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 cac^TS2 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 cac^S//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 (Ca_v2.1) and a1B (Ca_v2.2) 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 cac^TS2. Electrophysiological analysis at neuromuscular synapses has revealed that neurotransmitter release in cac^TS2 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 cac^S 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 cac^H18 mutant exhibits defects in visual physiology (including complete unresponsiveness to light in certain genetic combinations) and visually mediated behaviors; this mutant (originally nbA^H18) 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).

GENE STRUCTURE

There are many indications of alternative exon usage in cac. The ORF of clone c31 is interrupted by four unspliced introns. The introns are bounded by consensus splice-site sequences and contain no regions of similarity to overlapping cDNAs. Identity as introns was confirmed by comparison with genomic sequences. In addition, in the region immediately downstream of the IS6 transmembrane domain, the c31 ORF diverges from that of clone cS14a for 116 nucleotides, encoding a 38 amino acid sequence beginning at amino acid 315. Each of these divergent sequences is of the same length and is in frame with the Dmca1A ORF. The two divergent sequences (alternative cassettes) are encoded in separate (albeit nearby) genomic regions, where they each are flanked by consensus splice-site sequences. Comparison of the c31-encoded amino acid sequence with that of representative vertebrate sequences shows that the pattern of similarity to these sequences differs between the exons. The c31-encoded exon is more similar to vertebrate a1 subunits in this region (58%-84% identity) than is the cS14a sequence (37%-47% identity). The c31 form is most similar to the non-L-type isoforms A, B, and E in this region (Smith, 1996).

The c31 exon contains the first 17 amino acids of the conserved ß subