PHP) can be initiated by disruption of postsynaptic neurotransmitter receptors and is expressed as a change in presynaptic vesicle release. As such, PHP requires retrograde, transsynaptic signaling. The homeostatic potentiation of presynaptic release is mediated by increased presynaptic calcium influx without a change in the presynaptic action potential waveform. A remarkable property of presynaptic homeostatic plasticity is that it can be induced in a time frame of seconds to minutes and can be stably maintained throughout the life of an organism -- months in Drosophila and decades in humans. Equally remarkable, presynaptic homeostasis can precisely offset the magnitude of postsynaptic perturbations that vary widely in severity. This implies the existence of profoundly stable and remarkably precise homeostatic modifications to the presynaptic release apparatus. Transsynaptic signaling systems that are capable of achieving the rapid, accurate, and persistent control of presynaptic vesicle release are generally unknown (Wang, 2016).

In a large-scale forward genetic screen for homeostatic plasticity genes, mutations were identified in the α2δ-3 auxiliary subunit of the CaV2.1 calcium channel. α2δ genes encode a family of proteins that are post-translationally processed into a large glycosylated extracellular α2 domain that is linked through disulfide bonding to a short, membrane-associated δ domain. Existing loss-of-function data are consistent with the primary function of α2δ being the trafficking and synaptic stabilization of pore-forming α1 calcium channel subunits, with which they associate in the ER. There is also evidence that α2δ subunits control calcium channel kinetics in a channel-type- and cell-type-specific manner. However, the function of the α2δ gene family extends beyond calcium channel trafficking and membrane stabilization, including activities related to synapse formation and stability. As such, the large, glycosylated extracellular domain in α2δ may have additional, potent signaling activities at the active zone (Wang, 2016).

Importantly, the α2δ gene family is associated with a wide range of neurological diseases, including autism spectrum disorders (ASDs), neuropathic pain, and epilepsy. The α2δ-1 and α2δ-2 proteins are the primary targets of gabapentin and pregabalin, two major drugs used to treat neuropathic pain and epilepsy. This study demonstrates that α2δ-3 is essential for PHP. Thus, while α2δ-3 is an extracellular component of the extended presynaptic calcium channel complex (Davies, 2010), it nonetheless has a profound ability to modulate the intracellular neurotransmitter release mechanism. It is proposed that α2δ-3 relays signaling information from the synaptic cleft to the cytoplasmic face of the presynaptic active zone during PHP, an activity that could reasonably be related to the function of α2δ-3 during neurological disease (Wang, 2016).

This study demonstrates that α2δ-3 is essential for the rapid induction and sustained expression of presynaptic homeostatic potentiation (PHP). α2δ-3 encodes a glycosylated extracellular protein known to interact with matrix proteins that reside within the synaptic cleft. As such, it is proposed that α2δ-3 mediates homeostatic, retrograde signaling by connecting signaling within synaptic cleft to effector proteins within the presynaptic terminal, such as RIM. Since α2δ-3 associates with the pore-forming α1 subunit of calcium channels, it is ideally positioned to relay signaling to the site of high-release probability vesicle fusion adjacent to the presynaptic calcium channels (Wang, 2016).

It was previously demonstrated that PHP requires not only potentiation of presynaptic calcium influx but also a parallel homeostatic expansion of the readily releasable pool (RRP). Several lines of evidence argue against the possibility that the homeostatic potentiation of presynaptic calcium influx fully accounts for the observed potentiation of the RRP. First, it is well established in mammalian systems and the Drosophila NMJ (Müller, 2015) that the calcium-dependence of the RRP is sub-linear. Therefore, the relatively small change in presynaptic calcium influx that occurs during PHP (12%-25%) would not be sufficient to account for the observed doubling of the RRP, an effect that has been quantified across a wide range of extracellular calcium (0.3-15 mM [Ca2+]e) (Müller, 2015). Second, the homeostatic increase of presynaptic calcium influx and the homeostatic expansion of RRP are genetically separable processes (Harris, 2015). Since loss of α2δ-3 completely blocks the homeostatic expansion of the RRP, it appears that α2δ-3 has an additional activity that is directed at the homeostatic modulation of the RRP (Wang, 2016).

Collectively, these data argue that α2δ-3 functions with Rab3 interacting molecule (rim), either directly or indirectly, to achieve a homeostatic potentiation of the RRP. First, the loss of function phenotype of α2δ-3 is strikingly similar to that observed in rim mutants. Both mutations cause a deficit in presynaptic release that is associated with diminished baseline presynaptic calcium influx, diminished size of the baseline RRP, and enhanced sensitivity to application of EGTA-AM. Second, this study demonstrates a strong trans-heterozygous interaction between rim/+ and α2δ-3/+, suggesting that both genes function to control the same presynaptic processes during PHP. Since the rim mutation selectively disrupts the homeostatic modulation of the RRP, this genetic interaction could reflect a failure to homeostatically modulate the RRP (Wang, 2016).

Both RIM and α2δ-3 bind the pore-forming α1 subunit of the CaV2.1 calcium channel. As such, signaling could be relayed from α2δ-3 to RIM through molecular interactions within the extended CaV2.1 calcium channel complex. However, not all evidence is consistent with this possibility. For example, RNAi-mediated depletion of CaV2.1 channels, sufficient to decrease release by ∼80%, does not prevent presynaptic homeostasis. Thus, loss of α2δ-3 blocks PHP, whereas loss of the CaV2.1 α1 subunit does not. In addition, the double-heterozygous mutant of rim/+ and α2δ-3/+ blocks PHP but does not disrupt baseline vesicle release, arguing that this genetic interaction is not due to a decrease in the number or organization of presynaptic α1 calcium channel subunits. Thus, it is speculated that α2δ-3 conveys signaling through a co-receptor on the plasma membrane to participate in the homeostatic modulation of the RRP. There are very few extracellular proteins known to establish baseline levels of primed, fusion-competent synaptic vesicles. Since α2δ proteins should reside at chemical synapses throughout the nervous system, this signaling could reasonably be related to the neurological and psychiatric diseases associated with α2δ genes (Wang, 2016).


REGULATION

Functional Characterization of Cacophony

The neuropeptide CAP2b stimulates fluid transport obligatorily via calcium entry, nitric oxide, and cGMP in Drosophila melanogaster Malpighian (renal) tubules. The Drosophila L-type calcium channel alpha1-subunit genes Dmca1D and Dmca1A (nbA/cacophony) are both expressed in tubules. CAP2b-stimulated fluid transport and cytosolic calcium concentration ([Ca2+]i) increases are inhibited by the L-type calcium channel blockers verapamil and nifedipine. cGMP-stimulated fluid transport is verapamil and nifedipine sensitive. Furthermore, cGMP induces a slow [Ca2+]i increase in tubule principal cells via verapamil- and nifedipine-sensitive calcium entry; RT-PCR shows that tubules express Drosophila Cyclic nucleotide-gated channel (Cng). Additionally, thapsigargin-induced [Ca2+]i increase is verapamil sensitive. Phenylalkylamines bind with differing affinities to the basolateral and apical surfaces of principal cells in the main segment; however, dihydropyridine binds apically in the tubule initial segment. Immunocytochemical evidence suggests localization of alpha1-subunits to both basolateral and apical surfaces of principal cells in the tubule main segment. Roles for L-type calcium channels and cGMP-mediated calcium influx in both calcium signaling and fluid transport mechanisms in Drosophila are suggested (MacPherson, 2002).

mRNA editing of Cacophony

Messenger RNA editing of transcripts encoding voltage-sensitive ion channels has not been extensively analyzed -- least of all in Drosophila, for which several channel-encoding genes are known. Previous sequence studies of D. melanogaster's cacophony gene, which encodes an alpha 1 calcium-channel subunit called Dmca1A, suggested that several nucleotides within the ORF of the primary transcript are edited such that 'A-to-G' substitutions occur (these two nucleotides being the adenine that is found at the relevant sites in the sense strand of genomic DNA or the primary transcript, compared to the substitution of guanine that is detected at the level of cDNA analysis). Such A-to-G changes are the same kind of post-transcriptional variations originally discovered (in a neurobiological context) for a ligand-sensitive channel in vertebrates. RNA was extracted from adult flies and it has been revealed, by RT-PCR and restriction-enzyme analyses, that transcript heterogeneity exists in vivo for three distinct edited sites within the cac-encoded RNA. Each such nucleotide would lead to channel variability at the level of the Dmca1A polypeptide. Owing to cacophony being originally identified as a 'behavioral gene', the possible significance of Dmca1A RNA editing for influencing the relevant neuro-functional phenotypes is discussed (Smith, 1998a).

Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release

The molecular organization of presynaptic active zones during calcium influx-triggered neurotransmitter release is the focus of intense investigation. The Drosophila coiled-coil domain protein Bruchpilot (BRP) was observed in donut-shaped structures centered at active zones of neuromuscular synapses by using subdiffraction resolution STED (stimulated emission depletion) fluorescence microscopy. At brp mutant active zones, electron-dense projections (T-bars) are entirely lost, Ca2+ channels are reduced in density, evoked vesicle release is depressed, and short-term plasticity is altered. BRP-like proteins seem to establish proximity between Ca2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity (Kittel, 2006).

Synaptic communication is mediated by the fusion of neurotransmitter-filled vesicles with the presynaptic membrane at the active zone, a process triggered by Ca2+ influx through clusters of voltage-gated channels. The spacing between Ca2+ channels and vesicles at active zones is particularly thought to influence the dynamic properties of synaptic transmission (Kittel, 2006).

The larval Drosophila neuromuscular junction (NMJ) is frequently used as a model of glutamatergic synapses. The monoclonal antibody Nc82 specifically stains individual active zones by recognizing a coiled-coil domain protein of roughly 200 kD named Bruchpilot (Brp). Brp shows homologies to the mammalian active zone components CAST [cytoskeletal matrix associated with the active zone (CAZ)-associated structural protein], also called ERC (ELKS, Rab6-interacting protein 2, and CAST). Whereas confocal microscopy recognized diffraction limited spots, the subdiffraction resolution of stimulated emission depletion (STED) fluorescence microscopy revealed donut-shaped Brp structures at active zones. Viewed perpendicular to the plane of synapses, both single and multiple 'rings' were uncovered, of similar size to freeze-fracture-derived estimates of fly active zones. The donuts were up to 0.16 µm high, as judged by images taken parallel to the synaptic plane (Kittel, 2006).

Brp seems to demark individual active zones associated with clusters of Ca2+ channels. Transposon-mediated mutagenesis allowed isolation of a mutant chromosome (brp69) in which nearly the entire open reading frame of Brp was deleted. brp mutants [brp69/df(2R)BSC29] develop into mature larvae but do not form pupae. The Nc82 label is completely lost from the active zones of brp mutant NMJs but can be restored by re-expressing the brp cDNA in the brp mutant background with use of the neuron-specific driver lines ok6-GAL4. This also rescued larval lethality. Mutants had slightly smaller NMJs and somewhat fewer individual synapses. However, individual receptor fields, identified by the glutamate receptor subunit GluRIID, were enlarged in brp mutants. Thus, principal synapse formation occurred in brp mutants, with individual postsynaptic receptor fields increased in size but moderately decreased in number (Kittel, 2006).

In electron micrographs of brp mutant NMJs, synapses with pre- and postsynaptic membranes in close apposition were present at regular density, and consistent with the enlarged glutamate receptor fields postsynaptic densities appeared larger while otherwise normal. However, intermittent rufflings of the presynaptic membrane were noted, and brp mutants completely lacked presynaptic dense projections (T-bars). Occasionally, very little residual electron-dense material attached to the presynaptic active zone membrane was identified. After re-expressing the Brp protein in the mutant background, T-bar formation could be partially restored, although these structures were occasionally somewhat aberrant in shape. Thus, Brp assists in the ultrastructural assembly of the active zone and is essential for T-bar formation (Kittel, 2006).

In brp mutant larvae a drastic decrease was noted in evoked excitatory junctional current (eEJC) amplitudes by using two-electrode voltage clamp recordings of postsynaptic currents at low stimulation frequencies. This drop in current amplitude could be partially rescued through brp re-expression within the presynaptic motoneurons by using either elav-GAL4 or ok6-GAL4. In contrast, the amplitude of miniature excitatory junctional currents (mEJCs) in response to single, spontaneous vesicle fusion events was increased over control levels. This is consistent with the enlarged individual glutamate receptor fields of brp mutants and excludes a lack of postsynaptic sensitivity as the cause of the reduced eEJC amplitudes (Kittel, 2006).

It follows that the number of vesicles released per presynaptic action potential (AP) (quantal content) was severely compromised at brp mutant NMJs and could not be attributed solely to the moderate decrease in synapse number. The ultrastructural defects of brp mutant synapses may interfere with the proper targeting of vesicles to the active zone membrane and thereby impair exocytosis. The number of vesicles directly docked to active zone membranes was slightly decreased in brp mutants. However, the amplitude distribution and sustained frequency of mEJCs showed that brp mutant synapses did not appear to suffer from extrasynaptic release, as would be caused by a misalignment of vesicle fusion sites with postsynaptic receptors. Consistent with the appropriate deposition of exo- and endocytotic proteins, an apparently normal distribution of Syntaxin, Dap160, and Dynamin was observed at brp mutant synapses (Kittel, 2006).

The exact amplitude and time course of AP-triggered Ca2+ influx in the nerve terminal governs the amplitude and time course of vesicle. Nerve-evoked responses of brp mutants were delayed when compared with controls, whereas in contrast mEJC rise times were unchanged. Thus, evoked vesicle fusion events were less synchronized with the invasion of the presynaptic terminal by an AP. Spatiotemporal changes in Ca2+ influx have a profound effect on short-term plasticity. Whereas at 10 Hz controls exhibited substantial short-term depression of eEJC amplitudes, brp mutants showed strong initial facilitation before stabilizing at a slightly lower but frequency-dependent steady-state current. As judged by the initial facilitation at 10 Hz, neither a reduction in the number of releasable vesicles nor available release sites could fully account for the low quantal content of brp mutants at moderate stimulation frequencies. Furthermore, the altered short-term plasticity of brp mutant synapses suggested a change in the highly Ca2+-dependent vesicle release probability. Paired-pulse protocols were applied to the NMJ. Closely spaced stimuli lead to a buildup of residual Ca2+ in the vicinity of presynaptic Ca2+ channels, enhancing the probability of a vesicle within this local Ca2+ domain to undergo fusion after the next pulse. The absence of marked facilitation at control synapses could be explained by a depletion of release-ready vesicles. At brp mutant NMJs, however, the prominent facilitation at short interpulse intervals showed that the enhancement of release probability strongly outweighed the depletion of releasable vesicles. Thus, initial vesicle release probability was low, and release at brp synapses particularly benefited from the accumulation of intracellular Ca2+ (Kittel, 2006).

Vesicle fusion is highly sensitive to the spacing between Ca2+ channels and vesicles at release sites. It has been calculated that doubling this distance from 25 to 50 nm decreases the release probability threefold, and the larger this distance, the more effective the slow synthetic Ca2+ buffer EGTA should become in suppressing release. Indeed, after bath application of membrane permeable EGTA-AM, the reduction of evoked vesicle release was more pronounced at brp mutant than at control NMJs (Kittel, 2006).

The Ca2+-channel subunit Cacophony governs release at Drosophila NMJs. By using a fully functional, GFP (green fluorescent protein)-labeled variant (CacGFP), Ca2+ channels were visualized in vivo. Consistently, Ca2+ channel expression was severely reduced over the entire NMJ and at synapses lacking Brp (Kittel, 2006).

Thus, it is concluded that brp mutants suffer from a diminished vesicle release probability due to a decrease in the density of presynaptic Ca2+ channel clusters. It is conceivable that Brp tightly surrounds but is not part of the T-bar structure, contained within the unlabeled center of donuts. Brp may establish a matrix, required for both T-bar assembly as well as the appropriate localization of active zone components including Ca2+ channels, possibly by mediating their integration into a restricted number of active zone slots. Related mechanisms might underlie functional impairments of mammalian central synapses lacking active zone components and natural physiological differences between synapse types. Electron microscopy has identified regular arrangements at active zones of mammalian CNS synapses ('particle web') and frog NMJs ('ribs'), where these structures have also been proposed to organize Ca2+ channel clustering. At calyx of Held synapses (an axosomatic synapse in the auditory brainstem), both a fast and a slow component of exocytosis have been described. The fast component recovers slowly and is believed to owe its properties to vesicles attached to a matrix tightly associated with Ca2+ channels, whereas the slow component recovers faster and is thought to be important for sustaining vesicle release during tetanic stimulation. In agreement with this concept, the absence or impairment of such a matrix at brp synapses has a profound effect on vesicle release at low stimulation frequencies, but this effect subsides as the frequency increases. The sustained frequency of mEJCs at brp synapses could be explained if spontaneous fusion events arise from the slow release component or a pathway independent of evoked vesicle fusion (Kittel, 2006).

Synapses lacking Brp and T-bars exhibited a defective coupling of Ca2+ influx with vesicle fusion, whereas the vesicle availability did not appear rate-limiting under low frequency stimulation. The activity-induced addition of presynaptic dense bodies has been proposed to elevate vesicle release probability. This work supports this hypothesis and suggests an involvement of Brp or related factors in synaptic plasticity by promoting Ca2+ channel clustering at the active zone membrane (Kittel, 2006).

Maturation of active zone assembly by Drosophila Bruchpilot

Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel-clustering defects. This study used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, BRP was identified as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains (Fouquet, 2009).

This study addressed whether BRP signals T-bar formation (without being a direct component of the T-bar) or whether the protein itself is an essential building block of this electron-dense structure. Evidence is provided that BRP is a direct T-bar component. Immuno-EM identifies the N terminus of BRP throughout the whole cross section of the T-bar, and genetic approaches show that a truncated BRP, lacking the C-terminal 30% of the protein's sequence, forms truncated T-bars. Immuno-EM and light microscopy consistently demonstrate that N- and C-terminal epitopes of BRP are segregated along an axis vertical to the AZ membrane and suggest that BRP is an elongated protein, which directly shapes the T-bar structure (Fouquet, 2009).

In brp5.45 (predicted as aa 1-866), T-bars were not detected, whereas brp1.3 (aa 1-1,389) formed T-bar-like structures, although fewer and smaller than normal. Moreover, the BRPD1-3GFP construct (1-1,226) did not rescue T-bar assembly. Thus, domains between aa 1,226 and 1,390 of BRP may also be important for the formation of T-bars. Clearly, however, the assembly scheme for T-bars is expected to be controlled at several levels (e.g., by phosphorylation) and might involve further protein components. Nonetheless, it is highly likely that the C-terminal half of BRP plays a crucial role (Fouquet, 2009).

Since BRP represents an essential component of the electron-dense T-bar subcompartment at the AZ center, it might link Ca2+ channel-dependent release sites to the synaptic vesicle cycle. Interestingly, light and electron microscopic analysis has located CAST at mammalian synapses both with and without ribbons. Overall, this study is one of the first to genetically identify a component of an electron-dense synaptic specialization and thus paves the way for further genetic analyses of this subcellular structure (Fouquet, 2009).

The N terminus of BRP is found significantly closer to the AZ membrane than the C terminus, where it covers a confined area very similar to the area defined by the CacGFP epitope. Electron tomography of frog NMJs suggested that the cytoplasmic domains of Ca2+ channels, reminiscent of pegs, are concentrated directly beneath a component of an electron-dense AZ matrix resembling ribs. In addition, freeze-fracture EM identified membrane-associated particles at flesh fly AZs, which, as judged by their dimensions, might well be Ca2+ channels. Peg-like structures were observed beneath the T-bar pedestal. Similar to fly T-bars, the frog AZ matrix extends up to 75 nm into the presynaptic cytoplasm. Based on the amount of cytoplasmic Ca2+ channel protein it has been concluded that Ca2+ channels are likely to extend into parts of the ribs. Thus, physical interactions between cytoplasmic domains of Ca2+ channels and components of ribs/T-bars might well control the formation of Ca2+ channel clusters at the AZ membrane. However, a short N-terminal fragment of BRP (aa 1-320) expressed in the brp-null background was unable to localize to AZs efficiently and consistently failed to restore Cac clustering (unpublished data) (Fouquet, 2009).

The mean Ca2+ channel density at AZs is reduced in brp-null alleles. In vitro assays indicate that the N-terminal 20% of BRP can physically interact with the intracellular C terminus of Cacaphony (Cac). Notably, it was found that the GFP epitope at the very C terminus of CacGFP was closer to the AZ membrane than the N-terminal epitope of BRP. It is conceivable that parts of the Cac C terminus extend into the pedestal region of the T-bar cytomatrix to locally interact with the BRP N terminus. This interaction might play a role in clustering Ca2+ channels beneath the T-bar pedestal (Fouquet, 2009).

Clearly, additional work will be needed to identify the contributions of discrete protein interactions in the potentially complex AZ protein interaction scheme. This study should pave the way for a genetic analysis of spatial relationships and structural linkages within the AZ organization. Moreover, the current findings should integrate in the framework of mechanisms for Ca2+ channel trafficking, clustering, and functional modulation (Fouquet, 2009).

The imaging assays allowed a temporally resolved analysis of AZ assembly in vivo. BRP is a late player in AZ assembly, arriving hours after DLiprin-α and also clearly after the postsynaptic accumulation of DGluRIIA. Accumulation of Cac was late as well, although it slightly preceded the arrival of BRP, and impaired Cac clustering at AZs lacking BRP became apparent only from a certain synapse size onwards, form at sites distant from preexisting ones and grow to reach a mature, fixed size. Thus, the late, BRP-dependent formation of the T-bar seems to be required for maintaining high Ca2+ channel levels at maturing AZs but not for initializing Ca2+ channel clustering at newly forming sites. As the dominant fraction of neuromuscular AZs is mature at a given time point, the overall impression is that of a general clustering defect in brp mutants. In reverse, it will be of interest to further differentiate the molecular mechanisms governing early Ca2+ channel clustering. Pre- to postsynaptic communication via neurexin-neuroligin interactions might well contribute to this process. A further candidate involved in early Ca2+ channel clustering is the Fuseless protein, which was recently shown to be crucial for proper Cac localization at AZs (Fouquet, 2009).

In summary, during the developmental formation of Drosophila NMJ synapses, the emergence of a presynaptic dense body, which is involved in accumulating Ca2+ channels, appears to be a central aspect of synapse maturation. This is likely to confer mature release probability to individual AZs and contribute to matching pre- and postsynaptic assembly by regulating glutamate receptor composition. Whether similar mechanisms operate during synapse formation and maturation in mammals remains an open question (Fouquet, 2009).

This study concentrated on developmental synapse formation and maturation. The question arises whether similar mechanisms to those relevant for AZ maturation might control activity-dependent plasticity as well and whether maturation-dependent changes might be reversible at the level of individual synapses. Notably, experience-dependent, bidirectional changes in the size and number of T-bars (occurring within minutes) were implied at Drosophila photoreceptor synapses by ultrastructural means. Moreover, at the crayfish NMJ, multiple complex AZs with double-dense body architecture were produced after stimulation and were associated with higher release probability. In fact, a recent study has correlated the ribbon size of inner hair cell synapses with Ca2+ microdomain amplitudes. Thus, a detailed understanding of the AZ architecture might permit a prediction of functional properties of individual AZs (Fouquet, 2009).

Mutations in a Drosophila α2δ voltage-gated calcium channel subunit reveal a crucial synaptic function

Voltage-dependent calcium channels regulate many aspects of neuronal biology, including synaptic transmission. In addition to their α1 subunit, which encodes the essential voltage gate and selective pore, calcium channels also contain auxiliary α2δ, β, and γ subunits. Despite progress in understanding the biophysical properties of calcium channels, the in vivo functions of these auxiliary subunits remain unclear. Mutations were isolated in the gene encoding an α2δ calcium channel subunit (dα2δ-3) using a forward genetic screen in Drosophila. Null mutations in this gene are embryonic lethal and can be rescued by expression in the nervous system, demonstrating that the essential function of this subunit is neuronal. The photoreceptor phenotype of dα2δ-3 mutants resembles that of the calcium channel α1 mutant cacophony (cac), suggesting shared functions. Genotypes that survive to the third-instar stage have been examined in detail. Electrophysiological recordings demonstrate that synaptic transmission is severely impaired in these mutants. Thus the α2δ calcium channel subunit is critical for calcium-dependent synaptic function. As such, this Drosophila isoform is the likely partner to the presynaptic calcium channel α1 subunit encoded by the cac locus. Consistent with this hypothesis, cacGFP fluorescence at the neuromuscular junction is reduced in dα2δ-3 mutants. This is the first characterization of an α2δ-3 mutant in any organism and indicates a necessary role for α2δ-3 in presynaptic vesicle release and calcium channel expression at active zones (Dickman, 2008).

Calcium channels have well established roles in synaptic transmission, cell excitability, intracellular signaling, and disease. Voltage-gated calcium channels have a unique responsibility for converting electrical changes across the plasma membrane into intracellular changes in calcium concentration. Molecularly, they contain a pore-forming α1 subunit that confers many of the basic properties of the ion channel, including its voltage-sensitive gating, selectivity for calcium, and pharmacological properties. However, calcium channels also contain α2δ and β subunits that can have a substantial influence on the properties of calcium channels when expressed in heterologous systems. Both α2δ and β subunits can markedly increase surface expression of the channels and can also influence the gating properties of the channel. The β subunit is entirely intracellular and is the target for several pathways that modulate calcium channel function. The α2δ subunit, in contrast, lacks an intracellular domain. This subunit consists of two polypeptides that are transcribed as a single transcript and posttranslationally cleaved into the α2 and δ chains, which remain linked by a disulfide bond (Klugbauer, 2003). The α2 portion is entirely extracellular and heavily glycosylated, whereas the δ chain also includes a C-terminal transmembrane domain. In addition, there is a γ subunit whose role is controversial and that need not assemble with the calcium channel complex. Although much has been gained about the biophysical properties of calcium channels, the roles of the auxiliary subunits in regulating calcium channels in vivo is less clear (Dickman, 2008).

Some insights into the role of these accessory subunits in vivo come from a series of spontaneously occurring mutations in mice. These include mutations in a β subunit in lethargic, an α2δ subunit in ducky (Barclay, 2001; Brodbeck, 2002) and in a spontaneous variant of C57BL/10 strain mice, as well as a γ subunit in stargazer. Interestingly, each of these mutants displays ataxia and some form of epilepsy. Moreover, the α2δ calcium channel subunit has been shown to be a target of the anti-epileptic drug gabapentin, although the role of this subunit in the disease remains unclear (Dickman, 2008).

One complication in the genetic analysis of these accessory subunits has been the presence of multiple isoforms in the genome. With regard to α2δ subunits, the number of genes in an organism's genome has remained relatively constant: there are three α2δ isoforms in worms and flies and four isoforms in mammals; these have been classed as α2δ-1,2,3, and 4. In mammals, the α2δ-1 subunit is expressed ubiquitously, whereas the α2δ-2 subunit, the subunit mutated in ducky, is expressed in the brain, kidney, heart, and testes. The α2δ-3 subunit is expressed only in brain. In ducky mice, loss of the α2δ-2 subunit decreases the amplitude of calcium currents in Purkinje cells, in which it is highly expressed, but not in all neurons. Purkinje cells also have abnormal morphologies. At neuromuscular junctions, however, ducky mutations have little effect on transmitter release. Loss of the α2δ-4 subunit causes abnormalities in the outer plexiform layer of the retina. At present, it is uncertain which α1 calcium channel assembles with which α2δ subunit in vivo. In heterologous systems, various combinations promote channel expression, but their associations may be less promiscuous in vivo. Thus, it has not been determined whether synaptic calcium channels also require an α2δ subunit and, if so, what significance that subunit would hold for the physiology of the synapse (Dickman, 2008).

In a forward genetic screen for mutations affecting synaptic transmission, mutations were isolated in the Drosophila α2δ-3 calcium channel subunit. This subunit (dα2δ-3) is essential for viability in Drosophila and shares many of the phenotypes described in mutations of the α1 calcium channel subunit, cacophony. A critical role is demonstrated for dα2δ-3 in synaptic function in both photoreceptors and motorneurons (Dickman, 2008).

α2δ-2 mutant mice show no physiological defects at synapses beyond what can be attributed to the small size of the animals. α2δ-1 has not been studied genetically, but it is expressed in both neuronal and non-neuronal tissues and therefore is likely to have a more general function. Loss of the murine α2δ-4 subunit (Cacna2d4) results in a phenotype that comes closest to that which was observed for loss of dα2δ-3: defects in the synaptic endings of photoreceptors, as revealed by electroretinograms and histology (Wycisk, 2006). The Drosophila genome also contains predicted isoforms of α2δ-1 and α2δ-2, but they do not appear to be functionally redundant with α2δ-3 as α2δ-3 null alleles are lethal and mutations in this isoform produce severe phenotypes in both photoreceptors and neuromuscular junctions. Thus, despite studies in heterologous expression systems that indicate that each α2δ isoform will promiscuously promote the surface expression of any α1 subunit, their functions in vivo are sufficiently distinct that loss of a single subunit can cause a severe phenotype (Dickman, 2008).

Similarly, studies of mammalian channels have not resolved whether each α2δ isoform is associated in vivo with a particular α1 isoform, although there does appear to be some level of selective association. This pairing may derive primarily from the expression patterns of the α2δ and α1 subunits. However, from studies on gabapentin and on ducky mice, α2δ-2 and α2δ-3 appear to be the primary subunits in brain and preferentially associate with P- and N-type calcium channels. In Drosophila, it was found that loss of 2δ-3 gives an electrophysiological phenotype similar to loss of the cac-encoded presynaptic α1 subunit in the ERG and neuromuscular junction. cac is the only Drosophila member of the Cav2 family, homologous to N-, P- and Q-type channels of mammals. Indeed, the cac channel has been established by both electrophysiological studies and cytochemical localization to be the major calcium channel in active zones for driving vesicle release. The present data indicate that the α2δ-3 subunit is its partner and necessary for its proper localization to the active zone. A similar pairing may occur in C. elegans in which unc-36, an α2δ subunit mutant, displays an identical phenotype to the α1 subunit mutant unc-2. At murine photoreceptor synapses, L-type calcium channels mediate transmitter release and therefore, in a subset of mammalian synapses, α2δ-4 may play a similar role to α2δ-3, but partnering L-type rather than N-type channels (Wycisk, 2006), although this has not been investigated with direct recordings of synaptic properties. In the present study, transgenic rescue experiments demonstrated that the only essential function of α2δ-3 in Drosophila is in the nervous system. Other isoforms are thus likely to promote calcium channel expression in other cell types including muscle cells, which express the L-type α1 subunit Dmca1D (Dickman, 2008).

How does the dα2δ-3 subunit contribute to presynaptic function? The leading hypothesis from mammalian work and studies in heterologous systems is that α2δ subunits promote robust plasma membrane expression of the α1 subunit, at least in part by stabilizing them at the plasma membrane (Bernstein, 2007). The phenotype of 2δ-3k10814 is consistent with the hypothesis that 2δ-3 is similarly required for proper synaptic expression of the cacophony α1 subunit. Although it is not possible to record directly from these synaptic boutons to determine the amplitude of calcium currents, the reduction in quantal content per active zone and the decreased amplitude of the EJP are consistent with a decrease in calcium influx at the terminals attributable to decreased channel density. Because of the fourth-order dependence of release on calcium influx, even a 33% reduction in channel density could account for the ~5-fold observed reduction in vesicles released per active zone in the 2δ-3k10814 allele. By fluorescent imaging of the cacGFP transgene, a 25-60% reduction in the level of the cac α1 subunit was observed in 2 different allelic combinations that, because they are not completely null, survive to the third-instar stage. This degree of reduction in α1 subunits at the active zone is consistent with the physiological findings and the hypothesis that the synaptic role of 2δ-3 is to promote the expression, localization, or retention of the cac α1 subunit at active zones. Similarly, the ability of cac overexpression to extend the lifespan of 2δ-3DD196/Df(2)7128 mutants suggests that the 2δ-3 phenotype arises from an insufficiency of synaptic cac channels. In the context of the P-element insertion allele 2δ-3k10814, however, in which substantial amounts of the α1 subunit likely were already present in the terminals, this overexpression was not adequate to significantly increase synaptic transmission (Dickman, 2008).

The 20% decrease in active zones observed per neuromuscular junction, suggested by the decrease in nc82-immunoreactive puncta, is likely to account for a significant component of the 37% decrease in mEJP frequency, but a portion of the decrease may also arise from a change in calcium channel density. Similar decreases in mini frequency arise when neuromuscular junctions are placed in calcium-free salines, indicating that mEJPs are at least partially dependent on the entry of extracellular calcium. One possibility is that the ambient, resting calcium concentration in the synaptic cytosol depends on the amount of calcium that enters through spontaneous openings of calcium channels even at hyperpolarized potentials. Alternatively, sporadic, spontaneous calcium channel openings at the active zones of unstimulated terminals may cause brief, local increases in cytosolic calcium and trigger a portion of the observed minis (Dickman, 2008).

An unexpected feature of the dα2δ-3 mutants was their anatomical phenotype at the neuromuscular junction. In particular, mutants had slightly more boutons per junction, although the muscles themselves were smaller, and these boutons had a lower density of active zones, as scored by detectable puncta of nc82 immunoreactivity. This led to an overall 20% decrease in active zones per muscle (Dickman, 2008).

Changes in the size and morphology of the Drosophila neuromuscular junction have been shown to occur as a result of perturbations in both presynaptic activity. However, many other perturbations of synaptic function do not have these effects, including viable mutations in synaptotagmin, syntaxin and SNAP-25. Mutations in the calcium channel α1 subunit, cac, did not cause an overgrowth of boutons in these studies, and others have reported fewer than normal boutons in cac alleles (Rieckhof, 2003; Xing, 2005). The changes in bouton number and active zone density in 2δ-3 mutants therefore merit additional study. At present, these phenotypes may be direct consequences of the loss of this subunit or may include indirect consequences, possibly compensatory, in response to changes in the activation of the terminals, calcium influx, or transmitter release (Dickman, 2008).

In summary, although much attention has been paid to the pore-forming α1 subunits, the calcium channel is a multi-subunit complex whose other subunits can also serve essential functions. The profound synaptic consequences of the loss of the 2δ-3 subunit highlights the need to understand these subunits in their normal cellular milieu in which both physiological and developmental phenotypes may emerge that could not be appreciated in heterologous systems. These in vivo phenotypes should ultimately refine understanding of the calcium channel complex in neuronal development, function, and disease (Dickman, 2008).

TRP, TRPL and cacophony channels mediate Ca(2+) influx and exocytosis in photoreceptors axons in Drosophila

In Drosophila photoreceptors Ca(2+)-permeable channels TRP and TRPL are the targets of phototransduction, occurring in photosensitive microvilli and mediated by a phospholipase C (PLC) pathway. Using a novel Drosophila brain slice preparation, the distribution and physiological properties were studied of TRP and TRPL in the lamina of the visual system. Immunohistochemical images revealed considerable expression in photoreceptors axons at the lamina. Other phototransduction proteins are also present, mainly PLC and protein kinase C, while rhodopsin is absent. The voltage-dependent Ca(2+) channel Cacophony is also present there. Measurements in the lamina with the Ca(2+) fluorescent protein G-CaMP ectopically expressed in photoreceptors, revealed depolarization-induced Ca(2+) increments mediated by Cacophony. Additional Ca(2+) influx depends on TRP and TRPL, apparently functioning as store-operated channels. Single synaptic boutons resolved in the lamina by FM4-64 fluorescence revealed that vesicle exocytosis depends on Cacophony, TRP and TRPL. In the PLC mutant norpA bouton labeling was also impaired, implicating an additional modulation by this enzyme. Internal Ca(2+) also contributes to exocytosis, since this process was reduced after Ca(2+)-store depletion. Therefore, several Ca(2+) pathways participate in photoreceptor neurotransmitter release: one is activated by depolarization and involves Cacophony; this is complemented by internal Ca(2+) release and the activation of TRP and TRPL coupled to Ca(2+) depletion of internal reservoirs. PLC may regulate the last two processes. TRP and TRPL would participate in two different functions in distant cellular regions, where they are opened by different mechanisms. This work sheds new light on the mechanism of neurotransmitter release in tonic synapses of non-spiking neurons (Astorga, 2012).

Light transduction in Drosophila occurs in retinal microvillar arrangements running along the photoreceptor soma, termed rhabdomere (see Drosophila visual system and brain slices). The axon of this non-spiking neuron releases histamine in a tonic manner. It presents a T-bar ribbon synapse, a particular structure of the active zones specialized for fast and sustained multivesicular neurotransmitter release in response to graded membrane depolarizations. R1-R6 photoreceptors make multiple axo-axonic synaptic contacts with large monopolar (LI-L3) and amacrine cells in the lamina. Cell somata are located in the outermost part of this neuropile, leading to a particular situation where axonal arrays (named cartridges) are the predominant components of the lamina. The axons of centrifugal medullar neurons (C2-C3), a T-shaped centripetal neuron (T1) and a wide field tangential neuron (Tan) are also found in the lamina. In the rhabdomere, photon absorption triggers rhodopsin isomerization into an active state which, upon interaction with a Gq-protein, activates phospholipase C (PLCβ4). This enzyme, encoded by norpA, hydrolyses phosphatidylinositol biphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG). This signaling cascade has been widely implicated in the activation of TRP and TRPL. Although the mechanism of channel gating remains undetermined, there is evidence that under experimental conditions, DAG, polyunsaturated fatty acids (PUFAs), PIP2 and protons are involved in opening TRP and TRPL, whereas IP3 receptor does not. Interestingly, TRP and TRPL expressed in heterologous systems are activated by Ca2+ depletion of the endoplasmic reticulum (ER). This study confirmed the presence of TRP in the lamina, where TRPL is also expressed. For the first time, evidence is provided that these channels are implicated in neurotransmitter release in the lamina, where they apparently allow Ca2+ influx via a store-operated channel (SOC) mechanism and could also be regulated by a PLC-mediated cascade. Furthermore, it was shown that the voltage-dependent Ca2+ channel Cacophony, the only fly homologue of vertebrate N-, P/Q- and R-type, is present in the lamina where it plays an important role in photoreceptor synaptic transmission, probably as a first step in a complex cascade involving both intracellular and extracellular Ca2+ signalling (Astorga, 2012).

TRP and TRPL are the targets of Drosophila phototransduction in the rhabdomere, gated by an as yet undetermined PLC-dependent mechanism independent of internal membrane systems, which are absent in the microvilli. This study provides the first evidence that both channels additionally participate in exocytosis in photoreceptor synaptic terminals, where they can be activated by depletion of Ca2+ stores. It is also demonstrated that the voltage-dependent Ca2+ channel, Cacophony, plays a critical role in exocytosis (Astorga, 2012).

This study confirmed that, in addition to the rhabdomere, TRP localizes to the lamina and the medulla. Additionally, TRPL was found in these two neuropiles, where photoreceptors synapse with secondary neurons. The lamina, where most photoreceptors make synaptic connections into well-defined structures, was studied (Astorga, 2012).

A Drosophila slice preparation suitable for immunohistochemistry and functional experiments in the lamina was studied. In addition to TRP and TRPL, PLC and PKC exhibited high expression levels, while Gq and INAD were scarce and rhodopsin was absent. The four former proteins colocalized with ectopically expressed GFP, used as photoreceptor marker, whereas Gq and INAD colocalization with GFP was low. While TRP, TRPL and PLC were not restricted to photoreceptors, the relevant conclusion is that their presence in photoreceptors axons in the lamina suggests a participation in presynaptic events (Astorga, 2012).

The prominent cacophony immunostaining in the lamina is relevant. This Ca2+ channel is involved in synaptic transmission in Drosophila neuromuscular junction, brain and retina, suggesting a role in synaptic transmission in the lamina. A role of cacophony in photoreceptor synaptic transmission is supported by the observation that inhibition of this channel by PLTX-II affected bouton labeling. Although the possibility that PLTX-II could also affect other Ca2+ channels cannot be ruled out, the role of cacophony in vesicle release was further strengthened by the substantial reduction in FM4-64 fluorescence in the thermosensitive cacophony mutant cacTS at non-permissive temperature. In agreement with this, a mutation in the 2δ-3 gene encoding a cacophony subunit abolishes the ERG 'on' transient. In contrast depolarization-induced G-CaMP Ca2+ fluorescence changes in the photoreceptors were significantly decremented by PLTX-II, providing additional evidence involving cacophony in the synaptic events (Astorga, 2012).

The observations that TRP and TRPL are also in the photoreceptors axons and are considerably Ca2+-permeable (PCa:PNa ~100:1 and ~4:1, respectively) suggested a synaptic role. Accordingly, vesicle release was drastically impaired in the double mutant. Opening a Ca2+ pathway with the ionophore induced exocytosis in this mutant, an observation that opposes to a generalized degeneration of synaptic machinery. This evidence shows that TRP and TRPL are involved in exocytosis. Only one of these channels was sufficient for sustaining exocytosis (Astorga, 2012).

FM4-64 is presumably incorporated by all lamina neurons and therefore not only photoreceptor boutons should be labeled. However, it is expected that the dramatic changes in release observed include photoreceptor terminals, which represent the most numerous synaptic contacts in the lamina. Altogether, these results support the participation of TRP, TRPL and cacophony in synaptic transmission in photoreceptor terminals (Astorga, 2012).

What is PLC doing in photoreceptors synaptic terminals? Depolarization-induced exocytosis was markedly reduced in norpA mutant, suggesting a role of PLC in neurotransmitter release. An obvious possibility is that it mediates TRP/TRPL activation. In principle, PLC may act by either DAG or IP3. PUFAs can activate the light-dependent channels when added to intact ommatidia, as well as to excised rhabdomeric membrane patches, in which DAG can do the same. Thus, it is conceivable that these lipids may also activate TRP/TRPL channels in the lamina. Nevertheless, there is no evidence that PUFAs are generated in these photoreceptors (Astorga, 2012).

How is PLC activated? In Drosophila photoreceptors, a level of PLC activity has been observed both in vitro and in vivo. This basal activity is probably a property of the PLC molecule itself, as it is not affected by mutation of Gq-protein. In addition, a positive modulation of PLC activity by micromolar Ca2+ has been reported in Drosophila head membranes. Therefore, basal PLC activity could be boosted by Ca2+ influx through cacophony (and additional Ca2+ pathways described in this study) during depolarization-induced vesicle exocytosis, representing a feed-forward mechanism in this graded synapse. Alternatively, PLC activation may be a consequence of a direct activation of Gq by depolarization, as reported in other insects. In contrast, the substantial PKC expression in the terminals suggests that it may down-regulate PLC, as in the rhabdomere (Astorga, 2012).

Calcium reservoirs appear to be involved in exocytosis, since inhibition of SERCA with Thg deeply affected vesicle release. Moreover, exposure of sercaTS to the non-permissive temperature considerably decreased bouton labeling compared to permissive temperature, and this study shows that this decrease cannot be explained exclusively by a temperature effect. These results strongly implicate ER Ca2+ release in photoreceptors exocytosis (Astorga, 2012).

The robust Ca2+ signals in the lamina after Ca2+ depletion implicated TRP/TRPL, as it was absent in trpl;trp animals. This supports the function of TRP/TRPL as SOCs in the synaptic terminals, allowing Ca2+ influx. This mechanism drives exocytosis, as indicated by the Ca2+-depletion protocol, where bouton labeling was significant. Interestingly, TRP and TRPL function as SOCs in heterologous expression systems, but not in the rhabdomere (Astorga, 2012).

Mammalian homologues of Drosophila TRP, TRPC1, 2, 4 and 6, are proposed to function as SOCs in different cell types. Moreover, TRPC1 operating as SOC regulates Ca2+ influx related to neurotransmission in rods and cones. The Drosophila genome has one gene encoding STIMh, an ER Ca2+ sensor protein that forms functional SOCs in association with TRPC1. It remains to be determined whether TRP/TRPL could form equivalent presynaptic macromolecular complexes in photoreceptors (Astorga, 2012).

This study showed that it is improbable that in the Ca2+-depletion experiments TRPL/TRP opening could be induced by a PLC-dependent mechanism mediated by phospholipase activation by a cytoplasmic Ca2+ increase due to altered reticular release/uptake balance during Thg treatment. In these experiments PLC contribution to exocytosis was possibly by-passed. In normal conditions, this enzyme may elicit Ca2+ elevation in the synaptic terminals by DAG-mediated activation of TRP/TRPL and/or by inducing Ca2+ release (Astorga, 2012).

Photoreceptors synaptic transmission must accurately follow the fast photoresponses generated in the rhabdomere. As graded synapses support rapid changes in neurotransmitter release, they should undergo fast variations in internal free Ca2+. Small and fast Ca2+ increments induce correspondent changes in release, something that would be implausible if a threshold were involved, as in non-graded synapses (Astorga, 2012).

Besides cacophony contribution to exocytosis, the presence of the ryanodine receptor (RyR) in the lamina suggests the participation of Ca2+-induced Ca2+ release (CICR), but direct evidence for this is lacking. CICR regulates exocytosis in rods allowing high rates of neurotransmitter release. A reasonable expectation is that Drosophila photoreceptors use all available Ca2+ pathways (cacophony; TRP/TRPL; the IP3 receptor, IP3R and RyR) to satisfy the synaptic demands required by their extremely fast photoresponse. It has been speculated that the IP3R might reinforce transmitter release, but no direct evidence for it has been shown. This possibility is supported by the current results implicating PLC. Moreover, the observation that Ca2+ from the ER contributes to depolarization-induced exocytosis strengthens the possibility of internal release via IP3R and/or RyR (Astorga, 2012).

Bouton labeling experiments were conducted under prolonged depolarization, implying that vesicle exocytosis was at steady-state. Thg experiments under such conditions show that released Ca2+ plays an essential role in neurotransmission. In tonic synapses, this mechanism may be crucial to sustain synaptic transmission for extensive periods of time (Astorga, 2012).

The following model is proposed for the synaptic events at the axon terminals (see Model for photoreceptor synaptic events in the lamina): the receptor potential activates cacophony in the axon, allowing its propagation towards the axonal terminal, where Ca2+ enters through cacophony inducing vesicle release, perhaps enhanced by CICR. Additionally, PLC activated by an unknown mechanism which may be Ca2+ itself or depolarization, generates IP3, triggering Ca2+ release through IP3Rs. ER Ca2+ depletion in turn opens TRP/TRPL by a SOC mechanism, incrementing the Ca2+ supply. These channels may also be opened by lipid and pH changes resulting of PLC activity. This multi-source transient Ca2+ increment guarantees efficient, rapid and sustained neurotransmitter release. After depolarization, resting Ca2+ levels would be restored by extrusion by the Na+/Ca2+ exchanger and uptake by the ER (Astorga, 2012).

It is important to integrate the data into a plausible working model that could be helpful for designing further experiments. Although the model accounts for the data, it is by no means the only possible one. Accordingly, some aspects of it may be interpreted differently or given a different weight. For example, the relative contributions of cacophony, CICR, IP3-induced Ca2+ depletion and TRP/TRPL to presynaptic Ca2+ for vesicle release can vary widely. Also, the activation of TRP/TRPL may rely on ER depletion and/or lipids associated to PLC activity. It may be thought that the Ca2+ influx through cacophony should be sufficient to account for exocytosis, making Ca2+ release redundant and rather unnecessary. However, in this graded synapse the level of cacophony activation will follow the graded depolarization. The amplitude attained by the receptor potential are most likely within a small voltage range above the threshold for cacophony activation (-20 or -40 mV), inconsistent with a massive cacophony-dependent Ca2+ influx. Therefore, additional Ca2+ sources amplifying this initial signal are likely to be required for light-induced synaptic transmission (Astorga, 2012).

This study has provide novel evidence for TRP/TRPL function in Drosophila photoreceptors. For the first time, it was shown that these channels have dual roles in separate regions of the same cell, namely the rhabdomere and the synapse, apparently involving different mechanisms. More generally, the observations reported herein shed light on the mechanism controlling presynaptic events in graded synapses (Astorga, 2012).

Presynaptic DLG regulates synaptic function through the localization of voltage-activated Ca(2+) channels

The DLG-MAGUK subfamily of proteins plays a role on the recycling and clustering of glutamate receptors (GLUR) at the postsynaptic density. discs-large1 (dlg) is the only DLG-MAGUK gene in Drosophila and originates two main products, DLGA and DLGS97 which differ by the presence of an L27 domain. Combining electrophysiology, immunostaining and genetic manipulation at the pre and postsynaptic compartments, this study examined the DLG contribution to the basal synaptic-function at the Drosophila larval neuromuscular junction. The results reveal a specific function of DLGS97 in the regulation of the size of GLUR fields and their subunit composition. Strikingly the absence of any of DLG proteins at the presynaptic terminal disrupts the clustering and localization of the calcium channel DmCa1A subunit (Cacophony), decreases the action potential-evoked release probability and alters short-term plasticity. These results show for the first time a crucial role of DLG proteins in the presynaptic function in vivo (Astorga, 2016).

dlg1 is the only gene of the DLG-MAGUK subfamily in Drosophila. Similar to vertebrate genes, two forms of the gene are expressed as the result of two transcription start sites. DLGA (α form) and DLGS97 (β form) are distinguished by the inclusion of an L27 domain in beta forms located in the amino terminus of the protein. In vertebrates DLG4/PSD95 is predominantly expressed as α form while DLG1/SAP97 is mainly expressed as β form. DLGA is expressed in epithelial tissues, somatic muscle and neurons; in turn, DLGS97 is not expressed in the epithelial tissue. In the larval neuromuscular junction (NMJ), a glutamatergic synapse, both dlg products are expressed pre and postsynaptically. Hypomorphic dlg alleles display underdeveloped subsynaptic reticulum, bigger glutamate receptors fields and an increased size of synaptic boutons, active zones and vesicles. Additionally to these morphological defects, altered synaptic responses such as increased excitatory junction currents (EJC) and increased amplitude of miniature excitatory junction potentials have been observed. The strong morphological defects make difficult to distinguish developmental defects from the role of DLGs in the basal function of the mature synapse. Previously studies have reported form-specific null mutant strains for DLGA (dlgA40.2) and DLGS97, (dlgS975). These mutants do not show the gross morphological defects observed in hypomorphic mutants, although still show functional synaptic defects, supporting a role of DLG proteins in the mature synaptic function (Astorga, 2016).

Combining genetic, electrophysiology and immunostaining techniques this study dissected the role of DLG proteins at the pre and postsynaptic compartments. The results show the specific requirement of postsynaptic DLGS97 for normal glutamate receptor (GLUR) distribution. In turn, both DLG proteins increase the release probability by promoting voltage-dependent Ca2+ channel localization. The results demonstrate for the first time a relevant role to DLG proteins in the presynaptic function contributing to Ca2+ mediated short-term plasticity and probability of release (Astorga, 2016).

Flies carrying the severe hypomorph dlg1 mutant allele, dlgXI-2 and the isoform specific dlgS97 null mutant displayed increased amplitude of the spontaneous excitatory postsynaptic (junctional) potential (mEJP) without changes in frequency. In addition all mutants displayed a decreased quantal content as measured by evoked post-synaptic potentials. The specific defects behind these results were explored. To characterize the synaptic transmission in WT and dlg mutants, post synaptic currents were recorded in HL3.1 solution on muscles 6 or 7 of third instar male larvae of the various genotypes. Recordings of spontaneous excitatory postsynaptic currents (mEJC) were obtained after blocking the voltage activated sodium channels. Thereafter, histogram distributions were constructed with the amplitudes of the miniature events and the quantal size was estimated by the peak value obtained adjusting a Log-Normal distribution in each genotype. It is worth to emphasize that finding a phenotype on dlgA or dlgS97 mutants means that DLGA or DLGS97 proteins by themselves cannot replace DLG function (Astorga, 2016).

The average amplitude of spontaneous postsynaptic potentials were compared and, supporting previous results, it was found that the average amplitude of the mEJC of the mutants dlgXI-2 (0.99 ± 0.05 nA) and dlgS97 (0.98 ± 0.03 nA) were significantly larger compared to the average amplitudes of the mEJC of Canton-S strain used as WT control (0.81 ± 0.04 nA) and of dlgA (0.78 ± 0.02 nA) specific mutant. The same result was obtained comparing the quantal size. None of the mutants showed a significant change compared to the WT in the frequency of the mEJC. As an additional control, all mutants were recorded over a deficiency covering the dlg gene, finding similar results. These findings are in accordance with the idea that DLGS97 protein and not DLGA is necessary for the quantal size determination (Astorga, 2016).

Bigger quantal size could be of pre or postsynaptic origin as the result of increased neurotransmitter (NT) content in vesicles or increased glutamate receptor field's size respectively. First, to determine the pre or post-synaptic origin of this phenotype, a UAS-dsRNA construct that targets all dlg products, was expressed under the control of the motoneuron promoter OK6-GAL4 or the muscle promoter C57-GAL4. As expected for a post-synaptic defect, the increased quantal size observed in dlgS97 mutants was mimicked only by the decrease of DLG in the muscle. The specific role of DLGS97 in the muscle is supported by the rescue of the dlgS97 mutant phenotype only by the expression of DLGS97 in the muscle and not in the motor neuron. The effect of GAL4 expression was examined in the mutant background in all experiments; neither of the GAL4 lines without the specific UAS constructs changed the phenotype of the mutants. Again, none of the genotypes studied displayed differences with the WT in the frequency of the minis (Astorga, 2016).

Changes in quantal size of postsynaptic origin could be due to higher number of post-synaptic receptors and/or a different composition of the postsynaptic receptors. An increase in the size of glutamate receptors fields has been described in dlg hypomorphic alleles including dlgXI-2 mutants. Therefore, the size of the glutamate receptor fields was compared among the mutants and with WT, and also the active zones were measured using antibodies for the active zone protein Bruchpilot. Consistently with previous results bigger glutamate receptors fields were found compared to WT only in dlgXI-2 and dlgS97 mutants but not in dlgA mutants. Surprisingly, an increased number of active zones per bouton was also found in all mutants, a phenotype usually associated with an increase in the frequency of minis that were not observe. In addition, an increased active zone area was found in dlgA and dlgXI-2 mutants (Astorga, 2016).

As expected for a postsynaptic defect, the bigger size of the glutamate fields in dlgS97 mutants was rescued by the expression of DLGS97 in the muscle but not by its expression in the motor neuron. These results confirm that DLGS97, but not DLGA is responsible for the regulation of the size of the receptors fields in the muscle (Astorga, 2016).

The strict requirement of DLGS97 in the regulation of the size of GLUR fields supports results that have involved other DLGS97 interacting proteins in the regulation of the size of the glutamate receptors fields. METRO, an MPP-like MAGUK protein, has been shown to form a complex with DLGS97 and LIN-7 through the L27 domains present in each of the three proteins. metro mutants display decreased DLGS97 at the synapse and larger GLUR receptors fields than WT, even bigger than dlgS97 mutants. METRO and DLGS97 depend on each other for their stability on the synapse, thus, in dlgS97 mutants, METRO and dLIN-7 are highly reduced at the synapse. The similar post-synaptic phenotype of metro and dlgS97 and the reported interaction between these two proteins suggests the proposal that the increase size of GLUR fields is consequence of the loss of METRO due to the loss of DLGS97 protein (Astorga, 2016).

As stated before, changes in quantal size of postsynaptic origin can also reflect a different composition of the receptors. Drosophila NMJ GLUR receptors are tetramers composed by obligatory subunits and two alternative subunits, GLURIIA and GLURIIB. Receptors composed by one of these two subunits differ in their kinetics; GLURIIB receptors desensitizes faster than GLURIIA receptors. Thus, the kinetic of the spontaneous currents (mEJCs), is associated to the relative abundance of these two types of receptors in the GLUR fields. It has been shown that the abundance of GLURIIB but not of GLURIIA in the synapse is associated with the expression of dlg. To analyze if dlg mutants display a change in the composition of the subunits abundance relative to the control, the kinetics of the mEJCs were studied. Kinetics analyses of the mEJCs revealed that only dlgS97 and the double mutant display a slower kinetic in the off response, which is compatible with a different composition of the glutamate receptors fields regarding the proportion between GLURIIA and B receptors. The value of tau also increased in larvae expressing dsRNA-dlg in the muscle, but not by its expression in the motor neuron. Finally tau-off values recovered the WT value only with the expression of DLGS97 in the muscle. As slower mEJCs were observed, the results suggest an increase in the ratio of GLURIIA/GLURIIB. It is known that receptors containing the GLURIIA subunit display bigger conductance and slower inactivation kinetics than receptors containing the GLURIIB subunit. Thus, synapses with post-synaptic receptors fields containing proportionally less GLURIIB subunits would display bigger and slower mEJCs similar to the phenotype observed in dlgS97 mutants. To confirm this hypothesis, the abundance of GLURIIA and GLURIIB receptors was evaluated by immunofluorescence in the NMJ of WT and dlg mutant larvae. The immunofluorescence that allowed the detection and quantification of GLURIII and GLURIIB fields was performed with paraformaldehyde (PFA) fixative. However, the immunofluorescence to detect GLURIIA receptors only works fixating the tissue with Bouin reagent. Thus, in order to be able to compare between these two fixations, the size of the GLUR fields was normalized by the HRP staining that labels the whole presynaptic bouton. First, as a control, GLURIIA and GLURIII were double stained in the same larvae. The results show that using PFA fixative, GLURIII fields display bigger size only in dlgS97 mutants and not in dlgA mutants. Even more, as predicted from the kinetic data, only dlgS97 and not dlgA mutants display bigger GLURIIA fields while there are not difference in the size of GLURIIB fields between WT and the mutants. Additionally the results show no difference in the number of GLURIIA or GLURIIB clusters between WT and dlg mutants. Immunohistochemical results confirm the prediction from the electrophysiological data revealing that in dlgS97 mutants, GLURIIA subunits are proportionally more abundant in GLUR fields than in control larvae. In conclusion, the results show that dlgS97 mutants display larger quanta and mEJCs with slower kinetic establishing its participation in the regulation of the size of GLUR fields where the increased size is obtained mainly through the recruitment of receptors containing GLURIIA subunits. As a similar result was obtained in another study that observed that the loss of GLURIIB receptors in the NMJ of dlgXI-2 mutant embryos, these observation suggest that either of the two DLG proteins are necessary for the localization of GLURIIB in the synapse but only DLGS97 is actively limiting the size of the clusters by regulating the number of GLURIIA receptors (Astorga, 2016).

Taking into account previous reports that show the regulation of the synaptic localization of DLG by CAMKII, the regulation of the subunit composition by CAMKII and these results, a mechanism is proposed by which, after a strong activation of CAMKII, the phosphorylation of DLGS97 would detach it from the synapse allowing the increase of the size of the GLUR fields by the recruitment of GLURIIA over GLURIIB. These changes should increase the synaptic response by two different mechanisms (Astorga, 2016).

To determine if DLG proteins modulate the presynaptic release probability, excitatory junction currents (EJC) were recorded in the muscle by stimulating the nerve at 0.5 Hz in low extracellular Ca2+ (0.2 mM), both conditions to avoid synaptic depression. For all mutant genotypes the average peak amplitude and quantal content (EJC amplitude/quantal size) of the evoked responses were significantly smaller than WT. In congruence with previous results, the lower amplitude of the current response is accompanied by a decrease in the quantal content. Taking into account the results on the size of the GLUR fields in the mutant's muscles, these results are compatible with a reduction of the neurotransmitter release in dlg mutants. A decreased neurotransmitter release could be associated with a decreased number of release sites in the boutons. However, the number of active zones per bouton is increased in all dlg mutants with bigger active zones in dlgXI-2 and dlgA mutants (Astorga, 2016).

The decrease in the evoked response could be a consequence of the absence of the specific form of DLG in the postsynaptic side, transmitted by unknown mechanisms or, alternatively, it could be the result of an effect of DLG on the probability of release. In order to explore where this phenotype originates (pre or post-synaptically) DLG levels were downregulated by expressing dsRNA against all forms of dlg. Compatible with a presynaptic defect, the expression of UAS-dsRNA-dlg presynaptically decreases the amplitude of the evoked response while the same construct expressed postsynaptically using C57 promoter did not changed the amplitude of the EJCs. The presynaptic expression of the dsRNA-dlg also mimics the reduction in quantal content of the mutants, displaying a severe reduction in this parameter. On the other hand, the postsynaptic expression of the dsRNA-dlg associates to a moderate but significant decrease on the quantal content, as expected from the effect already reported of the postsynaptic dsRNA-dlg on the quantal size and the lack of effect on the amplitude of the EJCs. The presynaptic effect of DLG is supported further by the rescue experiments. Thus, the amplitude of the evoked response and the quantal content in dlgA40.2 mutant is completely rescued by the selective expression of DLGA in the presynaptic compartment but not by its expression in the postsynaptic compartment. The pre-synaptic expression of DLGS97 improves the synaptic function increasing the average size of the EJCs such that the difference between the WT and the presynaptic-rescue is not significant, suggesting a complete rescue. However, the average EJC in the presynaptic rescue is not different either from the control mutant animal, which is interpreted as the rescue not being complete and thus the term partial rescue is used. DLGS97 does not, however rescued at al the quantal content. This is explained because although the amplitude of the current increased, the quantal size remains unchanged by the presynaptic expression of DLGS97. In consequence the quantal content does not increase as much as the current. On the other hand, the postsynaptic expression did not increase the amplitude of the evoked current. However, since it does rescue the quantal size the quantal content augmented enough to be different from the mutant control. Notably, DLGA expressed presynaptically in dlgA40.2mutants not only rescued the EJC amplitude but also the number of active zones per bouton and the size of the active zones. On the other hand DLGS97 expression only partially rescued the increased number of active zones in dlgS975 mutants. These results support a role of DLG proteins in the presynaptic function where DLGA seems to regulate more aspects than DLGS97. Despite the fact that both forms of DLG share most of their protein domains, neither of the two-forms is able to fully rescue the absence of the other, suggesting that both of them participate in a complex. The binding between the SH3 and GUK domains of MAGUK proteins has been described; this interaction (at least in vitro) is able to form intra or intermolecular associations and offers a mechanism by which DLGA and DLGS97 proteins could be associated to recruit proteins to a complex (Astorga, 2016).

Changes in the overall quantal content at these synapses may reveal presynaptic defects. However, genetic background and other independent modification could alter apparent release. To independently scrutinize alteration in the presynaptic release probability two presynaptic properties were examined, the short-term plasticity and the calcium dependency of quantal release (Astorga, 2016).

To explore the EJC phenotype observed in dlg mutants, stimulation paradigms were carried out that allow characterization of aspects of the short term plasticity that are known to depend on presynaptic functionality and give clues about the mechanisms involved in the observed defects. First, the response were studied of the mutants to high frequency stimulation, 150 stimuli at 20 Hz. WT responses at high frequency stimulation show a fast increase in the amplitude of the response that then slows down. The fast initial increase is called facilitation and the second phase with smaller slope is called augmentation. The time constant of the facilitation is believed to reflex the calcium dynamics in the terminal and its slope to be the product of the accumulation of calcium and the consequent calcium dependent increase in the probability of release. The fractional increment in the mutants' responses showed an increased facilitation in all mutants, while an increased augmentation was only significant in dlgA mutants compared to WT. Additionally all mutants showed a trend toward steeper slopes than WT, but only the augmentation slope in dlgA mutants reached statistical significance. These results support that the mutants display a lower probability of release than WT, which could reflect defects in the calcium dynamics or in the response to calcium (Astorga, 2016).

Previous work in dlg mutants did not report defects in short-term plasticity. These works differ from the current one in methodological aspects, mainly that they were carried out in a media with high concentration of magnesium (20 mM) and calcium (1.5 mM). This work was carried out in a media containing low magnesium (4 mM) and calcium (0.2 mM) concentration. It is known that magnesium reduces neurotransmitter release, probably due to partial blockade of VGCC. Additionally, magnesium permeates more than sodium and potassium through GLURs (Astorga, 2016).

To better evaluate the calcium dynamics in the terminal pair pulse (PP) experiments, a well-known paradigm to evaluate presynaptic calcium dynamics, were carried out. In PP, a second depolarization shortly after the first one carried out in low extracellular calcium concentration elicits an increased release of neurotransmitter thought to reflect the increased calcium concentration in the terminal reached after the first stimulus. According to this, and posing as the working hypothesis that DLG affects presynaptic calcium dynamics, a second pulse would be expected to increase the release in a bigger proportion, since the first stimulus did not release much of the ready releasable pool. Conversely, a second stimulus given at high calcium concentration produces a decrease in the release of neurotransmitter, which is considered to originate in the partial depletion of the ready releasable pool at the release sites. Thus, a second pulse at high calcium concentration should elicit a smaller decrease of the release since an inferior entrance of calcium should produce less depletion of the ready releasable pool of vesicles (Astorga, 2016).

Consistently with a decreased calcium entrance, all mutants displayed increased pair pulse facilitation at low calcium concentration and decreased pair pulse depression at high calcium concentration. These results support a defect in the calcium entrance to the terminal as the underlying defect in dlg mutants causing the evoked stimuli defects. To characterize the calcium dependency of the release in the mutants the evoked responses were measured at different calcium concentrations. It can be observed that for all the mutants and at most calcium concentrations, the quantal content of the evoked response is lower than the control. The only exception is seen at 2 mM calcium where the quantal content of the dlgA mutants and the control are not different to each other. However, even at this calcium concentration the quantal content of dlgS97 and the double mutant dlgXI-2 are significantly lower than the control. To get insight about the release process the responses were fit to a Hill equation. This type of fitting better estimate the maximum responses and the EC50, which is masked in the overall release of different backgrounds. This is observed in the graph with the normalized responses by the maximal quantal predicted. The adjusted curves show that mutants reach the theoretical maximal quantal content at higher calcium concentration than the WT and that the EC50 for the mutants is diminished respect to the WT. To confirm the presynaptic origin of the defect in the calcium dependency, the calcium dependency was carried out in the mutant genotypes expressing DLGA or DLGS97 pre or postsynaptically. The quantal content analysis shows that only the presynaptic expression of DLGA in dlgA mutants completely rescued the calcium dependency, in line with previous results that show the importance of DLGA in the presynaptic compartment. On the other hand the presynaptic expression of DLGS97, although it rescued the calcium dependency, failed to rescue the maximal quantal content. Observing the graph with the normalized responses, DLGA as well as DLGS97 both are able to restore the WT calcium dependency. The inability to rescue the maximal quantal content could be explained by the existence of synaptic compensatory mechanisms that allow to counterweigh the bigger quantal size in dlgS97 mutants, which were shown before not to be rescued by the presynaptic expression of DLGS97 (Astorga, 2016).

Facilitation is thought to depend on the resultant of the calcium entrance, calcium release from intracellular stores and the clearance of cytosolic calcium. So, the defects in facilitation observed in the mutants could be due to a decreased calcium entrance but also they could be due to a defect on the clearance of calcium. In a preliminary experiment, the relative changes were measured of the total intracellular calcium concentration in the bouton using the genetically encoded calcium indicator GCamp6f. GCamp6f expressed in control flies (OK6-GAL4/UAS-GCamp6f) respond with a fast and transitory change in the cytoplasmic calcium of the boutons when they are exposed to a local pulse of potassium. The same experimental approach in dlgS97 mutant larvae reveals that the rise of the calcium response is significantly slower than the control; additionally the recovery of the response is also significantly slower. These preliminary experiments suggest a defect in calcium entrance in the mutants but they also support a defect in the extrusion that hint to additional defects. Further experiments are needed to clarify the calcium kinetics involved since these experiments were measuring the bulk of calcium change and in doing this approximation, the nanodomain changes that are known to be the ones that regulate the neurotransmitter release are being lost (Astorga, 2016).

Since the results described above including the calcium dependency of the release as well as the parameters of the short-term plasticity suggest that the calcium entrance to the terminal is impaired, a view that is supported by the preliminary data measuring the cytosolic calcium, the next experiments focused on the calcium entrance. The main calcium entry to the terminal is the voltage gated calcium channel (VGCC) encoded by the Drosophila gene cacophony. Advantage was taken of a UAS-cacophony1-EGFP transgenic fly (CAC-GFP) to study the distribution of the channel in WT and mutant genotypes. CAC-GFP overexpressed in WT background localizes in the synapse in a strictly plasma membrane-associated manner in big clusters closely associated with release sites. However, CAC-GFP overexpressed in dlgS97 or dlgA mutant background displays a significant decrease in the expression accompanied by a more disperse localization with significantly smaller clusters, suggesting that the Cacophony protein might not be properly delivered or anchored to the plasma membrane in dlg mutants (Astorga, 2016).

It was reasoned that if dlg mutants had a defect on calcium entrance, the over expression of calcium channels should rescue at least partially the phenotype. Advantage was taken of the fact that CAC-GFP construct encodes a functional channel, and recordings were taken from control and dlg mutants overexpressing CAC. As expected and supporting a decreased calcium entrance in the mutants, dlgS97 and dlgA mutants that overexpress CAC-GFP display significantly bigger evoked EJCs compared to dlg mutants, without a change of phenotype in the spontaneous currents. Additionally, the over expression of CAC-GFP partially rescued the pair pulse facilitation and the pair pulse depression as well as the calcium curve (Astorga, 2016).

The disrupted localization of CAC could result from the disturbance of a normal direct association to DLG or it could be affected indirectly. To test an immunoprecipitation assay was carried out using flies that express CAC-EGFP in all neurons. Antibodies against GFP were able to precipitate DLG together with Cacophony-GFP, supporting that Cacophony channel is part of the DLG complex in the boutons (Astorga, 2016).

A possible interaction between DLG and voltage-gated calcium channels (VGCCs is) the VGCC auxiliary subunits. The α2δ auxiliary subunit (Straightjacket in Drosophila) increases calcium channel activity and plasma membrane expression of CaV2 α1 subunits and Cacophony. The β auxiliary subunit increases plasma membrane expression of several mammalian VGCC classes. Intriguing β subunits are also MAGUK proteins and they are able to release the VGCC α subunit from the endoplasmic reticulum retention. It may be speculated that DLG through their SH3-GUK domain might be playing the role of the β subunit (Astorga, 2016).

On the other hand, in mammalian cultured neurons it has been proposed that a complex formed by the scaffold proteins LIN-2/CASK, LIN-10/MINT and LIN-7 is involved in the localization of VGCCs at the synapse and that SAP97 forms a complex with CASK. The association of DLGS97 with LIN7 has been reported in the postsynaptic compartment in the Drosophila NMJ. Furthermore, an association between the L27 domain of DLGS97 and the L27 domain of Drosophila CASK has been shown in vitro, however there are no reports of this type of association with DLGA. Another protein involved in the localization of calcium channels in the active zone is RIM. Drosophila rim has been involved in synaptic homeostasis and the modulation of vesicle pools. Surprisingly rim mutants, display low probability of release and altered responses to different calcium concentrations. Recently it was shown that spinophilin mutants display a phenotype with bigger quantal size and GLUR fields size with a higher proportion of GLURIIA subtype of receptors as well as decreased EJCs and decreased pair pulse facilitation. This is a phenotype very similar to the one described here for dlg mutants. The authors in this report did not explore the calcium channels abundance or distribution and the current study did not explore the link of DLG to Neuroligins, Neurexins and Syd. It would be interesting to determine if there is a link between Spinophilin and DLG (Astorga, 2016).

Taken together these results show that dlgS97 is the main isoform responsible for the postsynaptic defects in the dlgXI-2 mutants; which comprise the increase in the size of the receptors fields and the change in the ratio of GLURIIA/GLURIIB. The results as well support a model in which DLG forms a presynaptic complex that includes Cacophony where the absence of either form of DLG leads to defects in the localization of the voltage dependent calcium channel and to a decrease in the entrance of calcium to the bouton; which in turn affect the probability of release and the short-term plasticity in the mutants. The results described in this study highlight the specificity of the function of DLGS97 and DLGA proteins and describe for the first time an in vivo presynaptic role of DLG proteins (Astorga, 2016).

Motor neuron expression of the voltage-gated calcium channel cacophony restores locomotion defects in a Drosophila, TDP-43 loss of function model of ALS

Dysfunction of the RNA-binding protein, TDP-43, is strongly implicated as a causative event in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). TDP-43 is normally found in the nucleus and pathological hallmarks of ALS include the presence of cytoplasmic protein aggregates containing TDP-43 and an associated loss of TDP-43 from the nucleus. Loss of nuclear TDP-43 likely contributes to neurodegeneration. Using Drosophila melanogaster to model TDP-43 loss of function, this study shows that reduced levels of the voltage-gated calcium channel, Cacophony, mediate some of the physiological effects of TDP-43 loss. Null mutations in the Drosophila orthologue of TDP-43, named TBPH, resulted in defective larval locomotion and reduced levels of Cacophony protein in whole animals and at the neuromuscular junction. Restoring the levels of Cacophony in all neurons or selectively in motor neurons rescues these locomotion defects. Using TBPH immunoprecipitation, TBPH was shown to associate with cacophony transcript, indicating that it is likely to be a direct target for TBPH. Loss of TBPH leads to reduced levels of cacophony transcript, possibly due to increased degradation. In addition, TBPH also appears to regulate the inclusion of some alternatively spliced exons of cacophony. If similar effects of cacophony or related calcium channels are found in human ALS patients, these could be targets for the development of pharmacological therapies for ALS (Chang, 2013).


DEVELOPMENTAL BIOLOGY

There are three peaks of cac expression during development. The first peak begins to rise in mid-to-late embryo stages and reaches a peak during the first larval instar. Expression then declines over the remaining larval instars but begins to rise again after pupariation. There is a second peak in midpupal stages and a final peak in late pupae just before adult eclosion. cac RNA is expressed widely in the embryonic nervous system. Intense, dark staining is seen in the dorsal cerebral hemispheres as well as throughout the ventral nerve cord. In addition, lightly stained nerves can be seen extending anteriorly from the CNS toward the region of the antennomaxillary complex at the extreme anterior end of the animal (Smith, 1996).

Active zone localization of presynaptic calcium channels encoded by the cacophony locus of Drosophila

Presynaptic calcium channels play a central role in chemical synaptic transmission by providing the calcium trigger for evoked neurotransmitter release. These voltage-gated calcium channels are composed of a primary structural subunit, alpha1, as well as auxiliary ß and alpha2delta subunits. The cacophony (cac) gene encodes a primary presynaptic calcium channel alpha1 subunit in Drosophila. Transgenic expression of a cac-encoded alpha1 subunit fused with enhanced green fluorescent protein efficiently rescues cac lethal mutations and allows in vivo analysis of calcium channel localization at active zones. The results reported in this study further characterize the primary role of cac-encoded calcium channels in neurotransmitter release. In addition, these studies provide a unique genetic tool for live imaging of functional presynaptic calcium channels in vivo and define a molecular marker for immunolocalization of other presynaptic proteins relative to active zones. These findings are expected to facilitate additional analysis of synaptic development and function in this important model system (Kawasaki, 2004).

To characterize the presynaptic localization of cac-encoded calcium channels, confocal immunofluorescence imaging was used at larval neuromuscular synapses to examine the distribution of EGFP-tagged 1 subunits. Double labeling using a monoclonal anti-GFP antibody and a neuronal plasma membrane-specific antibody, anti-HRP, was performed in a cac lethal mutant rescued by neural expression of CAC1-EGFP. These experiments confirm the presence of calcium channel puncta within presynaptic boutons of rescued larvae but not wild-type controls lacking the transgene. The likely possibility that these CAC1-EGFP puncta correspond to active zones was examined by double labeling with an antibody against DPAK, a well characterized marker for the postsynaptic densities closely apposed to presynaptic active zones. These experiments revealed extensive colocalization of CAC1-EGFP and DPAK, confirming active zone localization of cac-encoded calcium channel 1 subunits. Consistent with previous studies defining the three-dimensional ultrastructure of larval neuromuscular boutons and the surrounding postsynaptic membrane, active zones exhibited an approximately even distribution over the entire surface of a bouton. This was revealed in a series of optical sections through the z-axis. This study therefore confirms that transgenic expression of a EGFP-tagged calcium channel 1 subunit is targeted to active zones and also retains its function in neurotransmitter release (Kawasaki, 2004).

The role of central parts of the brain in the control of sound production during courtship in Drosophila melanogaster

The question of the roles of the two main parts of the insect brain, the mushroom bodies and the central complex, in controlling motor coordination and triggering a variety of behavioral programs, including sound production, remains controversial. With the aim of improving understanding of this question, the parameters of songs used by five-day-old males during courtship for fertilized wild-type females (Canton-S, C-S) were studied over 5-min periods at 25°C; males were of two wild-type Drosophila melanogaster lines (Berlin and C-S). Berlin males lacking mushroom bodies because of treatment with hydroxyurea during development (chemical removal of the mushroom bodies) were used, along with two mutants with defects in the mushroom bodies (mbm1 and mud1), two mutants with defects in the central complex (ccbKS127 and cexKS181), and mutant cxbN71 with defects in both the mushroom bodies and the central complex. The experiments reported here show that courtship songs in males lacking mushroom bodies were virtually identical to those of wild-type males. The main parameters of pulsatile song in mutants mbm1 and mud1 (interpulse interval and train duration) were insignificantly different from those of the songs of wild-type flies, though the stability of the pulse oscillator was the same. Flies of these lines were no different from wild-type flies in terms of courtship success (percentage of copulating pairs in 10-min tests). Conversely, the songs of mutants with defects in the central complex differed from those of wild-type males: (1) there was degradation of the stability of the pulse oscillator and interpulse intervals were very variable; (2) pulses were often significantly longer and appeared multicyclic, as in the well-known cacophony mutant, while the mean train duration was significantly shorter. Males of the line cexKS181 usually courted very intensely, though abnormal sounds were generally emitted. Mutants cexKS181 and ccbKS127 were significantly less successful in courtship than wild-type flies. These data show that the central complex appears to play a very important role in controlling song, while the mushroom bodies are not related to this function (Popov, 2003).


EFFECTS OF MUTATION

Phenogenetic analysis reveals that cac specifies an essential gene product that is involved in the operation of the visual system and thoracic neuromuscular systems as well as being required for specific behavioral and physiological functions. The cac transcription unit maps to l(1)L13-associated chromosomal lesions, and the cacS and cacH18 mutants have sequence polymorphisms that cause significant changes in the predicted Cac protein (Smith, 1998).

It is still conceivable that this nbA (H18) mutation and the cac one (S) define two different functions (or even genes); but the fact that both are mutated in the same ORF, which encodes a protein that can be considered highly relevant to both song-related and visual-system functions, increases the weight of evidence in favor of these two different kinds of mutants having identified the same molecular-genetic entity. Furthermore, the phenotypic interrelatedness of cac and nbA-defined functions has been boosted by elements of the current results. Thus, several genetic combinations involving cacP73 (originally isolated on the basis of visual defects) give reduced courtship-song pulse amplitude, and several genotypes involving the cacS mutation (identified initially with respect to an anomalous courtship song) have subtle ERG defects. The coupling of courtship-song and visual phenotypes, previously thought to be strictly separated between mutually exclusive classes of these interacting mutants, further suggests that all these mutations are allelic. It is concluded that the cac gene encodes the Dmca1A calcium channel a1 subunit protein, that this protein is an important factor mediating behaviorally-related functions of excitable cells, and that mutations in this gene are responsible for the various phenotypes of cac mutants (Smith, 1998)

The cacS mutation does not lead to pathological abnormalities in the courtship song, but causes quantitative changes in elements of the song, leaving these acoustical signals nicely patterned. The cacS mutation causes analogous (meaning nonpathological) changes in visual system physiology: it reduces the amplitude of the ERG transients (but does not eliminate them) and causes a novel but low amplitude aberration in the ERG. Also, the cacS/cacL-6 heteroallelic combination has defective Y-tube phototaxis. The cacS mutation is in Dmca1A exon 19; this exon seems not to be subject to alternative splicing, so it would be expected to be included in all products expressed from the cac locus. These results imply that the cacS mutation damages Dmca1A channel functions common to physiological processes underlying the generation of courtship song and of a normal ERG, but not sufficiently to disrupt most visually-mediated behaviors (Smith, 1998).

Mutations in ion-channel genes have often been associated with temperature-sensitive phenotypes such as paralysis. In this context, an additional cacophony phenotype, temperature-sensitive convulsions, was recently discovered to be a feature of the 'song allele' cacS but not the 'vision alleles' cacH18 and cacEE171 (Peixoto, 1998). This raised the question of whether other temperature-sensitive ion-channel mutants would sing abnormally. Indeed, mutations at the slowpoke (slo) locus in D. melanogaster, which encodes a calcium-activated potassium channel cause severe song defects. Calcium and potassium currents are involved in the function of pacemaker cells. A simple, speculative scheme is presented for how the products of cac and slo could work together to form a pacemaker that would underlie the tone-pulse component of Drosophila's courtship song (Smith, 1998).

The lovesongs of these flies are thought to be involved in species recognition as well as stimulation of females to copulate and hence are hypothesized to be a component of prezygotic isolation during speciation. It is intriguing that changes in courtship song caused by the cacS mutation -- specifically affecting the number of cycles per pulse, the interpulse interval, and the pulse amplitude -- are similar to the differences in song between several closely related species. The quasi-separability of cac phenotypes implies that it might be possible to 'tune' the courtship song with relatively small evolutionary changes in this one gene. It will be interesting to examine the homologous channel protein from species known to differ in their song components for differences that might contribute to evolutionary divergences of these species' signatures. A gene such as slo may harbor song-related interspecific variations as well (Smith, 1998).

A phenylalanine (analogous to the cacS-mutated residue) in the transmembrane domain of the minK subunit of the IsK potassium channel has been subjected to in vitro mutagenesis (Wilson, 1994). This protein change (F to C at residue 57) led to an IsK potassium current that was normal in terms of half-maximal activation voltage and effect on membrane potential; that is, indistinguishable in these parameters from IsK currents stemming from the expression of molecularly unaltered transcripts. However, the evolutionary and structural divergence of the minK protein makes it difficult to extrapolate these results to the Dmca1A calcium channel. These results draw attention to the role of this transmembrane region (IIIS6) (and in particular to the cac-defined phenylalanine within it, which is now accessible for electrophysiological bioassay via analysis of calcium currents in cacS mutant flies) in the function of calcium channels in general (Smith, 1998).

The cacH18 mutant has defects in most visual phenotypes assayed but no defects in courtship song, and it carries a mutation that creates a stop codon within the alternative exon I/IIa, indicating that Dmca1A isoforms containing at least this variant motif are necessary for and specific to normal visual function. In this context 'variant' means this exon encodes a stretch of amino acids that is hypothesized not to be able to interact with the typical ß subunit (Smith, 1998).

What might be the specific etiology of the vision defects in cac mutants? The mutations could cause developmental or degenerative defects in the optic ganglia or in retinula cells; indeed, degeneration has been reported for many mutations that affect visual transduction. The presence was verified of a deep pseudopupil, an indicator of intact eye structure, during preparation for ERG recording in all genotypes tested. However, a subtle morphological or degenerative defect in the genotypes examined in this report cannot be ruled out (Smith, 1998).

The absence or reduced amplitude of ERG transients in cac mutants, even in genotypes with robust (if aberrant) light coincident receptor potentials, indicates a probable defect in transmission from the retinula cells to postsynaptic cells in the lamina. Synaptic neurotransmitter secretion is known to be dependent on calcium influx mediated by voltage-dependent calcium channels. While the defect in transmission could be pre- or postsynaptic, an attractive hypothesis is that a class of synapse-specific Dmca1A channel isoforms is affected by these mutations (Smith, 1998).

That the light coincident receptor potential in the most severely affected cac mutants is almost completely eliminated implies that such a genotype (visual mutation heterozygous with a lethal) causes an almost total failure of photoreceptor excitation -- with the proviso that an increased stimulus intensity might have coaxed a small degree of depolarization from these mutant types. In Drosophila and other invertebrates, photoexcitation of rhodopsin molecules leads to G-protein-mediated activation of phospholipase-C and generation of inositol phosphates, followed by a light-activated inward current carried predominantly by Ca2+ and thought to be mediated by cation channels formed by the transient receptor potential (TRP) and TRP-like (TRPL) proteins. Ca2+-CaM-regulated Ca2+ release from ryanodine-sensitive stores is believed to be involved in generation of the light-activated current. Inactivation of phototransduction appears to require an influx of extracellular Ca2+, hypothetically involving calcium-regulated phosphorylation mechanisms. Adaptation, or variation of the gain of phototransduction in varying light levels, is controlled by light-dependent changes in intracellular calcium levels, likely mediated by an eye-specific protein kinase C encoded by the inaC gene. Given the regulatory role of calcium in phototransduction, it seems that aberrant calcium regulation due to defective Dmca1A calcium channel function could disrupt phototransduction. Indeed, the ERG of trp mutants exhibit a transient near-normal light coincident receptor potential followed by a rapid decay, and intense light stimulation has been shown to completely but reversibly inactivate trp-mutant photoreceptors; these phenotypes have been suggested to be due to exhaustion of intracellular Ca2+ stores secondary to the defect in TRP-mediated calcium influx. Regardless of etiology, it is clear that defects in proteins involved in calcium influx can have profound effects on phototransduction (Smith, 1998).

Voltage-activated Dmca1A-encoded calcium currents should now be considered as contributing under physiological conditions to the predominantly calcium-mediated light-activated current, subsequent to the light-dependent initiation of retinula depolarization, which is mediated by TRP and TRPL currents. The current phenogenetic and molecular results suggest that further experiments -- in particular, the analysis of Dmca1A and other ion currents in photoreceptors of cac-mutant flies -- could decipher the contributions of Dmca1A calcium currents to membrane excitability or calcium regulation of phototransduction (Smith, 1998).

The multiple phenotypes, complicated genetic interactions, and extensive intragenic complementation imply that cac mutations affect Dmca1A channel functions that are at least partially separable. Consideration of complementation patterns between viable and lethal cac alleles supports this idea. A deletion that removes the cac locus fails to complement all viable alleles for all phenotypes assayed. The cacL-6 allele has allele-specific effects on light coincident receptor potential amplitude, in that it dramatically worsens the amplitude defect of cacH18 and cacEE171 but complements the amplitude defect of cacP73. The cacL-6 allele also partially complements the Y-tube phototaxis defect of cacP73, and its heteroallelic combination with cacEE171 caused blindness in the Y-tube phototaxis assay rather than negative phototaxis. The cacL-10 allele complements cacH18 for phototaxis only. The effects of the cacL-13 allele are identical to those of the deletion, indicating that cacL-13 is null for all functions assayed. The cacL-20 allele partially complements cacP73 for optomotor behavior and fully complements cacP73 in both phototaxis assays. The cacL-24 allele complements every visual phenotype, but not courtship song (Smith, 1998).

The ensemble of these phenogenetic analyses indicates that (1) cacH18 and cacP73 are hypomorphic for phototaxis, in that homozygotes with two copies of the mutant gene give normal behavior, but one copy of either (when heteroallelic with the deletion) reveals a mutant phenotype; (2) only one of these lethal cac alleles (cacL-13) is null, in that the others each complement cac phenotypes that the deletion does not; (3) the lethal cac alleles (except cacL-13) must each have different, putatively separable undamaged functions, in that they each are able to complement (and therefore retain functions required for) different subsets of phenotypes and of the viable cac alleles; and, as a corollary, (4) the viable cac alleles must have different separable damaged functions, in that they each exhibit a different pattern of complementation by the several lethal alleles (Smith, 1998).

Other results also reveal the separability of these phenotypes. The cacH18 mutation leaves courtship song and light coincident receptor potential kinetics intact. There are heteroallelic genotypes that cause aberrant courtship song and ERG transients but normal LCRP amplitude and kinetics (cacS, when heterozygous with any of several cac lethal alleles) or aberrant kinetics but normal LCRP amplitude and normal courtship song (cacP73/cacL-6). Similar examples exist for most of the assayed phenotypes. While the complexity of the interactions precludes a simple definition of functional classes, it is clear that the etiology of the various phenotypes must involve multiple Dmca1A functions that are at least partially separable (Smith, 1998).

Previous analyses of Dmca1A transcripts identified pairs of mutually exclusive alternative exons at two different sites and a third site that generates four transcript variants by differential inclusion of three- and six-bp exons; additional transcript complexity is thought to be generated by RNA editing at 11 identified nucleotides in the transcript. Functional complexity could be mediated by several mechanisms, including temporal, tissue-specific, or subcellular spatial regulation of Dmca1A expression; one imagines that at least some distinct cac-mediated functions might correspond directly to distinct Dmca1A isoforms. Indeed, the cacH18 mutation creates a stop codon within the intriguingly variant alternative exon I/IIa, indicating that Dmca1A isoforms containing this variant motif are required for normal visual function, but not for viability or normal courtship song. Additional molecular and phenogenetic analyses will continue to unravel the links between the molecular complexity and the varied and functionally separable biological functions of these Dmca1A calcium channels (Smith, 1998).

When exposed to high temperatures (37 degrees), cac flies show frequent convulsions and pronounced locomotor defects. This TS phenotype seems consistent with the idea that cac is a mutation in a calcium-channel gene; it maps to the same X-chromosomal locus that encodes the polypeptide comprising the alpha-1 subunit of this membrane protein: Dmca1A. Previously, only one other voltage-sensitive calcium channel (Dmca1D) was known in Drosophila, but no behavioral defects have as yet been associated with variations at the autosomal locus encoding Dmca1D. Analysis of the courtship song of some other TS physiological mutants that are independent of cac shows that slowpoke mutations, which affect a calcium-activated potassium channel, cause severe song abnormalities. Certain additional TS mutants, in particular paralytic (parats1) and no-action-potential (napts1), exhibit subtler song defects. The results therefore suggest that genes involved in ion-channel function are a potential source of intraspecific genetic variation for song parameters, such as the number of cycles present in 'pulses' of tone or the rate at which pulses are produced by the male's wing vibrations during courtship. The implications of these findings from the perspective of interspecific lovesong variations in Drosophila are discussed. cacophony is one of the most interesting song mutations from an evolutionary point of view, at least in part because its abnormal pulses are nicely patterned, as in the case of wild-type males from various Drosophila species, and do not appear to be pathologically defective. A similar statement is possible about the songs of slowpoke males, although perhaps some of these mutant song bouts are more properly categorized as erratic and messy. Nevertheless, it is hard to believe that the song produced by double mutants cac;slo1/slo1 comes from D. melanogaster males, so striking are the differences from the wild-type patterns (Peixoto, 1998).

cacophony is a temperature-sensitive mutant: When exposed to high temperatures (~37°) cac flies show frequent convulsions and pronounced locomotor defects. This convulsion phenotype is characterized by flies turning upside-down or on their sides, shaking their legs for a few seconds, and then turning right-side up. The flies also curl their abdomen severely, either when on their backs or when walking, and twist their bodies at the same time. In addition, occasionally the cac adults will walk sideways, spin around on the same spot for a couple of seconds (apparently completely disoriented), leap across the chamber, or jump and tumble up and down out of control. There was no obvious sequence in the occurrence of these phenotypes. After long exposures at 37°, cac flies spend more and more time on their backs, shaking their legs until they seem to collapse. This typically requires more than 1 hr of heating for 1-day-old flies, but much less for older ones. As long as leg movement is still occurring, the mutant individuals usually recover in a few minutes after transfer to room temperature (Peixoto, 1998).

Only pulse song was examined in this report (courtship hums, or sine-song, being another type of song). Usually, all the pulses of the song of a given fly are logged, that is, marked for storage in the relevant file using the computer as an event-recorder, while scanning the visual record of the song along with the video image of the flies' behavior. Logging of some songs extended for only 2 min, and more than 500 pulses were typically logged. Songs with less than 40 pulses were not included in the analysis. Four parameters of the flies' pulse song were measured: interpulse interval (IPI), Cycles-per-Pulse (CPP), amplitude, and intrapulse frequency (IPF). CPP and IPF values can vary together among Drosophila types, but there is no way to predict one value from knowledge of the other; thus, these were treated as separate song parameters. The pulse amplitude measurements were attempts to quantify a song's loudness. This is difficult to measure reliably, and the units specified are arbitrary (Peixoto, 1998).

To examine the effects that temperature variation might have on the pulse song produced by cac, a song analysis of cac and wild-type flies was carried out at temperatures ranging from 15 to 30 degrees in steps of 2.5 degrees. Also included in this analysis was the mutant parats1, because a preliminary analysis had found it to have an effect on song at 25 degrees. Four pulse-song parameters were examined: amplitude of sound, IPI, CPP, and IPF. Temperature has a major effect on amplitude and IPI of all three genotypes, although it is far less clear in the case of CPP and IPF, even though the temperature effect is significant for the latter. Significant genotype differences were observed for amplitude, IPI, and CPP but not for IPF. The results also show the basic differences between cac mutation songs and wild-type (normal) songs, that is, higher amplitude and CPP, as well as longer IPIs in the former compared to the latter. IPFs are similar between these two genotypes. Although the overall trend observed for amplitude and IPI is similar for wild-type, cac, and para (as the temperature rises, there is an increase in the former and a decrease in the latter), differences were revealed in the way the various types of males react to temperature. These differences are responsible for the significant genotype x temperature interactions observed. The difference in IPI between cac and wild type shows a significant negative correlation with temperature. The difference is actually larger at lower temperatures, a result that is somewhat counterintuitive if one considers that the convulsion phenotype of this mutant occurs at elevated temperatures. It is possible that this reflects in part the nonlinear nature of the IPI change with temperature. No significant correlation with temperature was observed for the amplitude differences. The difference in IPI between parats1 and wild type shows the opposite trend observed for cac mutants. There is a significant positive correlation of temperature with the larger IPI difference at 30°. In the case of amplitude, however, the differences between parats1 and wild type show a significant negative correlation (Peixoto, 1998).

Genetic analysis of synaptic mechanisms in Drosophila has identified a temperature-sensitive paralytic mutant of the voltage-gated calcium channel alpha1 subunit gene, cacophony (cac). Electrophysiological studies in this mutant, designated cacTS2, indicate cac encodes a primary calcium channel alpha1 subunit functioning in neurotransmitter release. To further examine the functions and interactions of cac-encoded calcium channels, a genetic screen was performed to isolate new mutations that modify the cacTS2 paralytic phenotype. The screen recovered 10 mutations that enhance or suppress cacTS2, including second-site mutations in cac (intragenic modifiers) as well as mutations mapping to other genes (extragenic modifiers). Molecular characterization of three intragenic modifiers is reported and the consequences of these mutations for temperature-sensitive behavior, synaptic function, and processing of cac pre-mRNAs, is reported. These mutations may further define the structural basis of calcium channel alpha1 subunit function in neurotransmitter release (Brooks, 2003).

The effects of slo mutation on courtship song

The mutant alleles slo1 and slo2 define slowpoke as a new courtship-song gene. The pulse songs produced by these two mutants are clearly aberrant and they are in fact often difficult to log due to the low-amplitude or polycyclic nature of pulses (at a given moment of singing). Using the same criteria and IPI cutoffs used with the other mutants, all four song parameters examined are affected by these two slo alleles, which cause somewhat distinct song abnormalities. Males homozygous for the slo1 mutation produce very low-amplitude songs with long IPIs, and low CPP and IPF values. Isolated putative pulses, usually monocyclic signals, often occur in slo1 song records; however, they were not logged because they did not occur in pulse trains. In the case of the slo2 allele, the IPIs of homozygous mutant males are not as long, and the sound amplitude not as low, as in the case of slo1. A train of pulses in the song produced by flies homozygous for slo2 often ends with a highly polycyclic pulse. In fact, the mean number of cycles per pulse of slo2 flies is higher than the wild-type control. Isolated pulses were also often observed, but in this case (cf. slo1) they are usually highly polycyclic. Heterozygous flies slo1/slo2 show effects intermediate between the two homozygotes. The differences in the phenotypes between the two mutants obviously suggest differences in the molecular nature of the lesions that are unknown. slo1 is a chemically induced mutation, while slo2 was generated using gamma rays (Peixoto, 1998). Neither shows any gross chromosomal rearrangements (N. S. Atkinson, personal communication to Peixoto, 1998).

A fair fraction of the song mutants resulting from changes in genes that have been characterized at the molecular level involve membrane excitability. Not surprisingly, these basic functions, when mutated, lead to grossly appreciable defects in behavior. Only some of these mutants are song-defective as well. cacophony now finds itself in this category, that is, the courtship variant mutant that started out as a song mutant but is now known to have other phenotypic defects, such as heat-induced convulsions. This kind of general impairment could be at least as detrimental to fitness as the song abnormalities produced by cac mutants. Other pleiotropic song mutants with molecular correlates involve the regulation of gene expression (considered in general terms: transcription or RNA processing). In addition to the period and dissonance mutants in this category, consider the fruitless gene and its mutants. These courtship mutations defined a locus encoding a transcription factor. fru mutations affect courtship song, as well as other aspects of the fly's reproductive behavior, including fertility. Pleiotropies of these sorts place important constraints on the evolution of these behavioral genes (Peixoto, 1998 and references).

Genetic variation for features of the Drosophila courtship song have been reported from natural populations. It is possible that the level of genetic variability observed is influenced not only by sexual selection acting on the song parameters themselves, but also by selection on the pleiotropic effects of these putative song genes. These pleiotropic effects could even include other aspects of the mate recognition system. For example, there are smellblind mutations at the para locus that affect the response of males to female pheromones. It is also conceivable that directional selection acting on some of these pleiotropic effects, for example, selection for temperature tolerance and ion-channel genes, could drive changes in the song repertoire that could eventually lead to reproductive isolation between different populations (Peixoto, 1998 and references).

While the constraints associated with pleiotropy certainly do not prevent the rapid evolution of Drosophila courtship songs, it might explain why there is little evidence for genes with major effects on song found in crosses between closely related species. It is likely that the lovesong differences between most such species are based on the cumulative effect of very mild and subtle changes in several genes, at least a handful of them involving, for example, interspecific variations at the cac, slo, and mle (nap) loci. The major innovations in song production in the genus Drosophila seem to have occurred among Hawaiian flies for which founder-effect models of speciation have been proposed. These include, for example, the idea of fixation of a mutation in a major locus, via genetic drift, followed by selection for modifiers on its deleterious effects. Pleiotropy and epistasis have major roles in these models. Epistasis between conspecific genes is a key component of this sexually related phenotype. Epistatic interactions among song genes, such as the one found between cac and napts1 within D. melanogaster, could also have important implications for sexual selection on the phenotypes they control and on their potential role in speciation. Because of the role acoustic signals (such as the Drosophila's lovesong) play in female receptivity, mating preferences, and sexual isolation between species, song factors are among the best candidates for the so-called 'speciation genes'. The behavioral analysis presented here reveals that mutations in loci affecting ion-channel function might be a source of genetic variation in the fly's lovesong. Because of their enormous diversity, channel genes might turn out to be among the most common classes of song genes (Peixoto, 1998 and references).

The N-ethylmaleimide-sensitive fusion protein (NSF) has been implicated in vesicle trafficking in perhaps all eukaryotic cells. The Drosophila comatose (comt) gene encodes an NSF homolog, dNSF1. Work with temperature-sensitive (TS) paralytic alleles of comt has revealed a function for dNSF1 at synapses, where it appears to prime synaptic vesicles for neurotransmitter release. To further examine the molecular basis of dNSF1 function and to broaden the analysis of synaptic transmission to other gene products, a genetic screen was performed for mutations that interact with comt. Four mutations that modify TS paralysis in comt are described, including two intragenic modifiers (one enhancer and one suppressor) and two extragenic modifiers (both enhancers). The intragenic mutations will contribute to structure-function analysis of dNSF1 and the extragenic mutations identify gene products with related functions in synaptic transmission. Both extragenic enhancers result in TS behavioral phenotypes when separated from comt, and both map to loci not previously identified in screens for TS mutants. One of these mutations is a TS paralytic allele of the calcium channel 1-subunit gene, cacophony (Dellinger, 2000).

Identification of cacTS2 as a modifier of comt raises a number of interesting issues. The original cacophony mutant (now known as cacS) was named on the basis of an aberrant male courtship song. The courtship song is produced by a patterned beating of the wings, and this pattern as well as the wing beat amplitude are altered in cacS mutants. The finding that cac-encoded calcium channels function in neurotransmitter release suggests that impairment of central synapses may contribute to altered song patterning in cacS. Given that cac-encoded alpha1-subunits function at flight muscle neuromuscular synapses, peripheral synaptic defects may contribute to the song phenotype as well. A second issue is whether the genetic interaction of cacTS2 and comt reflects direct or indirect interactions of the encoded gene products. Electrophysiological analysis indicates that the cac-encoded alpha1-subunit mediates fast neurotransmitter release and that dNSF1 functions in maintaining the readily releasable pool of synaptic vesicles. Thus the observed genetic interaction may reflect simply that both the comt and cac gene products function in neurotransmitter release. Alternatively, the genetic interaction may result from the well-characterized biochemical interactions of SNAREs with both NSF and calcium channels. While this issue remains unresolved, two observations favor the former possibility. (1) No sequence homology has been detected between the cac-encoded alpha1-subunit and SYNPRINT sequences thought to mediate direct interactions with other synaptic proteins. (2) Preliminary synaptic electrophysiology in cacTS2 comtST17 double mutants is consistent with independent actions of comt and cac mutations in neurotransmitter release (Dellinger, 2000).

Courtship and other behaviors affected by a heat-sensitive, molecularly novel mutation in the cacophony calcium-channel gene of Drosophila

The cacophony locus of Drosophila, which encodes a calcium-channel subunit, has been mutated to cause courtship-song defects or abnormal responses to visual stimuli. However, the most recently isolated cac mutant was identified as an enhancer of a comatose mutation's effects on general locomotion. The cacTS2 mutation was analyzed in terms of its intragenic molecular change and its effects on behaviors more complex than the fly's elementary ability to move. The molecular etiology of this mutation is a nucleotide substitution that causes a proline-to-serine change in a region of the polypeptide near its EF hand. Given that this motif is involved in channel inactivation, it was intriguing that cacTS2 males generate song pulses containing larger-than-normal numbers of cycles -- provided that such males are exposed to an elevated temperature. Similar treatments caused only mild visual-response abnormalities and generic locomotor sluggishness. These results are discussed in the context of calcium-channel functions that subserve certain behaviors and of defects exhibited by the original cacophony mutant. Despite its different kind of amino-acid substitution, compared with that of cacTS2, cacS males sing abnormally in a manner that mimics the new mutant's heat-sensitive song anomaly (Chan, 2002).

The newest cacophony mutant is a courtship variant, as is the original cac mutant. Thus, cacTS2 males are somewhat impaired in their overall courtship performance, including mating ability. However, cacTS2 males courted more vigorously and effectively than one might expect from monitoring their generic locomotor activity. One component of the courtship performance of cacTS2 males implies a behavioral problem that goes beyond the nature of the sounds they communicate to females. They performed worse than wingless wild-type males did, which indicates that this mutant is more pleiotropically defective than a 'song only' variant (Chan, 2002).

Nevertheless, the most sharply defined courtship defect exhibited by a cacTS2 male is its heat-sensitive anomalies of tone pulses that emanate from the wing vibrations it directs at a female. These abnormalities of cycles per pulse and pulse amplitude were found to be similar to the nonconditional courtship-song peculiarities exhibited by the original cacS mutant. That the respective mutant phenotypes are alike is important, because cacTS2 males did not have to exhibit any kind of singing eccentricity: inasmuch as the isolation of this mutant involved behavioral criteria that had nothing to do with courtship, the outcome of song-testing cacTS2 could have left cacS as the only singing variant associated with this gene. But both the original and the newest cacophony mutations cause courtship-song peculiarities, and it is interesting that the anomalously loud and polycyclic pulses produced by both cacS and cacTS2 males do not involve an appreciable derangement of such sounds: each mutant type remains nicely patterned with respect to the qualities of individual 'clicks' and their rate of production. Once again, if cacTS2 turned out to be song defective it was not a foregone conclusion that such males would produce these sounds in a manner more salutary than that of other singing variants, such as those expressing slowpoke (slo) mutations. In this regard, slo potassium-channel mutants were identified using generic behavioral criteria (as was cacTS2) and were found later to sing aberrantly and to exhibit erratic patterns of anomalous tone pulses (Chan, 2002).

This brings us to the question of why it might be that the songs of cacS and of cacTS2 (at 30° C) males are not only song defective, but also similarly so in their tone-pulse qualities. As was introduced in conjunction with documenting cacTS2's intragenic site change, this amino-acid substitution is very near the EF hand within Dmca1A, directly C-terminal to the IVS6 transmembrane domain. The highly conserved EF hand and adjacent residues among calcium-channel alpha1 subunits of various species are involved in channel inactivation mediated by Ca2+ binding. Thus, this form of inactivation involves a calcium-influenced conformational change that occurs via cation binding within the EF hand's helix-loop-helix. Given the P-to-S substitution in cacTS2 immediately C-terminal to the EF hand (where this evolutionarily conserved proline is changed to a polar serine that has more conformational freedom) one imagines that the local three-dimensional structure in which the EF hand finds itself is altered in the mutant. The function of this domain would be altered accordingly but not ruined at permissive temperatures. Thus, the amino-acid substitution in cacTS2 near the EF hand suggests that this protein change could cause the Dmca1A calcium channel to exhibit altered inactivation kinetics. Whereas inactivation features of the alpha1 subunit encoded by cac are unknown, it is reasonable to speculate that that process becomes less robust than normal in the cacTS2 mutant as the flies are heated from 20° to 30°. Why the dynamics of inactivation may be subtly heat sensitive over the temperature range just stated is difficult to surmise, although perhaps it is the case that this process can barely occur at all at 37°, accounting for the grossly subnormal synaptic neurotransmission that occurs at that extreme temperature (Chan, 2002).

This hypothesis, as it relates to cacTS2's behavioral phenotype within a 'physiological' range of temperatures, goes on to suggest that anomalously polycyclic pulses in the songs of males expressing this mutation smack of a channel-inactivation change that would alter the contribution of calcium currents to the overall behavioral process in question. Thus, the repetitive-pattern phenotype, which is a reasonable descriptor for trains of Drosophila song pulses, would not have the intrapulse cycles inactivated as 'tightly' as in wild type (Chan, 2002).

What about the songs of cacS males, whose pulses are similarly polycyclic (albeit without the temperature sensitivity that accompanies the cacTS2 phenotype)? The cacS mutant is accounted for by an amino-acid substitution within the sixth membrane-embedded region of the penultimate intra-Dmca1A repeat, a.k.a. IIIS6. Certain types of calcium channels prevent excessive influx of calcium when the channel opens by voltage-mediated inactivation. Pore-forming S6 transmembrane domains play a role in modulating voltage-dependent calcium-channel inactivation. This has been revealed (1) by creating chimeric alpha1-subunit polypeptides in which portions of IIIS6 from fast-inactivating channels replaced those of a slow-inactivating one, leading to inactivation kinetics characteristic of the donor calcium-channel type and (2) by physiological disruptions of channel functions that are pointed to by the etiology of certain patho-physiological mutants in humans; certain such S6 mutations slow and others accelerate the development of inactivation. Therefore, a mnemonic device for apprehending the song abnormality exhibited by cacS mutant males is, again, subnormal inactivation of intratone-pulse sounds, owing to their inappropriate polycyclicity. However, in this case the putative inactivation defect would have a different mechanistic etiology compared with that hypothesized for the cacTS2-mutated polypeptide (Chan, 2002).

Presynaptic N-type calcium channels regulate synaptic growth

Voltage-gated calcium channels couple changes in membrane potential to neuronal functions regulated by calcium, including neurotransmitter release. Presynaptic N-type calcium channels not only control neurotransmitter release but also regulate synaptic growth at Drosophila neuromuscular junctions. In a screen for behavioral mutants that disrupt synaptic transmission, an allele of the N-type calcium channel locus (Dmca1A) was identified that caused synaptic undergrowth. The underlying molecular defect was identified as a neutralization of a charged residue in the third S4 voltage sensor. RNA interference reduction of N-type calcium channel expression also reduced synaptic growth. Hypomorphic mutations in syntaxin-1A or n-synaptobrevin, which also disrupt neurotransmitter release, did not affect synapse proliferation at the neuromuscular junction, suggesting calcium entry through presynaptic N-type calcium channels, not neurotransmitter release per se, is important for synaptic growth. The reduced synapse proliferation in Dmca1A mutants is not due to increased synapse retraction but instead reflects a role for calcium influx in synaptic growth mechanisms. These results suggest N-type channels participate in synaptic growth through signaling pathways that are distinct from those that mediate neurotransmitter release. Linking presynaptic voltage-gated calcium entry to downstream calcium-sensitive synaptic growth regulators provides an efficient activity-dependent mechanism for modifying synaptic strength (Rieckhof, 2003).

Dmca1A is abundantly expressed in the Drosophila nervous system and encodes the presynaptic N-type calcium channel responsible for calcium influx that triggers synaptic vesicle fusion. Null mutations in Dmca1A are embryonic lethal [lethal(1)L13], whereas partial loss-of-function mutations disrupt synaptic transmission, leading to defects in various behaviors, including courtship (cacaphony) and phototaxis (nightblind-A). In addition to defects in neurotransmitter release, Dmca1A hypomorphic mutants show altered morphology at the mature larval NMJ. There is a decrease in both terminal branching and varicosity number compared with wild-type controls and hypomorphic alleles of syntaxin and n-synaptobrevin. The reduced synaptic proliferation is not secondary to defective synaptic transmission because syntaxin and synaptobrevin show more profound defects in transmitter release but have normal synaptic proliferation. Similar results have also been reported in synaptotagmin mutants as well as SNARE mutants. Thus, mutations in Dmca1A affect calcium-regulated synaptic pathways separate from those that regulate transmitter release (Rieckhof, 2003).

No evidence was found for a role of presynaptic calcium entry through either N- or L-type calcium channels in the early stages of synapse formation during late embryogenesis. It was also determined that the morphological defects in Dmca1A mutants are not due to an increase in terminal retraction, suggesting active growth rather than synapse stability is defective. Therefore, the structural defects observed in Dmca1A mutants occur between the establishment of the initial synaptic field and its final larval maturation. Previous work has demonstrated that the overall shape and branching pattern at the Drosophila NMJ is established early in development. Subsequent growth largely requires the addition of new varicosities to previously formed terminal branches. It is within this second activity-dependent growth phase that is thought to be presynaptic that calcium entry is required to promote synaptic maturation. The 35% reduction observed in varicosity number in Dmca1ANT27 mutants at the end of larval development is likely an underestimate of the actual contribution of presynaptic calcium entry to synaptic growth regulation. (1) The initial activity-independent elaboration of synapses during late embryogenesis does not require calcium channel function, allowing the establishment of the initial synaptic field. (2) The Dmca1ANT27 mutant is a hypomorphic allele, reducing calcium channel function but not eliminating it. Previously isolated alleles of the Dmca1A locus that are more severe than Dmca1ANT27 are embryonic lethal, preventing an analysis of the activity-dependent phase of synaptic growth in more severe alleles. Further studies with mosaic animals will be required to fully characterize the persistence of synaptic growth mechanisms in the complete absence of presynaptic calcium influx (Rieckhof, 2003).

The opening of presynaptic N-type channels during robust synaptic activity may allow calcium to influence varicosity sprouting mechanisms to locally control synaptic remodeling. Changes in intracellular calcium have been shown to affect growth cone motility and neurite outgrowth. Indeed, filopodial protrusions from neuronal growth cones are triggered by altered calcium concentrations. Synaptic activity results in calcium-dependent CaMKII activation via binding of calcium/calmodulin and subsequent auto-phosphorylation. Activated CaMKII phosphorylates the synaptic MAGUK protein DLG, causing release of FAS2 from its synaptic scaffold and subsequent modulation of synaptic growth in Drosophila. In addition, CaMKII activation also regulates the activity of the ether-a-go-go (eag) family of potassium channels in Drosophila, altering aspects of nerve excitability that could contribute to synaptic growth. Intracellular calcium levels directly regulate cAMP signaling through the activation of adenylate cyclase by calmodulin, enhancing cAMP-dependent pathways implicated in synaptic growth. It is likely that disruptions in presynaptic calcium entry in Dmca1ANT27 mutants leads to alterations in several presynaptic signaling cascades that modulate growth. Further genetic analysis should begin to elucidate how the regulation of calcium entry modulates these activity-dependent synaptic growth pathways (Rieckhof, 2003).

Voltage-gated calcium channels consist of four repeated units (I-IV) containing six alpha-helical transmembrane segments (S-S6). The fourth transmembrane segment (S4) of voltage-gated ion channels has been shown to function as a voltage sensor. It is thought that the S4 sensor, a transmembrane alpha-helix in which every third or fourth residue is basic and carries a positive charge, undergoes conformational changes during depolarization that result in channel opening. Sequence analysis of Dmca1ANT27 identified a charge-neutralizing mutation in a highly conserved arginine residue in the S4 voltage sensor, supporting an essential role for S4 helix movement during channel gating and subsequent calcium influx. The altered S4 charged amino acid likely explains the TS phenotype of the mutant; channel gating requires conformational changes in the S4 helix that are temperature-dependent. Mutations in mammalian Dmca1A homologs have been linked to a variety of neuronal disorders, including episodic and spinocerebellar ataxias, hemiplegic migraine, blindness, hypokalemic periodic paralysis, and epilepsy. Although it seems paradoxical that a hyperexcitability phenotype such as seizures could arise from a reduction in calcium channel function, the Drosophila giant fiber pathway is extremely sensitive to changes in both inhibition and excitation. The decrease in calcium channel function in the inhibitory pathways may bear more weight in the overall output of the circuit, even though the excitatory outputs have reduced release as well. Similar seizure defects are seen in mammalian N-type calcium channel mutants (tottering, lethargic, and rocker) where overall calcium channel function is also reduced. Similar to what is observed in Drosophila N-type mutants, mutations in the mouse alpha1a calcium channel locus (rocker) that disrupt the pore region of the channel and reduce calcium influx cause a profound reduction in Purkinje cell dendritic arborization. In addition, pharmacological disruption of calcium channel function in salamander rod photoreceptors has been shown to inhibit varicosity formation. The reduced varicosity number in calcium channel mutants suggests an important role for normal levels of calcium entry during synaptic activity for the proper modulation of synaptic growth, in addition to its well established role in neurotransmitter release. Modulation of N-type calcium channel function via alterations in the rates of presynaptic action potential firing could provide an efficient mechanism for the regulation of activity-dependent synaptic growth (Rieckhof, 2003).

Cacophony and exocytosis: Ca2+ influx through distinct routes controls exocytosis and endocytosis at Drosophila presynaptic terminals

Endocytosis of synaptic vesicles follows exocytosis, and both processes require external Ca2+. However, it is not known whether Ca2+ influx through one route initiates both processes. At larval Drosophila neuromuscular junctions, exocytosis and endocytosis were separately measured using the fluorescent dye FM1-43. In a temperature-sensitive Ca2+ channel mutant, cacophonyTS2, exocytosis induced by high K+ decreases at nonpermissive temperatures, while endocytosis remains unchanged. In wild-type larvae, a spider toxin Ca2+ blocker, PLTXII, preferentially inhibits exocytosis, whereas the T-type Ca2+ channel blocker flunarizine and the blocker La3+ selectively depresses endocytosis. None of these blockers affect exocytosis or endocytosis induced by a Ca2+ ionophore. Evoked synaptic potentials are depressed regardless of stimulus frequency in cacophonyTS2 at nonpermissive temperatures and in wild-type by PLTXII, whereas flunarizine or La3+ gradually depressed synaptic potentials only during high-frequency stimulation, suggesting depletion of synaptic vesicles due to blockade of endocytosis. In shibirets1, a dynamin mutant, flunarizine or La3+ inhibit assembly of clathrin at the plasma membrane during stimulation without affecting dynamin function (Kuromi, 2004).

To maintain synaptic transmission during intense neuronal activities, synaptic vesicles (SVs) are effectively recycled by endocytosis. Ca2+ influx through voltage-gated Ca2+ channels plays a crucial role in exocytosis. It has also been found, in early studies at frog neuromuscular junctions, that external Ca2+ is essential for SV recycling. Subsequent studies have confirmed that Ca2+ influx is also required for endocytosis in various types of central synapses and secretory cells. In contrast, endocytosis has been shown to occur even in the absence of external Ca2+ after cessation of stimulation in rat hippocampal neurons and in presynaptic boutons of a Drosophila temperature-sensitive mutant, shibirets1 (shits1), at room temperature after depletion of SVs at nonpermissive temperatures. These studies suggest that endocytosis can be triggered independently of the Ca2+ influx that initiates exocytosis. To reconcile these seemingly contradictory findings, it has been postulated that Ca2+ influx during stimulation that causes exocytosis also triggers the formation of intermediates for SV recycling, and once they are formed, the following steps of endocytosis proceed without Ca2+. However, because of difficulties in separating Ca2+ influx routes for exocytosis and endocytosis during stimulation, this hypothesis remains unproven (Kuromi, 2004 and references therein).

Immunostaining studies suggest that sites for endocytosis are distinct from those for exocytosis at nerve terminals. It is then possible that Ca2+ influx routes for these two processes are separate. Along the line of this idea, multiple subtypes of Ca2+ channels are demonstrated in nerve terminals. Those subtypes of Ca2+ channels are spatially segregated in presynaptic terminals, and their roles in transmitter release have been subject to speculation. Specific roles of these Ca2+ channel subtypes in exocytosis and endocytosis, however, have not been identified (Kuromi, 2004).

Does Ca2+ influx through one route trigger both exocytosis and endocytosis? To address this question, a temperature-sensitive Ca2+ channel mutant, cacophonyTS2 (cacTS2), and various Ca2+ channel blockers have been used at larval Drosophila neuromuscular junctions. A fluorescent dye, FM1-43, is incorporated into SVs in nerve terminals by endocytosis, and FM1-43 loaded in SVs is released by exocytosis. By measuring the amount of FM1-43 released from or taken up into nerve terminals, exocytosis and endocytosis were separately determined. Thus, it has been revealed that distinct Ca2+ influx routes separately regulate exocytosis and endocytosis. Taking advantage of drugs that selectively block endocytosis, it has been further shown in shits1 that selective blockade of the Ca2+ influx route linked to endocytosis inhibits clathrin assembly on the plasma membrane of nerve terminals. It is suggested that Ca2+ influx during stimulation through this route forms an intermediate complex, which leads to endocytosis (Kuromi, 2004).

A widely accepted model of endocytosis is that the clathrin coat assembles first on the presynaptic membrane, forming a shallow coated pit, which then invaginates to generate a bud with a constricted neck and eventually a free clathrin-coated vesicle by fission of the neck. A model has been proposed with two steps in SV recycling in which a Ca2+-dependent step (step I), which occurs during stimulation, is followed by a Ca2+-independent, shibire-dependent step (step II). In shits1 it has been shown that when FNZ or La3+ is added after high K+ stimulation, endocytosis at permissive temperatures occurs normally, indicating that FNZ or La3+ have no effect on step II. In contrast, when FNZ or La3+ is present during high K+ stimulation, endocytosis is not observed although exocytosis is unaffected. These observations strongly support the hypothesis that FNZ or La3+ selectively block Ca2+ influx through the route designated for endocytosis (step I) (Kuromi, 2004).

Immunostaining experiments with shits1 at nonpermissive temperatures reveal that synaptotagmin I is transferred to the plasma membrane during high K+ stimulation regardless of the presence of La3+, confirming that La3+ has no effect on exocytosis. However, it was noted that in the absence of La3+, clusters of clathrin immmunoreactivity are detected at the periphery of boutons after high K+ stimulation, while in the presence of La3+, clathrin remains in the cytosol of boutons after high K+ stimulation. These observations suggest that La3+ inhibits clathrin assembly at the plasma membrane. Dynamin plays an essential role in the fission of a clathrin-coated bud, and this process occurs in the absence of external Ca2+. It is suggested that the part of Ca2+ influx sensitive to FNZ or La3+ during stimulation (step I), plays a crucial role in clathrin assembly at the plasma membrane (Kuromi, 2004).

Presynaptic calcium channel localization and calcium-dependent synaptic vesicle exocytosis regulated by the Fuseless protein

A systematic forward genetic Drosophila screen for electroretinogram mutants lacking synaptic transients identified the fuseless (fusl) gene, which encodes a predicted eight-pass transmembrane protein in the presynaptic membrane. Null fusl mutants display >75% reduction in evoked synaptic transmission but, conversely, an approximately threefold increase in the frequency and amplitude of spontaneous synaptic vesicle fusion events. These neurotransmission defects are rescued by a wild-type fusl transgene targeted only to the presynaptic cell, demonstrating a strictly presynaptic requirement for Fusl function. Defects in FM dye turnover at the synapse show a severely impaired exo-endo synaptic vesicle cycling pool. Consistently, ultrastructural analyses reveal accumulated vesicles arrested in clustered and docked pools at presynaptic active zones. In the absence of Fusl, calcium-dependent neurotransmitter release is dramatically compromised and there is little enhancement of synaptic efficacy with elevated external Ca2+ concentrations. These defects are causally linked with severe loss of the Cacophony voltage-gated Ca2+ channels, which fail to localize normally at presynaptic active zone domains in the absence of Fusl. These data indicate that Fusl regulates assembly of the presynaptic active zone Ca2+ channel domains required for efficient coupling of the Ca2+ influx and synaptic vesicle exocytosis during neurotransmission (Long, 2008).

Null fuseless mutants have an increased number of vesicles clustered and docked at the presynaptic density. These phenotypes are consistent with direct disruption of Ca2+-triggered vesicle exocytosis. Similar vesicle accumulation characterizes syntaxin1A, dunc-13, comatose, and rolling blackout mutants, for example, each of which has a specific exocytosis deficit. It has been shown that a specific block in SV exocytosis leads to a secondary accumulation of vesicles in pools upstream of the presynaptic membrane. FM dye studies complement the ultrastructural analyses. As is expected for any defect in the SV cycle, there is a decrease in the overall rate of endo-exo cycling in fusl mutants. In addition, there is a particularly severe defect in acute FM dye release in the absence of Fusl function, consistent with a specific defect in SV exocytosis. these fusl mutant defects are attributed wholly to the loss of the appropriate Ca2+ influx trigger to signal release of otherwise fusion-competent vesicles (Long, 2008).

Together, this study strongly supports a mechanistic role for the Fusl protein in regulating the voltage-gated Ca2+ channels that trigger synaptic vesicle fusion. In the absence of this regulation, active zone Ca2+ channels domains fail to form or be properly organized. Transmission is severely impaired but certainly not eliminated, indicating that Fusl facilitates Ca2+ channel domain assembly, but is not absolutely required. Ultrastructurally normal active zones persist in the complete absence of Fusl but lack the localized Ca2+ trigger for vesicle exocytosis and therefore only inefficiently manage neurotransmitter release. One possible role for the Fusl protein might be to serve as a direct interacting partner with the Ca2+ channel to enable its correct trafficking and/or localized maintenance at the active zone. Fusl is not restricted to active zone domains, like the Ca2+ channel, but rather shows a diffuse presynaptic plasma membrane localization, similar to the tSNARE Syntaxin. Tests for direct association between Fusl and Cac channels have so far been inconclusive. Another model, consistent with the predicted transmembrane transporter function, is that Fusl might regulate the presynaptic environment in a way that facilitates Ca2+ channel localization. Arguing for this model, the closest human sequence homolog, Sialin, transports sialic acid. This modified sugar group is a component of transmembrane glycoproteins, typically as the terminal residue of cell surface oligosaccharides. Misregulation of this cargo transport could be easily envisioned to cause defects in Ca2+ channel trafficking and/or maintenance during presynaptic active zone assembly. Future work will examine the molecular function of the Fuseless protein by attempting to determine whether it functions in synaptic glycosylation, perhaps as a transmembrane transporter, as predicted, and, if so, the exact nature of the transported cargo and its effects on synaptogenesis (Long, 2008).

Flight and seizure motor patterns in Drosophila mutants: Simultaneous acoustic and electrophysiological recordings of wing beats and flight muscle activity

Tethered flies allow studies of biomechanics and electrophysiology of flight control. Microelectrode recordings were performed of spikes in an indirect flight muscle (the dorsal longitudinal muscle, DLMa) coupled with acoustic analysis of wing beat frequency (WBF) via microphone signals. Simultaneous electrophysiological recording of direct and indirect flight muscles has been technically challenging; however, the WBF is thought to reflect in a one-to-one relationship with spiking in a subset of direct flight muscles, including muscle m1b. Therefore, this approach enables systematic mutational analysis for changes in temporal features of electrical activity of motor neurons innervating subsets of direct and indirect flight muscles. This paper reports the consequences of specific ion channel disruptions on the spiking activity of myogenic DLMs (firing at approximately 5 Hz) and the corresponding wing beat frequency (approximately 200 Hz). Mutants were examined of: 1) voltage-gated Ca2+ channels (cacophony, cac), 2) Ca2+-activated K+ channels (slowpoke, slo), and 3) voltage-gated K+ channels (Shaker, Sh) and their auxiliary subunits (Hyperkinetic, Hk and quiver, qvr). Flight initiation in response to an air puff was severely disrupted in both cac and slo mutants. However, once initiated, slo flight was largely unaltered, whereas cac displayed disrupted DLM firing rates and WBF. Sh, Hk, and qvr mutants were able to maintain normal DLM firing rates, despite increased WBF. Notably, defects in the auxiliary subunits Hk and qvr could lead to distinct consequences, i.e. disrupted DLM firing rhythmicity, not observed in Sh. This mutant analysis of direct and indirect flight muscle activities indicates that the two motor activity patterns may be independently modified by specific ion channel mutations, and that this approach can be extended to other dipteran species and additional motor programs, such as electroconvulsive stimulation-induced seizures (Iyengar, 2014).

Role of Drosophila calcium channel Cacophony in dopaminergic neurodegeneration and neuroprotection

One of the most important questions in Parkinson's disease (PD) regards the selective vulnerability of a specific population of dopaminergic (DA) neurons. Recent reports identify Ca2+ channel as a potential source of this vulnerability. This work uses a Drosophila primary neuronal cell culture system as an in vitro PD model to explore the role of Ca2+ homeostasis in DA neurodegeneration and protection. The data showed that the Ca2+ chelator EGTA is neuroprotective against a PD toxin MPP+ (40mμM). The genetic tools available in Drosophila were used to manipulate Ca2+ channel activity. DA neurons lacking functional Ca2+ channels (i.e., cacophony mutant) are inherently protected against MPP+ toxicity. Furthermore, overexpression of wild type Ca2+ channels in DA neurons blocks the rescue effect of a D2 agonist quinpirole on DA neurodegeneration. The findings support the idea that Ca2+ is a source of vulnerability for DA neurons and that the modulation of Ca2+ levels in DA neurons could be a potential neuroprotective treatment (Wiemerslage, 2014).

Neuronal processing of noxious thermal stimuli mediated by dendritic Ca influx in somatosensory neurons

Adequate responses to noxious stimuli causing tissue damages are essential for organismal survival. Class IV neurons in Drosophila larvae are polymodal nociceptors responsible for thermal, mechanical, and light sensation. Importantly, activation of Class IV provoked distinct avoidance behaviors, depending on the inputs. Noxious thermal stimuli, but not blue light stimulation, was shown to cause a unique pattern of Class IV, which were composed of pauses after high frequency spike trains and a large Ca2+ rise in the dendrite (the Ca2+ transient). Both of these responses depended on two TRPA channels and the L-type voltage-gated calcium channel (L-VGCC), showing that the thermosensation provokes Ca2+ influx. The precipitous fluctuation of firing rate in Class IV neurons enhanced the robust heat avoidance. It is hypothesized that the Ca2+ influx can be a key signal encoding a specific modality (Terada, 2016).


EVOLUTIONARY HOMOLOGS

Calcium channel expression

A site-directed anti-peptide antibody (anti-CNA1) directed against the alpha 1 subunit of class A calcium channels (alpha 1A) recognizes a protein of approximately 190-200 kDa in immunoblot and immunoprecipitation analyses of rat brain glycoproteins. Calcium channels recognized by anti-CNA1 are distributed throughout the brain with a high concentration in the cerebellum. Calcium channels having alpha 1A subunits are concentrated in presynaptic terminals making synapses on cell bodies and on dendritic shafts and spines of many classes of neurons and are especially prominent in the synapses of the parallel fibers of cerebellar granule cells on Purkinje neurons where their localization in presynaptic terminals was confirmed by double labeling with the synaptic membrane protein syntaxin or the microinjected postsynaptic marker Neurobiotin. Calcium channels are present in lower density in the surface membrane of dendrites of most major classes of neurons. There was substantial labeling of Purkinje cell bodies, but less intense staining of the cell bodies of hippocampal pyramidal neurons, layer V pyramidal neurons in the dorsal cortex, and most other classes of neurons in the forebrain and cerebellum. Scattered cell bodies elsewhere in the brain were labeled at low levels. These results define a unique pattern of localization of class A calcium channels in the cell bodies, dendrites, and presynaptic terminals of most central neurons. Compared to class B N-type calcium channels, class A calcium channels are concentrated in a larger number of presynaptic nerve terminals implying a more prominent role in neurotransmitter release at many central synapses (Westenbroek, 1995).

Calcium channel alternative splicing

The N channel is critical for regulating release of neurotransmitter at many synapses, and even subtle differences in its activity would be expected to influence the efficacy of synaptic transmission. Although several splice variants of the N channel are expressed in the mammalian nervous system, their biological importance is presently unclear. Variants of the alpha1B subunit of the N channel are expressed in sympathetic ganglia and alternative splicing within IIIS3-S4 and IVS3-S4 generate kinetically distinct channels. A striking difference is shown between the expression pattern of the S3-S4 variants in brain and peripheral ganglia; it is concluded that the brain-dominant form of the N channel gates 2-to-4-fold more rapidly than that predominant in ganglia (Lin, 1997).

P-type and Q-type calcium channels mediate neurotransmitter release at many synapses in the mammalian nervous system. The alpha 1A calcium channel has been implicated in the etiologies of conditions such as episodic ataxia, epilepsy and familial migraine, and shares several properties with native P- and Q-type channels. However, the exact relationship between alpha 1A and P- and Q-type channels is unknown. Alternative splicing of the alpha 1A subunit gene results in channels with distinct kinetic, pharmacological and modulatory properties. Overall, the results indicate that alternative splicing of the alpha 1A gene generates P-type and Q-type channels as well as multiple phenotypic variants (Bourinet, 1999).

The N-type Ca channel alpha1B subunit is localized to synapses throughout the nervous system and couples excitation to release of neurotransmitters. Two functionally distinct variants of the alpha1B subunit have been identified, rnalpha1B-b and rnalpha1B-d, that differ at two loci; four amino acids [SerPheMetGly (SFMG)] in IIIS3-S4 and two amino acids [GluThr (ET)] in IVS3-S4. These variants are reciprocally expressed in rat brain and sympathetic ganglia. The slower activation kinetics of rnalpha1B-b (DeltaSFMG/+ET) compared with rnalpha1B-d (+SFMG/DeltaET) channels are fully accounted for by the insertion of ET in IVS3-S4 and not by the lack of SFMG in IIIS3-S4. The inactivation kinetics of these two variants are indistinguishable. Through genomic analysis a six-base cassette exon is identified that encodes the ET site. With ribonuclease protection assays it has been demonstrated that the expression of this mini-exon is essentially restricted to alpha1B RNAs of peripheral neurons. Evidence is shown for regulated alternative splicing of a six-base exon encoding NP in the IVS3-S4 linker of the closely related alpha1A gene; residues NP can functionally substitute for ET in domain IVS3-S4 of alpha1B. The selective expression of functionally distinct Ca channel splice variants of alpha1B and alpha1A subunits in different regions of the nervous system adds a new dimension of diversity to voltage-dependent Ca signaling in neurons that may be important for optimizing action potential-dependent transmitter release at different synapses (Lin, 1999).

Two splice variants of the human homolog of the alpha1A subunit of voltage-gated Ca2+ channels have been cloned. The sequences of human alpha1A-1 and alpha1A-2 code for proteins of 2510 and 2662 amino acids, respectively. Human alpha1A-2alpha2bdeltabeta1b Ca2+ channels expressed in HEK293 cells activate rapidly (tau+10mV = 2.2 ms), deactivate rapidly (tau-90mV = 148 micros), inactivate slowly (tau+10mV = 690 ms), and have peak currents at a potential of +10 mV with 15 mM Ba2+ as charge carrier. In HEK293 cellsm transient expression of Ca2+ channels containing alpha1A/B(f), an alpha1A subunit containing a 112 amino acid segment of alpha1B-1 sequence in the IVS3-IVSS1 region, resulted in Ba2+ currents that were 30-fold larger compared to wild-type (wt) alpha1A-2-containing Ca2+ channels, and had inactivation kinetics similar to those of alpha1B-1-containing Ca2+ channels. Cells transiently transfected with alpha1A/B(f)alpha2bdeltabeta1b expressed higher levels of the alpha1, alpha2bdelta, and beta1b subunit polypeptides as detected by immunoblot analysis. By mutation analysis two locations were identified in domain IV within the extracellular loops S3-S4 (N1655P1656) and S5-SS1 (E1740) that influence the biophysical properties of alpha1A. alpha1AE1740R resulted in a threefold increase in current magnitude, a -10 mV shift in steady-state inactivation, and an altered Ba2+ current inactivation, but did not affect ion selectivity. The deletion mutant alpha1ADeltaNP shifted steady-state inactivation by -20 mV and increased the fast component of current inactivation twofold. The potency and rate of block by omega-Aga IVA was increased with alpha1ADeltaNP. These results demonstrate that the IVS3-S4 and IVS5-SS1 linkers play an essential role in determining multiple biophysical and pharmacological properties of alpha1A-containing Ca2+ channels (Hans, 1999).

The calcium channel alpha(1A) subunit gene codes for proteins with diverse structure and function. This diversity may be important for fine tuning neurotransmitter release at central and peripheral synapses. The alpha(1A) C terminus, which serves a critical role in processing information from intracellular signaling molecules, is capable of undergoing extensive alternative splicing. The purpose of this study was to determine the extent to which C-terminal alternative splicing affects some of the fundamental biophysical properties of alpha(1A) subunits. Specifically, the biophysical properties of two alternatively spliced alpha(1A) subunits were compared. One variant was identical to an isoform identified previously in human brain, and the other was a novel isoform isolated from human spinal cord. The variants differed by two amino acids (NP) in the extracellular linker between transmembrane segments IVS3 and IVS4 and in two C-terminal regions encoded by exons 37 and 44. Expression in Xenopus oocytes demonstrated that the two variants were similar with respect to current-voltage relationships and the voltage dependence of steady-state activation and inactivation. However, the rates of activation, inactivation, deactivation, and recovery from inactivation were all significantly slower for the spinal cord variant. A chimeric strategy demonstrated that the inclusion of the sequence encoded by exon 44 specifically affects the rate of inactivation. These findings demonstrate that C-terminal structural changes alone can influence the way in which alpha(1A) subunits respond to a depolarizing stimulus and add to the developing picture of the C terminus as a critical domain in the regulation of Ca2+ channel function (Krovetz, 2000).

Structural diversity of voltage-gated Ca channels underlies much of the functional diversity in Ca signaling in neurons. Alternative splicing is an important mechanism for generating structural variants within a single gene family. In vivo expression pattern of an alternatively spliced 21 amino acid encoding exon in the II-III cytoplasmic loop region of the N-type Ca channel alpha(1B) subunit is shown and its functional impact is assessed. Exon-containing alpha(1B) mRNA dominates in sympathetic ganglia and is present in approximately 50% of alpha(1B) mRNA in spinal cord and caudal regions of the brain and in the minority of alpha(1B) mRNA in neocortex, hippocampus, and cerebellum (<20%). The II-III loop exon affects voltage-dependent inactivation of the N-type Ca channel. Steady-state inactivation curves are shifted to more depolarized potentials without affects on either the rate or voltage dependence of channel opening. Differences in voltage-dependent inactivation between alpha(1B) splice variants are most clearly manifested in the presence of Ca channel beta(1b) or beta(4), rather than beta(2a) or beta(3), subunits. These results suggest that exon-lacking alpha(1B) splice variants that associate with beta(1b) and beta(4) subunits will be susceptible to voltage-dependent inactivation at voltages in the range of neuronal resting membrane potentials (-60 to -80 mV). In contrast, alpha(1B) splice variants that associate with either beta(2a) or beta(3) subunits will be relatively resistant to inactivation at these voltages. The potential to mix and match multiple alpha(1B) splice variants and beta subunits probably represents a mechanism for controlling the plasticity of excitation-secretion coupling at different synapses (Pan, 2000).

The localization of voltage-gated calcium channel (VGCC) alpha(1) subunits in cultured GABAergic mouse cortical neurons was examined by immunocytochemical methods. Ca(v)1.2 and Ca(v)1.3 subunits of L-type VGCCs were found in cell bodies and dendrites of GABA-immunopositive neurons. Likewise, the Ca(v)2.3 subunit of R-type VGCCs is expressed in a somatodendritic pattern. Ca(v)2.2 subunits of N-type channels are found exclusively in small varicosities that were identified as presynaptic nerve terminals based on their expression of synaptic marker proteins. Two splice variants of the Ca(v)2.1 subunit of P/Q-type VGCCs show widely differing expression patterns. The rbA isoform displays a purely somatodendritic staining pattern, whereas the BI isoform is confined to axon-like fibers and nerve terminals. The nerve terminals of these cultured GABAergic neurons express Ca(v)2.2 either alone or in combination with Ca(v)2.1 (BI isoform) but never express Ca(v)2.1 alone. The functional association between VGCCs and the neurotransmitter release machinery was probed using the FM1-43 dye-labeling technique. N-type VGCCs are found to be tightly coupled to exocytosis in these cultured cortical neurons, and P-type VGCCs are also important in a fraction of the cells. The predominant role of N-type VGCCs in neurotransmitter release and the specific localization of the BI isoform of Ca(v)2.1 in the nerve terminals of these neurons distinguish them from previously studied central neurons. The complementary localization patterns observed for two different isoforms of the Ca(v)2.1 subunits provide direct evidence for alternative splicing as a means of generating functional diversity among neuronal calcium channels (Timmermann, 2002).

The Ca(V)2 family of voltage-gated calcium channels, present in presynaptic nerve terminals, regulates exocytosis and synaptic transmission. Cumulative inactivation of these channels occurs during trains of action potentials, and this may control short-term dynamics at the synapse. Inactivation during brief, repetitive stimulation is primarily attributed to closed-state inactivation, and several factors modulate the susceptibility of voltage-gated calcium channels to this form of inactivation. Alternative splicing of an exon in a cytoplasmic region of the Ca(V)2.2 channel modulates its sensitivity to inactivation during trains of action potential waveforms. The presence of this exon, exon 18a, protects the Ca(V)2.2 channel from entry into closed-state inactivation specifically during short (10 ms to 3 s) and small depolarizations of the membrane potential (-60 mV to -50 mV). The reduced sensitivity to closed-state inactivation within this dynamic range likely underlies the differential responsiveness of Ca(V)2.2 splice isoforms to trains of action potential waveforms. Regulated alternative splicing of Ca(V)2.2 represents a possible mechanism for modulating short-term dynamics of synaptic efficacy in different regions of the nervous system (Thaler, 2004).

The CaV2.2 gene encodes the functional core of the N-type calcium channel. This gene has the potential to generate thousands of CaV2.2 splice isoforms with different properties. However, the functional significance of most sites of alternative splicing is not established. The IVS3-IVS4 region contains an alternative splice site that is conserved evolutionarily among CaValpha1 genes from Drosophila to human. In CaV2.2, inclusion of exon 31a in the IVS3-IVS4 region is restricted to the peripheral nervous system, and its inclusion slows the speed of channel activation. To investigate the effects of exon 31a in more detail, four tsA201 cell lines were generated that stably express CaV2.2 splice isoforms. Coexpression of auxiliary CaVbeta and CaValpha2delta subunits is required to reconstitute currents with the kinetics of N-type channels from neurons. Channels including exon 31a activate and deactivate more slowly at all voltages. Current densities were high enough in the stable cell lines co-expressing CaValpha2delta to resolve gating currents. The steady-state voltage dependence of charge movement is not consistently different between splice isoforms, but on gating currents from the exon 31a-containing CaV2.2 isoform decay with a slower time course, corresponding to slower movement of the charge sensor. Exon 31a-containing CaV2.2 is restricted to peripheral ganglia; and the slower gating kinetics of CaV2.2 splice isoforms containing exon 31a correlate reasonably well with the properties of native N-type currents in sympathetic neurons. These results suggest that alternative splicing in the S3-S4 linker influences the kinetics but not the voltage dependence of N-type channel gating (Lin, 2004).

Calcium channel activity is modulated by G-proteins

The inhibition of presynaptic calcium channels via G-protein-dependent second messenger pathways is a key mechanism of transmitter release modulation. The calyx-type nerve terminal of the chick ciliary ganglion was used to examine which G-proteins are involved in the voltage-sensitive inhibition of presynaptic N-type calcium channels. Adenosine causes a prominent inhibition of the calcium current that is totally blocked by pretreatment with pertussis toxin (PTX), consistent with an exclusive involvement of Go/Gi in the G-protein pathway. Immunocytochemistry was used to localize these G-protein types to the nerve terminal and its transmitter release face. Two approaches were used to test for modulation by other G-protein types. (1) The terminals were treated with ligands for a variety of G-protein-linked neurotransmitter receptor types that have been associated with different G-protein families. Although small inhibitory effects are observed, these can all be eliminated by PTX, indicating that in this terminal the Gi family is the sole transmitter-induced G-protein inhibitory pathway. (2) The kinetics of calcium channel inhibition was examined by uncaging the nonselective and irreversible G-protein activator GTPgammaS, bypassing the receptors. A large fraction of the rapid GTPgammaS-induced inhibition persists, consistent with a Go/Gi-independent pathway. Immunocytochemistry identified Gq, G11, G12, and G13 as potential PTX-insensitive second messengers at this terminal. Thus, these results suggest that whereas neurotransmitter-mediated calcium channel inhibition is mainly, and possibly exclusively, via Go/Gi, other rapid PTX-insensitive G-protein pathways exist that may involve novel, and perhaps transmitter-independent, activating mechanisms (Mirotznik, 2000).

Voltage-dependent G-protein inhibition of presynaptic Ca2+ channels is a key mechanism for regulating synaptic efficacy. G-protein betagamma subunits produce such inhibition by binding to and shifting channel opening patterns from high to low open probability regimes, known respectively as 'willing' and 'reluctant' modes of gating. Recent macroscopic electrophysiological data hint that only N-type, but not P/Q-type channels can open in the reluctant mode, a distinction that could enrich the dimensions of synaptic modulation arising from channel inhibition. Here, using high-resolution single-channel recording of recombinant channels, this core contrast in the prevalence of reluctant openings is directly distinguished. Single, inhibited N-type channels manifest relatively infrequent openings of submillisecond duration (reluctant openings), which differ sharply from the high-frequency, millisecond gating events characteristic of uninhibited channels. By contrast, inhibited P/Q-type channels are electrically silent at the single-channel level. The functional impact of the differing inhibitory mechanisms is revealed in macroscopic Ca2+ currents evoked with neuronal action potential waveforms (APWs). Fitting with a change in the manner of opening, inhibition of such N-type currents produces both decreased current amplitude and temporally advanced waveform, effects that would not only reduce synaptic efficacy, but also influence the timing of synaptic transmission. However, inhibition of P/Q-type currents evoked by APWs show diminished amplitude without shape alteration, as expected from a simple reduction in the number of functional channels. Variable expression of N- and P/Q-type channels at spatially distinct synapses therefore offers the potential for custom regulation of both synaptic efficacy and synchrony, by G-protein inhibition (Colecraft, 2001).

N-type Ca2+ channels can be inhibited by neurotransmitter-induced release of G protein betagamma subunits. Two isoforms of Ca(v)2.2 alpha1 subunits of N-type calcium channels from rat brain [Ca(v)2.2a and Ca(v)2.2b; initially termed rbB-I and rbB-II] have different functional properties. Unmodulated Ca(v)2.2b channels are in an easily activated 'willing' (W) state with fast activation kinetics and no prepulse facilitation. Activating G proteins, shifts Ca(v)2.2b channels to a difficult to activate 'reluctant' (R) state with slow activation kinetics; they can be returned to the W state by strong depolarization resulting in prepulse facilitation. This contrasts with Ca(v)2.2a channels, which are tonically in the R state and exhibit strong prepulse facilitation. Activating or inhibiting G proteins has no effect. Thus, the R state of Ca(v)2.2a and its reversal by prepulse facilitation are intrinsic to the channel and independent of G protein modulation. Mutating G177 in segment IS3 of Ca(v)2.2b to E as in Ca(v)2.2a converts Ca(v)2.2b tonically to the R state, insensitive to further G protein modulation. The converse substitution in Ca(v)2.2a, E177G, converts it to the W state and restores G protein modulation. It is proposed that negatively charged E177 in IS3 interacts with a positive charge in the IS4 voltage sensor when the channel is closed and produces the R state of Ca(v)2.2a by a voltage sensor-trapping mechanism. G protein betagamma subunits may produce reluctant channels by a similar molecular mechanism (Zhong, 2001).

Presynaptic Ca2+ channels are inhibited by neurotransmitters acting through G protein-coupled receptors via a membrane-delimited pathway. Inhibition is reversed by strong depolarization, resulting in prepulse facilitation. Activated G protein betagamma subunits (Gbetagamma) are required for maximal prepulse facilitation. Gbetagamma binds to multiple sites on Ca(v)2.1, Ca(v)2.2, and Ca(v)2.3 alpha1 subunits. The functional relevance of a C-terminal binding site for Gbetagamma on Ca(v)2.2b channels, which mediate N-type Ca2+ currents, were examined. In vitro binding studies showed that Gbetagamma subunits bind to the intracellular loop connecting domains I and II and the C-terminal domain of Ca(v)2.2b but not the intracellular loops connecting domains II and III or III and IV. Deletion analysis revealed that the binding site is located near the C terminus, within amino acid residues 2257 to 2336. Directed yeast two-hybrid analysis confirmed this specific binding interaction in vivo in yeast cells. Ca(v)2.2b channels with this site deleted have normal function properties, and they are inhibited essentially normally by strong activation of G proteins with guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) and are facilitated nearly normally by depolarizing prepulses. Similarly deletion of this site has small, statistically insignificant effects on inhibition of Ca2+ current and on prepulse facilitation in the presence of somatostatin to stimulate receptor-mediated activation of G proteins. In contrast, deletion of the C-terminal Gbetagamma site substantially reduces the low level of intrinsic prepulse facilitation present at the basal level of G protein activation in tsA-201 cells. Thus, this C-terminal Gbetagamma binding site contributes to the affinity or efficacy of Gbetagamma regulation at basal levels of G protein activation. The simplest interpretation of these results is that the C-terminal binding site increases the affinity of Gbetagamma for the channel but is not required for Gbetagamma action. C-terminal binding of Gbetagamma may influence the physiological responsiveness of Ca2+ channels to low-level G protein activation (Li, 2004).

Presynaptic calcium influx at most excitatory central synapses is carried by both Cav2.1 and Cav2.2 channels. The kinetics and modulation of Cav2.1 and Cav2.2 channels differ and may affect presynaptic calcium influx. Release dynamics at CA3/CA1 synapses in rat hippocampus after selective blockade of either channel subtype and subsequent quantal content restoration were compared. Selective blockade of Cav2.1 channels enhanced paired-pulse facilitation, whereas blockade of Cav2.2 channels decreased it. This effect was observed at short (50 msec) but not longer (500 msec) intervals and was maintained during prolonged bursts of presynaptic activity. It did not reflect differences in the distance of the channels from the calcium sensor. The suppression of this effect by preincubation with the Go/i-protein inhibitor pertussis toxin suggests instead that high-frequency stimulation relieves inhibition of Cav2.2 by Go/i, thereby increasing the number of available channels (Scheuber, 2004).

L-type dihydropyridine-sensitive voltage dependent Ca2+ channels [L-VDCCs; alpha(1C)] are crucial in cardiovascular physiology. Currents via L-VDCCs are enhanced by hormones and transmitters operating via G(q), such as angiotensin II (AngII) and acetylcholine (ACh). It has been proposed that these modulations are mediated by protein kinase C (PKC). However, reports on effects of PKC activators on L-type channels are contradictory: inhibitory and/or enhancing effects have been observed. Attempts to reproduce the enhancing effect of AngII in heterologous expression systems failed. PKC modulation of the channel depends on alpha(1C) isoform used; only a long N-terminal (NT) isoform is up-regulated. This study reports the reconstitution of the AngII- and ACh-induced enhancement of the long-NT isoform of L-VDCC expressed in Xenopus oocytes. The current initially increases over several minutes but later declines to below baseline levels. Using different NT deletion mutants and human short- and long-NT isoforms of the channel, it was found the the initial segment of the NT is crucial for the enhancing, but not for the inhibitory, effect. Using blockers of PKC and of phospholipase C (PLC) and a mutated AngII receptor lacking G(q) coupling, it was demonstrated that the signaling pathway of the enhancing effect includes the activation of G(q), PLC, and PKC. The inhibitory modulation, present in both alpha(1C) isoforms, is G(q)- and PLC-independent and Ca2+-dependent, but not Ca2+-mediated, because only basal levels of Ca2+ are essential. Reconstitution of AngII and ACh effects in Xenopus oocytes will advance the study of molecular mechanisms of these physiologically important modulations (Weiss, 2004).

Calcium channel interaction with calmodulin

Neurotransmitter release at many central synapses is initiated by an influx of calcium ions through P/Q-type calcium channels, which are densely localized in nerve terminals. Because neurotransmitter release is proportional to the fourth power of calcium concentration, regulation of its entry can profoundly influence neurotransmission. N- and P/Q-type calcium channels are inhibited by G proteins, and recent evidence indicates feedback regulation of P/Q-type channels by calcium. Although calcium-dependent inactivation of L-type channels is well documented, little is known about how calcium modulates P/Q-type channels. A calcium-dependent interaction between calmodulin and a novel site in the carboxy-terminal domain of the alpha1A subunit of P/Q-type channels is reported. In the presence of low concentrations of intracellular calcium chelators, calcium influx through P/Q-type channels enhances channel inactivation, increases recovery from inactivation and produces a long-lasting facilitation of the calcium current. These effects are prevented by overexpression of a calmodulin-binding inhibitor peptide and by deletion of the calmodulin-binding domain. These results reveal an unexpected association of Ca2+/calmodulin with P/Q-type calcium channels that may contribute to calcium-dependent synaptic plasticity (Lee, 1999).

L-type Ca2+ channels are unusual in displaying two opposing forms of autoregulatory feedback, Ca2+-dependent inactivation and facilitation. Previous studies suggest that both involve direct interactions between calmodulin (CaM) and a consensus CaM-binding sequence (IQ motif) in the C terminus of the channel's alpha(1C) subunit. This study reports the functional effects of an extensive series of modifications of the IQ motif aimed at dissecting the structural determinants of the different forms of modulation. Although the combined substitution by alanine at five key positions [Ile(1624), Gln(1625), Phe(1628), Arg(1629), and Lys(1630)] abolishes all Ca2+ dependence, corresponding single alanine replacements behaves similarly to the wild-type channel (77wt) in four of five cases. The mutant I1624A stands out in displaying little or no Ca2+-dependent inactivation, but clear Ca2+- and frequency-dependent facilitation. An even more pronounced tilt in favor of facilitation is seen with the double mutant I1624A/Q1625A: overt facilitation is observed even during a single depolarizing pulse, as confirmed by two-pulse experiments. Replacement of Ile(1624) by 13 other amino acids produces graded and distinct patterns of change in the two forms of modulation. The extent of Ca2+-dependent facilitation is monotonically correlated with the affinity of CaM for the mutant IQ motif, determined in peptide binding experiments in vitro. Ca2+-dependent inactivation also depends on strong CaM binding to the IQ motif, but shows an additional requirement for a bulky, hydrophobic side chain at position 1624. Abolition of Ca2+-dependent modulation by IQ motif modifications mimics and occludes the effects of overexpressing a dominant-negative CaM mutant (Zuhlke, 2000).

Acute modulation of P/Q-type (alpha1A) calcium channels by neuronal activity-dependent changes in intracellular Ca2+ concentration may contribute to short-term synaptic plasticity, potentially enriching the neurocomputational capabilities of the brain. An unconventional mechanism for such channel modulation has been proposed in which calmodulin (CaM) may exert two opposing effects on individual channels, initially promoting ('facilitation') and then inhibiting ('inactivation') channel opening. Such dual regulation arises from three surprising Ca2+-transduction capabilities of CaM. (1) Although facilitation and inactivation are two competing processes, both require Ca2+-CaM binding to a single 'IQ-like' domain on the carboxy tail of alpha1A; a previously identified 'CBD' CaM-binding site has no detectable role. (2) Expression of a CaM mutant with impairment of all four of its Ca2+-binding sites (CaM1234) eliminates both forms of modulation. This result confirms that CaM is the Ca2+ sensor for channel regulation, and indicates that CaM may associate with the channel even before local Ca2+ concentration rises. (3) The bifunctional capability of CaM arises from bifurcation of Ca2+ signaling by the lobes of CaM: Ca2+ binding to the amino-terminal lobe selectively initiates channel inactivation, whereas Ca2+ sensing by the carboxy-terminal lobe induces facilitation. Such lobe-specific detection provides a compact means to decode local Ca2+ signals in two ways, and to separately initiate distinct actions on a single molecular complex (DeMaria, 2001).

Among the most intriguing forms of Ca2+ channel modulation is the regulation of L-type and P/Q-type channels by intracellular Ca2+, acting via unconventional channel-calmodulin (CaM) interactions. In particular, overexpressing Ca2+-insensitive mutant CaM abolishes Ca2+-dependent modulation, hinting that Ca2+-free CaM may 'preassociate' with these channels to enhance detection of local Ca2+. Despite the far-reaching consequences of this proposal, in vitro experiments testing for preassociation provide conflicting results. A three filter-cube fluorescence resonance energy transfer method (three-cube FRET) has been developed to directly probe for constitutive associations between channel subunits and CaM in single living cells. This FRET assay detects Ca2+-independent associations between CaM and the pore-forming alpha(1) subunit of L-type, P/Q-type, and, surprisingly, R-type channels. These results now definitively demonstrate channel-CaM preassociation in resting cells and underscore the potential of three-cube FRET for probing protein-protein interactions (Erickson, 2001).

Ca(v)2.1 channels, which mediate P/Q-type Ca2+ currents, undergo Ca2+/calmodulin (CaM)-dependent inactivation and facilitation that can significantly alter synaptic efficacy. The neuronal Ca2+-binding protein 1 (CaBP1) modulates Ca(v)2.1 channels in a manner that is markedly different from modulation by CaM. CaBP1 enhances inactivation, causes a depolarizing shift in the voltage dependence of activation, and does not support Ca2+-dependent facilitation of Ca(v)2.1 channels. These inhibitory effects of CaBP1 do not require Ca2+, but depend on the CaM-binding domain in the alpha1 subunit of Ca(v)2.1 channels (alpha12.1). CaBP1 binds to the CaM-binding domain, co-immunoprecipitates with alpha12.1 from transfected cells and brain extracts, and colocalizes with alpha12.1 in discrete microdomains of neurons in the hippocampus and cerebellum. These results identify an interaction between Ca2+ channels and CaBP1 that may regulate Ca2+-dependent forms of synaptic plasticity by inhibiting Ca2+ influx into neurons (Lee, 2002).

L-type (CaV1.2) and P/Q-type (CaV2.1) calcium channels possess lobe-specific CaM regulation, where Ca2+ binding to one or the other lobe of CaM triggers regulation, even with inverted polarity of modulation between channels. Other major members of the CaV1-2 channel family, R-type (CaV2.3) and N-type (CaV2.2), have appeared to lack such CaM regulation. R- and N-type channels undergo Ca2+-dependent inactivation, which is mediated by the CaM N-terminal lobe and present only with mild Ca2+ buffering (0.5 mM EGTA) characteristic of many neurons. These features, together with the CaM regulatory profiles of L- and P/Q-type channels, are consistent with a simplifying principle for CaM signal detection in CaV1-2 channels -- independent of channel context, the N- and C-terminal lobes of CaM appear invariably specialized for decoding local versus global Ca2+ activity, respectively (Liang, 2003).

Calcium channel interaction with syntaxin

N-type Ca2+ channels bind directly to the synaptic core complex of VAMP/synaptobrevin, syntaxin, and SNAP-25. Peptides containing the synaptic protein interaction ('synprint') site cause dissociation of N-type Ca2+ channels from the synaptic core complex. Introduction of synprint peptides into presynaptic superior cervical ganglion neurons reversibly inhibits synaptic transmission. Fast EPSPs due to synchronous transmitter release are inhibited, while late EPSPs arising from asynchronous release following a train of action potentials are increased and paired-pulse facilitation is increased. The corresponding peptides from L-type Ca2+ channels have no effect, and the N-type peptides have no effect on Ca2+ currents through N-type Ca2+ channels. These results are consistent with the hypothesis that binding of the synaptic core complex to presynaptic N-type Ca2+ channels is required for Ca2+ influx to elicit rapid, synchronous neurotransmitter release (Mochida, 1996).

Presynaptic N-type calcium channels interact with syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) through a binding site in the intracellular loop connecting domains II and III of the alpha1 subunit. This binding region was loaded into embryonic spinal neurons of Xenopus by early blastomere injection. After culturing, synaptic transmission of peptide-loaded and control cells was compared by measuring postsynaptic responses under different external Ca2+ concentrations. The relative transmitter release of injected neurons was reduced by approximately 25% at physiological Ca2+ concentration, whereas injection of the corresponding region of the L-type Ca2+ channel had virtually no effect. When applied to a theoretical model, these results imply that 70% of the formerly linked vesicles have been uncoupled after action of the peptide. These data suggest that severing the physical interaction between presynaptic calcium channels and synaptic proteins will not prevent synaptic transmission at this synapse but will make it less efficient by shifting its Ca2+ dependence to higher values (Rettig, 1997).

Syntaxin is a key presynaptic protein that binds to N- and P/Q-type Ca2+ channels in biochemical studies and affects gating of these Ca2+ channels in expression systems and in synaptosomes. The present study was aimed at understanding the molecular basis of syntaxin modulation of N-type channel gating. Mutagenesis of either syntaxin 1A or the pore-forming alpha(1B) subunit of N-type Ca2+ channels was combined with functional assays of N-type channel gating in a Xenopus oocyte coexpression system and in biochemical binding experiments in vitro. This analysis showed that the transmembrane region of syntaxin and a short region within the H3 helical cytoplasmic domain of syntaxin, containing residues Ala-240 and Val-244, appears critical for the channel modulation but not for biochemical association with the 'synprint site' in the II/III loop of alpha(1B). These results suggest that syntaxin and the alpha(1B) subunit engage in two kinds of interactions: an anchoring interaction via the II/III loop synprint site and a modulatory interaction via another site located elsewhere in the channel sequence. The segment of syntaxin H3 found to be involved in the modulatory interaction would lie hidden within the four-helix structure of the SNARE complex, supporting the hypothesis that syntaxin's ability to regulate N-type Ca2+ channels would be enabled after SNARE complex disassembly after synaptic vesicle exocytosis (Bezprovzvanny, 2000).

Syntaxin, a membrane protein vital in triggering vesicle fusion, interacts with voltage-gated N- and P/Q-type Ca2+ channels. This biochemical association is proposed to colocalize Ca2+ channels and presynaptic release sites, thus supporting rapid and efficient initiation of neurotransmitter release. The syntaxin channel interaction may also support a novel signaling function, to modulate Ca2+ channels according to the state of the associated release machinery. Syntaxin 1A (syn1A) coexpressed with N-type channels in Xenopus oocytes greatly promotes slow inactivation gating, but has little or no effect on the onset of and recovery from fast inactivation. Accordingly, the effectiveness of syntaxin depends strongly on voltage protocol. Slow inactivation is found for N-type channels even in the absence of syntaxin and can be distinguished from fast inactivation on the basis of its slow kinetics, distinct voltage dependence (voltage-independent at potentials higher than the level of half-inactivation), and temperature independence [Q(10), approximately 0.8]. Trains of action potential-like stimuli are more effective than steady depolarizations in stabilizing the slowly inactivated condition. Agents that stimulate protein kinase C decrease the inhibitory effect of syntaxin on N-type channels. Application of BoNtC1 to cleave syntaxin sharply attenuates the modulatory effects on Ca2+ channel gating, consistent with structural analysis of syntaxin modulation, supporting use of this toxin to test for the impact of syntaxin on Ca2+ influx in nerve terminals (Degtiar, 2000)

N-type Ca2+ channels are modulated by a variety of G-protein-coupled pathways. Some pathways produce a transient, voltage-dependent (VD) inhibition of N channel function and involve direct binding of G-protein subunits; others require the activation of intermediate enzymes and produce a longer-lasting, voltage-independent (VI) form of inhibition. The ratio of VD:VI inhibition differs significantly among cell types, suggesting that the two forms of inhibition play unique physiological roles in the nervous system. Mechanisms capable of altering the balance of VD and VI inhibition in chick dorsal root ganglion neurons have been explored. VD:VI inhibition is critically dependent on the Gbetagamma concentration, with VI inhibition dominant at low Gbetagamma concentrations. Syntaxin-1A (but not syntaxin-1B) shifts the ratio in favor of VD inhibition by potentiating the VD effects of Gbetagamma. Variations in expression levels of G-proteins and/or syntaxin provide the means to alter over a wide range both the extent and the rate of Ca2+ influx through N channels (Lü, 2001).

Syntaxin 1A, a component of the presynaptic SNARE complex, directly modulates N-type calcium channel gating in addition to promoting tonic G-protein inhibition of the channels, whereas syntaxin 1B affects channel gating but does not support G-protein modulation. The molecular determinants that govern the action of syntaxin 1 isoforms on N-type calcium channel function have been investigated. In vitro evidence shows that both syntaxin 1 isoforms physically interact with the G-protein beta subunit and the synaptic protein interaction (synprint) site contained within the N-type calcium channel domain II-III linker region. Moreover, in vitro evidence suggests that distinct domains of syntaxin participate in each interaction, with the COOH-terminal SNARE domain (residues 183-230) binding to Gbeta and the N-terminal (residues 1-69) binding to the synprint motif of the channel. Electrophysiological analysis of chimeric syntaxin 1A/1B constructs reveals that the variable NH(2)-terminal domains of syntaxin 1 are responsible for the differential effects of syntaxin 1A and 1B on N-type calcium channel function. Because syntaxin 1 exists in both 'open' and 'closed' conformations during exocytosis, a constitutively open form of syntaxin 1A was produced; it still promoted G-protein inhibition of the channels, but it did not affect N-type channel availability. This state dependence of the ability of syntaxin 1 to mediate N-type calcium channel availability suggests that syntaxin 1 dynamically regulates N-type channel function during various steps of exocytosis. Finally, syntaxin 1A appears to compete with Ggamma for the Gbeta subunit both in vitro and under physiological conditions, suggesting that syntaxin 1A may contain a G-protein gamma subunit-like domain (Jarvis, 2002).

When the presynaptic membrane protein syntaxin is coexpressed in Xenopus oocytes with N- or P/Q-type Ca2+ channels, it promotes their inactivation. These findings led to the hypothesis that syntaxin influences Ca2+ channel function in presynaptic endings, in a reversal of the conventional flow of information from Ca2+ channels to the release machinery. This effect was examined in isolated mammalian nerve terminals (synaptosomes). Botulinum neurotoxin type C1 (BoNtC1), which cleaves syntaxin, was applied to rat neocortical synaptosomes at concentrations that completely block neurotransmitter release. This treatment alters the pattern of Ca2+ entry monitored with fura-2. Whereas the initial Ca2+ rise induced by depolarization with K(+)-rich solution is unchanged, late Ca2+ entry is strongly augmented by syntaxin cleavage. Similar results were obtained when Ca2+ influx arose from repetitive firing induced by the K(+)-channel blocker 4-aminopyridine. Cleavage of vesicle-associated membrane protein with BoNtD or SNAP-25 with BoNtE failed to produce a significant change in Ca2+ entry. The BoNtC1-induced alteration in Ca2+ signaling is specific to voltage-gated Ca2+ channels, not Ca2+ extrusion or buffering, and it involves N-, P/Q- and R-type channels, the high voltage-activated channels most intimately associated with presynaptic release machinery. The modulatory effect of syntaxin is not immediately manifest when synaptosomes have been K(+)-predepolarized in the absence of external Ca2+, but develop with a delay after admission of Ca2+, suggesting that vesicular turnover may be necessary to make syntaxin available for its stabilizing effect on Ca2+ channel inactivation (Bergsman, 2000).

Calcium channel interaction with RGS proteins

Activation of GABAB receptors in chick DRG neurons inhibits the Cav2.2 calcium channel in both a voltage-dependent and voltage-independent manner. The voltage-independent inhibition requires activation of a tyrosine kinase which phosphorylates the alpha 1 subunit of the channel and thereby recruits RGS12, a member of the "regulator of G protein signaling" (RGS) proteins. RGS12 binds to the SNARE-binding or synprint region (amino acids 726-985) in loop II-III of the calcium channel alpha 1 subunit. Recombinant protein encompassing the N-terminal PTB domain of RGS12 binds to the synprint region in protein overlay and surface plasmon resonance binding assays; this interaction is dependent on tyrosine phosphorylation yet within a sequence that differs from the canonical N-P-x-Y motif targeted by other PTB domains. In electrophysiological experiments, microinjection of DRG neurons with synprint-derived peptides containing the tyrosine residue Y804 alters the rate of desensitization of neurotransmitter-mediated inhibition of the Cav2.2 calcium channel while peptides centered about a second tyrosine residue, Y815, are without effect. RGS12 from DRG neuron lysate was precipitated using synprint peptides containing phosphorylated Y804. The high degree of conservation of Y804 in the SNARE binding region of Cav2.1 and Cav2.2 calcium channels suggests that this region, in addition to the binding of SNARE proteins, is also important for determining the time course of modulation of calcium current via tyrosine phosphorylation (Richman, 2004).

Calcium channel interactions with accessory subunits

Calcium channel beta subunits are key modulators of calcium channel function and membrane targeting of the pore-forming alpha1 subunit. An invertebrate (Lymnaea stagnalis) homolog of P/Q- and N-type calcium channels (LCav2), although colocalized with beta subunits in synapses of mature neurons, is physically uncoupled from the beta subunits in the leading edge of growth cones of outgrowing neurons. Moreover, LCav2 channels that mediate transmitter release in mature synapses also participate in neuronal outgrowth in growth cones. The differential association of beta subunits with synaptic calcium channels and those expressed in emergent neuronal growth suggests that beta subunits may play a role in the transformation of Cav2 calcium channel function in immature neurons and mature synapses (Spafford, 2004).

The omega-conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The omega-conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The beta and alpha(2)delta-1 auxiliary subunits influence the expression and characteristics of the alpha(1B) subunit of N-type channels and are differentially regulated in disease states, including pain. This study examined the influence of these auxiliary subunits on the ability of the omega-conotoxins GVIA, MVIIA, CVID and their analogues to inhibit peripheral and central forms of the rat N-type channels. Although the beta3 subunit has little influence on the on- and off-rates of omega-conotoxins, coexpression of alpha(2)delta with alpha(1B) significantly reduces on-rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The alpha(2)delta also enhances the selectivity of MVIIA, but not CVID, for the central isoform. Similar but less pronounced trends were also observed for N-type channels expressed in human embryonic kidney cells. The influence of alpha(2)delta is not affected by oocyte deglycosylation. The extent of recovery from the omega-conotoxin block is least for GVIA, intermediate for MVIIA, and almost complete for CVID. Application of a hyperpolarizing holding potential (-120 mV) does not significantly enhance the extent of CVID recovery. Interestingly, [R10K]MVIIA and [O10K]GVIA have greater recovery from the block, whereas [K10R]CVID has reduced recovery from the block, indicating that position 10 has an important influence on the extent of omega-conotoxin reversibility. Recovery from CVID block is reduced in the presence of alpha(2)delta in human embryonic kidney cells and in oocytes expressing alpha(1B-b). These results may have implications for the antinociceptive properties of omega-conotoxins, given that the alpha(2)delta subunit is up-regulated in certain pain states (Mould, 2004).

Complex interactions result in modulation of calcium channel activity

N-type and P/Q-type Ca2+ channels are inhibited by neurotransmitters acting through G protein-coupled receptors in a membrane-delimited pathway involving Gbetagamma subunits. Inhibition is caused by a shift from an easily activated 'willing' (W) state to a more-difficult-to-activate 'reluctant' (R) state. This inhibition can be reversed by strong depolarization, resulting in prepulse facilitation, or by protein kinase C (PKC) phosphorylation. Comparison of regulation of N-type Ca2+ channels containing Cav2.2a alpha(1) subunits and P/Q-type Ca2+ channels containing Ca(v)2.1 alpha(1) subunits revealed substantial differences. In the absence of G protein modulation, Ca(v)2.1 channels containing Ca(v)beta subunits are tonically in the W state, whereas Ca(v)2.1 channels without beta subunits and Ca(v)2.2a channels with beta subunits are tonically in the R state. Both Ca(v)2.1 and Ca(v)2.2a channels can be shifted back toward the W state by strong depolarization or PKC phosphorylation. These results show that the R state and its modulation by prepulse facilitation, PKC phosphorylation, and Ca(v)beta subunits are intrinsic properties of the Ca2+ channel itself in the absence of G protein modulation. A common allosteric model of G protein modulation of Ca2+-channel activity is presented incorporating an intrinsic equilibrium between the W and R states of the alpha(1) subunits and modulation of that equilibrium by G proteins, Ca(v)beta subunits, membrane depolarization, and phosphorylation by PKC accommodates these findings. Such regulation will modulate transmission at synapses that use N-type and P/Q-type Ca2+ channels to initiate neurotransmitter release (Herlitze, 2001).

Syntaxin 1A mediates two effects on N-type channels transiently expressed in tsA-201 cells: a hyperpolarizing shift in the steady-state inactivation curve as well as a tonic inhibition of the channel by G-protein betagamma subunits. Some of the molecular determinants and factors that modulate the action of syntaxin 1A on N-type calcium channels have been examined. With the additional coexpression of SNAP25, the syntaxin 1A-induced G-protein modulation of the channel becomes reduced in magnitude by approximately 50% but nonetheless remains significantly higher than the low levels of background inhibition seen with N-type channels alone. In contrast, coexpression of nSec-1 does not reduce the syntaxin 1A-mediated G-protein inhibition; however, interestingly, nSec-1 is able to induce tonic G-protein inhibition even in the absence of syntaxin 1A. Both SNAP25 and nSec-1 block the negative shift in half-inactivation potential that is induced by syntaxin 1A. Activation of protein kinase C via phorbol esters or site-directed mutagenesis of three putative PKC consensus sites in the syntaxin 1A binding region of the channel (S802, S896, S898) to glutamic acid (to mimic a permanently phosphorylated state) did not affect the syntaxin 1A-mediated G-protein modulation of the channel. However, in the S896E and S898E mutants, or after PKC-dependent phosphorylation of the wild-type channels, the susceptibility of the channel to undergo shifts in half-inactivation potential was removed. Thus, separate molecular determinants govern the ability of syntaxin 1A to affect N-type channel gating and its modulation by G-proteins (Jarvis, 2001).

Calcium channels are directly responsive to intracellular calcium levels

L-type [alpha(1C)] calcium channels inactivate rapidly in response to localized elevation of intracellular Ca2+, providing negative Ca2+ feedback in a diverse array of biological contexts. The dominant Ca2+ sensor for such Ca2+-dependent inactivation is calmodulin, which appears to be constitutively tethered to the channel complex. This Ca2+ sensor induces channel inactivation by Ca2+-dependent CaM binding to an IQ-like motif situated on the carboxyl tail of alpha(1C). Apart from the IQ region, another crucial site for Ca2+ inactivation appears to be a consensus Ca2+-binding, EF-hand motif, located approximately 100 amino acids upstream on the carboxyl terminus. However, the importance of this EF-hand motif for channel inactivation has become controversial. This study demonstrates not only that the consensus EF hand is essential for Ca2+ inactivation, but that a four-amino acid cluster (VVTL) within the F helix of the EF-hand motif is itself essential for Ca2+ inactivation. Mutating these amino acids to their counterparts in non-inactivating alpha(1E) calcium channels (MYEM) almost completely ablates Ca2+ inactivation. In fact, only a single amino acid change of the second valine within this cluster to tyrosine (V1548Y) supports much of the functional knockout. However, mutations of presumed Ca2+-coordinating residues in the consensus EF hand reduce Ca2+ inactivation by only approximately 2-fold, fitting poorly with the EF hand serving as a contributory inactivation Ca2+ sensor, in which Ca2+ binds according to a classic mechanism. It is therefore suggested that while CaM serves as Ca2+ sensor for inactivation, the EF-hand motif of alpha(1C) may support the transduction of Ca2+-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed Ca2+-inactivation properties of wild-type and mutant V1548Y channels, as gauged by a novel inactivation model incorporating multivalent Ca2+ binding of CaM (Peterson, 2000).

Calcium channel mutation

The leaner (tgla) mutation in mice results in severe ataxia and an overt neurodegeneration of the cerebellum. Positional cloning has revealed that the tgla mutation occurs in a gene encoding the voltage-activated calcium channel alpha1A subunit. The alpha1A subunit is highly expressed in the cerebellum and is thought to be the pore-forming subunit of P- and Q-type calcium channels. In this study both whole-cell and single-channel patch-clamp recordings were used to examine the functional consequences of the tgla mutation on P-type calcium currents. High-voltage-activated (HVA) calcium currents were recorded from acutely dissociated cerebellar Purkinje cells of homozygous leaner (tgla/tgla) and age-matched wild-type (+/+) mice. In whole cell recordings, a marked reduction of peak current density is observed in tgla/tgla Purkinje cells (-35.0 +/- 1.8 pA/pF) relative to that in +/+ (-103.1 +/- 5.9 pA/pF). The reduced whole-cell current in tgla/tgla cells is accompanied by little to no alteration in the voltage dependence of channel gating. In both genotypes, HVA currents are predominantly of the omega-agatoxin-IVA-sensitive P-type. Cell-attached patch-clamp recordings revealed no differences in single-channel conductance between the two genotypes and confirmed the presence of three distinct conductance levels (9, 13-14, and 17-18 pS) in cerebellar Purkinje cells. Analysis of patch open-probability (NPo) revealed a threefold reduction in the open-probability of channels in tgla/tgla patches (0.04 +/- 0.01) relative to that in +/+ (0.13 +/- 0.02), which may account for the reduced whole-cell current in tgla/tgla Purkinje cells. These results suggest that the tgla mutation can alter native P-type calcium channels at the single-channel level and that these alterations may contribute to the neuropathology of the leaner phenotype (Dove, 1998).

The Ca2+ channel alpha(1A)-subunit is a voltage-gated, pore-forming membrane protein positioned at the intersection of two important lines of research: one exploring the diversity of Ca2+ channels and their physiological roles, and the other pursuing mechanisms of ataxia, dystonia, epilepsy, and migraine. Alpha(1A)-Subunits are thought to support both P- and Q-type Ca2+ channel currents, but the most direct test, a null mutant, has not been described, nor is it known which changes in neurotransmission might arise from elimination of the predominant Ca2+ delivery system at excitatory nerve terminals. Alpha(1A)-deficient mice [alpha(1A)(-/-)] have been generated; they developed a rapidly progressive neurological deficit with specific characteristics of ataxia and dystonia before dying approximately 3-4 weeks after birth. P-type currents in Purkinje neurons and P- and Q-type currents in cerebellar granule cells are eliminated completely whereas other Ca2+ channel types, including those involved in triggering transmitter release, also undergo concomitant changes in density. Synaptic transmission in alpha(1A)(-/-) hippocampal slices persists despite the lack of P/Q-type channels but shows enhanced reliance on N-type and R-type Ca2+ entry. The alpha(1A)(-/-) mice provide a starting point for unraveling neuropathological mechanisms of human diseases generated by mutations in alpha(1A) (Jun, 1999).

Mutations of the alpha1A calcium channel subunit have been shown to cause such human neurological diseases as familial hemiplegic migraine, episodic ataxia-2, and spinocerebellar ataxia 6 and also to cause the murine neurological phenotypes of tottering and leaner. The leaner phenotype is recessive and characterized by ataxia with cortical spike and wave discharges (similar to absence epilepsy in humans) and a gradual degeneration of cerebellar Purkinje and granule cells. The mutation responsible is a single-base substitution that produces truncation of the normal open reading frame beyond repeat IV and expression of a novel C-terminal sequence. Whole-cell recordings have been used to determine whether the leaner mutation alters calcium channel currents in cerebellar Purkinje cells, both because these cells are profoundly affected in leaner mice and because they normally express high levels of alpha1A. In Purkinje cells from normal mice, 82% of the whole-cell current was blocked by 100 nM omega-agatoxin-IVA. In Purkinje cells from homozygous leaner mice, this omega-agatoxin-IVA-sensitive current was 65% smaller than in control cells. Although attenuated, the omega-agatoxin-IVA-sensitive current in homozygous leaner cells has properties indistinguishable from that of normal Purkinje neurons. Additionally, the omega-agatoxin-IVA-insensitive current is unaffected in homozygous leaner mice. Thus, the leaner mutation selectively reduces P-type currents in Purkinje cells, and the alpha1A subunit and P-type current appear to be essential for normal cerebellar function (Lorenzon, 1999).

alpha(1) subunit of the voltage-dependent Ca2+ channel is essential for channel function and determines the functional specificity of various channel types. alpha(1E) subunit was originally identified as a neuron-specific one, but the physiological function of the Ca2+ channel containing this subunit [alpha(1E) Ca2+ channel] was not clear compared with other types of Ca2+ channels because of the limited availability of specific blockers. To clarify the physiological roles of the alpha(1E) Ca2+ channel, alpha(1E) mutant [alpha(1E)-/-] mice were generated by gene targeting. The lacZ gene was inserted in-frame and used as a marker for alpha(1E) subunit expression. alpha(1E)-/- mice show reduced spontaneous locomotor activities and signs of timidness, but other general behaviors are apparently normal. As involvement of alpha(1E) in pain transmission is suggested by localization analyses with 5-bromo-4-chloro-3-indolyl beta-d-galactopyranoside staining, several pain-related behavioral tests were conducted using the mutant mice. Although alpha(1E)+/- and alpha(1E)-/- mice exhibit normal pain behaviors against acute mechanical, thermal, and chemical stimuli, they both show reduced responses to somatic inflammatory pain. alpha(1E)+/- mice show reduced response to visceral inflammatory pain, whereas alpha(1E)-/- mice showed apparently normal response compared with that of wild-type mice. Furthermore, alpha(1E)-/- mice that had been presensitized with a visceral noxious conditioning stimulus showed increased responses to a somatic inflammatory pain, in marked contrast with the wild-type mice in which long-lasting effects of descending antinociceptive pathway were predominant. These results suggest that the alpha(1E) Ca(2 +) channel controls pain behaviors by both spinal and supraspinal mechanisms (Saegusa, 2000).

N-type voltage-dependent Ca2+ channels (VDCCs), predominantly localized in the nervous system, have been considered to play an essential role in a variety of neuronal functions, including neurotransmitter release at sympathetic nerve terminals. As a direct approach to elucidating the physiological significance of N-type VDCCs, mice genetically deficient in the alpha(1B) subunit [Ca(v) 2.2] have been generated. The alpha(1B)-deficient null mice, surprisingly, have a normal life span and are free from apparent behavioral defects. A complete and selective elimination of N-type currents, sensitive to omega-conotoxin GVIA, was observed without significant changes in the activity of other VDCC types in neuronal preparations of mutant mice. The baroreflex response, mediated by the sympathetic nervous system, is markedly reduced after bilateral carotid occlusion. In isolated left atria prepared from N-type-deficient mice, the positive inotropic responses to electrical sympathetic neuronal stimulation are dramatically decreased compared with those of normal mice. In contrast, parasympathetic nervous activity in the mutant mice is nearly identical to that of wild-type mice. Interestingly, the mutant mice showed sustained elevation of heart rate and blood pressure. These results provide direct evidence that N-type VDCCs are indispensable for the function of the sympathetic nervous system in circulatory regulation and indicate that N-type VDCC-deficient mice will be a useful model for studying disorders attributable to sympathetic nerve dysfunction (Ino, 2001).

The expansion of polyglutamine tracts encoded by CAG trinucleotide repeats is a common mutational mechanism in inherited neurodegenerative diseases. Spinocerebellar ataxia type 6 (SCA6), an autosomal dominant, progressive disease, arises from trinucleotide repeat expansions present in the coding region of CACNA1A (chromosome 19p13). This gene encodes alpha(1A), the principal subunit of P/Q-type Ca2+ channels, which are abundant in the CNS, particularly in cerebellar Purkinje and granule neurons. Ion channel function was assayed by introduction of human alpha(1A) cDNAs in human embryonic kidney 293 cells that stably coexpressed beta(1) and alpha(2)delta subunits. Immunocytochemical analysis showed a rise in intracellular and surface expression of alpha(1A) protein when CAG repeat lengths reach or exceed the pathogenic range for SCA6. This gain at the protein level is not a consequence of changes in RNA stability. The electrophysiological behavior of alpha(1A) subunits containing expanded (EXP) numbers of CAG repeats (23, 27, and 72) was compared against that of wild-type subunits (WT) (4 and 11 repeats) using standard whole-cell patch-clamp recording conditions. The EXP alpha(1A) subunits yield functional ion channels that support inward Ca2+ channel currents, with a sharp increase in P/Q Ca2+ channel current density relative to WT. These results show that Ca2+ channels from SCA6 patients display near-normal biophysical properties but increased current density attributable to elevated protein expression at the cell surface (Piedras-Renteria, 2001).

Ca2+-dependent inactivation (CDI) of L-type Ca2+ channels plays a critical role in controlling Ca2+ entry and downstream signal transduction in excitable cells. Ca2+-insensitive forms of calmodulin (CaM) act as dominant negatives to prevent CDI, suggesting that CaM acts as a resident Ca2+ sensor. However, it is not known how the Ca2+ sensor is constitutively tethered. The tethering of Ca2+-insensitive CaM has been localized to the C-terminal tail of alpha(1C), close to the CDI effector motif, and it depends on nanomolar Ca2+ concentrations, likely attained in quiescent cells. Two stretches of amino acids are found to support the tethering and to contain putative CaM-binding sequences close to or overlapping residues previously shown to affect CDI and Ca2+-independent inactivation. Synthetic peptides containing these sequences display differences in CaM-binding properties, both in affinity and Ca2+ dependence, leading to the proposal of a novel mechanism for CDI. In contrast to a traditional disinhibitory scenario, it is suggested that apoCaM is tethered at two sites and signals actively to slow inactivation. When the C-terminal lobe of CaM binds to the nearby CaM effector sequence (IQ motif), the braking effect is relieved, and CDI is accelerated (Pitt, 2001).

Rocker (gene symbol rkr), a new neurological mutant phenotype, was found in descendents of a chemically mutagenized male mouse. Mutant mice display an ataxic, unstable gait accompanied by an intention tremor, typical of cerebellar dysfunction. These mice are fertile and appear to have a normal life span. Segregation analysis reveals rocker to be an autosomal recessive trait. The overall cytoarchitecture of the young adult brain appears normal, including its gross cerebellar morphology. Golgi-Cox staining, however, reveals dendritic abnormalities in the mature cerebellar cortex characterized by a reduction of branching in the Purkinje cell dendritic arbor and a 'weeping willow' appearance of the secondary branches. Using simple sequence length polymorphism markers, the rocker locus was mapped to mouse chromosome 8 within 2 centimorgans of the calcium channel alpha1a subunit (Cacna1a, formerly known as tottering) locus. Complementation tests with the leaner mutant allele (Cacna1ala) produced mutant animals, thus identifying rocker as a new allele of Cacna1a (Cacna1arkr). Sequence analysis of the cDNA revealed rocker to be a point mutation resulting in an amino acid exchange: T1310K between transmembrane regions 5 and 6 in the third homologous domain. Important distinctions between rocker and the previously characterized alleles of this locus include the absence of aberrant tyrosine hydroxylase expression in Purkinje cells and the separation of the absence seizures (spike/wave type discharges) from the paroxysmal dyskinesia phenotype. Overall these findings point to an important dissociation between the seizure phenotypes and the abnormalities in catecholamine metabolism, and they emphasize the value of allelic series in the study of gene function (Zwingman, 2001).

Differential properties of voltage-dependent Ca2+ channels have been primarily ascribed to the alpha1 subunit, of which 10 different subtypes are currently known. For example, channels that conduct the N-type Ca2+ current possess the alpha1B subunit (Cav2.2), which has been localized, inter alia, to the piriform cortex, hippocampus, hypothalamus, locus coeruleus, dorsal raphe, thalamic nuclei, and granular layer of the cortex. Some of these regions have been implicated in metabolic and vigilance state control, and selective block of the N-type Ca2+ channel causes circadian rhythm disruption. In this study of Cav2.2-/- knock-out mice, potential differences in feeding behavior, spontaneous locomotion, and the sleep-wake cycle were examined. Cav2.2-/- mice do not display an overt metabolic phenotype but are hyperactive, demonstrating a 20% increase in activity under novel conditions and a 95% increase in activity under habituated conditions during the dark phase, compared with wild-type littermates. Cav2.2-/- mice also display vigilance state differences during the light phase, including increased consolidation of rapid-eye movement (REM) sleep and increased intervals between non-REM (NREM) and wakefulness episodes. EEG spectral power is increased during wakefulness and REM sleep and is decreased during NREM sleep in Cav2.2-/- mice. These results indicate a role for the N-type Ca2+ channel in activity and vigilance state control, that is interpreted in terms of effects on neurotransmitter release (Beuckmann, 2003).

N-type calcium channels are modulated by acute and chronic ethanol exposure in vitro at concentrations known to affect humans, but it is not known whether N-type channels are important for behavioral responses to ethanol in vivo. In mice lacking functional N-type calcium channels, voluntary ethanol consumption is reduced and place preference is developed only at a low dose of ethanol. The hypnotic effects of ethanol are also substantially diminished, whereas ethanol-induced ataxia is mildly increased. These results demonstrate that N-type calcium channels modulate acute responses to ethanol and are important mediators of ethanol reward and preference (Newton, 2004).

Calcium channels and exocytosis

Presynaptic N-type Ca2+ channels (CaV2.2, alpha1B) are thought to bind to SNARE (SNAP-25 receptor) complex proteins through a synaptic protein interaction (synprint) site on the intracellular loop between domains II and III of the alpha1B subunit. Whether binding of syntaxin to the N-type Ca2+ channels is required for coupling Ca2+ ion influx to rapid exocytosis has been the subject of considerable investigation. In this study, the synprint site was deleted from a recombinant alpha1B Ca2+ channel subunit and either the wild-type alpha1B or the synprint deletion mutant was transiently transfected into mouse pheochromocytoma (MPC) cell line 9/3L, a cell line that has the machinery required for rapid stimulated exocytosis but lacks endogenous voltage-dependent Ca2+ channels. Secretion was elicited by activation of exogenously transfected Ca2+ channel subunits. The current-voltage relationship was similar for the wild-type and mutant alpha1B-containing Ca2+ channels. Although total Ca2+ entry was slightly larger for the synprint deletion channel, compared with the wild-type channel, when Ca2+ entry was normalized to cell size and limited to cells with similar Ca2+ entry (approximately 150 x 106 Ca2+ ions/pF cell size), total secretion and the rate of secretion, determined by capacitance measurements, were significantly reduced in cells expressing the synprint deletion mutant channels, compared with wild-type channels. Furthermore, the amount of endocytosis was significantly reduced in cells with the alpha1B synprint deletion mutant, compared with the wild-type subunit. These results suggest that the synprint site is necessary for efficient coupling of Ca2+ influx through alpha1B-containing Ca2+ channels to exocytosis (Harkins, 2004).

Calcium channel function in C. elegans

Processing and storage of information by the nervous system requires the ability to modulate the response of excitable cells to neurotransmitter. A simple process of this type, known as adaptation or desensitization, occurs when prolonged stimulation triggers processes that attenuate the response to neurotransmitter. The C. elegans gene unc-2 is required for adaptation to two neurotransmitters, dopamine and serotonin. A loss-of-function mutation in unc-2 results in failure to adapt either to paralysis by dopamine or to stimulation of egg laying by serotonin. In addition, unc-2 mutants display behaviors similar to those induced by serotonin treatment. unc-2 encodes a homolog of a voltage-sensitive calcium-channel alpha-1 subunit. Expression of unc-2 occurs in two types of neurons implicated in the control of egg laying, a behavior regulated by serotonin. Unc-2 appears to be required in modulatory neurons to downregulate the response of the egg-laying muscles to serotonin. It is proposed that adaptation to serotonin occurs through activation of an Unc-2-dependent calcium influx, which modulates the postsynaptic response to serotonin, perhaps by inhibiting the release of a potentiating neuropeptide (Schafer, 1995).

Calcium signaling is known to be important for regulating the guidance of migrating neurons, yet the molecular mechanisms underlying this process are not well understood. Two different voltage-gated calcium channels are important for the accurate guidance of postembryonic neuronal migrations in the nematode C. elegans. In mutants carrying loss-of-function alleles of the calcium channel gene unc-2, the touch receptor neuron AVM and the interneuron SDQR often migrate inappropriately, leading to misplacement of their cell bodies. However, the AVM neurons in unc-2 mutant animals extended axons in a wild-type pattern, suggesting that the UNC-2 calcium channel specifically directs migration of the neuronal cell body and is not required for axonal pathfinding. In contrast, mutations in egl-19, which affect a different voltage-gated calcium channel, affect the migration of the AVM and SDQR bodies, as well as the guidance of the AVM axon. Thus, cell migration and axonal pathfinding in the AVM neurons appear to involve distinct calcium channel subtypes. Mutants defective in the unc-43/CaM kinase gene show a defect in SDQR and AVM positioning that resembles that of unc-2 mutants; thus, CaM kinase may function as an effector of the UNC-2-mediated calcium influx in guiding cell migration (Tam, 2000).

Regulation of structural plasticity by different channel types in rod and cone photoreceptors

In response to retinal disease and injury, the axon terminals of rod photoreceptors demonstrate dramatic structural plasticity, including axonal retraction, neurite extension, and the development of presynaptic varicosities. Cone cell terminals, however, are relatively inactive. Similar events are observed in primary cultures of salamander photoreceptors. To investigate the mechanisms underlying these disparate presynaptic responses, antagonists to voltage-gated L-type and cGMP-gated channels, known to be present on rod and cone cell terminals, respectively, were used to block calcium influx during critical periods of plasticity in vitro. In rod cells, L-type channel antagonists nicardipine and verapamil inhibited not only the outgrowth of processes and the formation of varicosities, but also the synthesis of vesicle proteins, SV2 and synaptophysin. In contrast, the synthesis of opsin in rod cells was unaffected. In cone cells, L-type channel antagonists caused only modest changes. However, cobalt bromide, which blocks all calcium channels, and l-cis-diltiazem, a potent antagonist of cGMP-gated channels, significantly inhibited varicosity formation and synthesis of SV2 in cone cells. Moreover, the cGMP-gated channel agonist 8-bromo-cGMP caused a significant increase in varicosity formation by cone but not rod cells. Thus voltage-gated L-type channels in rod cells and cGMP-gated channels in cone cells are the primary calcium channels required for structural plasticity and the accompanying upregulation of synaptic vesicle synthesis. The differing responses of rod and cone terminals to injury and disease may be determined by these differences in the regulation of Ca2+ influx (Zhang, 2002).

Endocannabinoids and calcium channels

At many central synapses, endocannabinoids released by postsynaptic cells inhibit neurotransmitter release by activating presynaptic cannabinoid receptors. The mechanisms underlying this important means of synaptic regulation are not fully understood. It has been shown at several synapses that endocannabinoids inhibit neurotransmitter release by reducing calcium influx into presynaptic terminals. One hypothesis maintains that endocannabinoids indirectly reduce calcium influx by modulating potassium channels and narrowing the presynaptic action potential. An alternative hypothesis is that endocannabinoids directly and selectively inhibit N-type calcium channels in presynaptic terminals. These hypotheses were tested at the granule cell to Purkinje cell synapse in cerebellar brain slices. By monitoring optically the presynaptic calcium influx (Cainflux) and measuring the EPSC amplitudes, it was found that cannabinoid-mediated inhibition arises solely from reduced presynaptic Cainflux. Next it was found that cannabinoid receptor activation does not affect the time course of presynaptic calcium entry, indicating that the reduced Cainflux reflects inhibition of presynaptic calcium channels. Finally, the classes of presynaptic calcium channels inhibited by cannabinoid receptor activation was assessed via peptide calcium channel antagonists. Previous studies established that N-type, P/Q-type, and R-type calcium channels are all present in granule cell presynaptic boutons. It was found that cannabinoid activation reduced Cainflux through N-type, P/Q-type, and R-type calcium channels to 29%, 60%, and 55% of control, respectively. Thus, rather than narrowing the presynaptic action potential or exclusively modulating N-type calcium channels, CB1 receptor activation inhibits synaptic transmission by modulating all classes of calcium channels present in the presynaptic terminal of the granule cell to Purkinje cell synapse (Brown, 2004).


REFERENCES

Search PubMed for articles about Drosophila cacophony

Astorga, G., Härtel, S., Sanhueza, M. and Bacigalupo, J. (2012). TRP, TRPL and cacophony channels mediate Ca(2+) influx and exocytosis in photoreceptors axons in Drosophila. PLoS One 7(8): e44182. PubMed Citation: 22952921

Astorga, C., Jorquera, R. A., Ramirez, M., Kohler, A., Lopez, E., Delgado, R., Cordova, A., Olguin, P. and Sierralta, J. (2016). Presynaptic DLG regulates synaptic function through the localization of voltage-activated Ca(2+) channels. Sci Rep 6: 32132. PubMed ID: 27573697

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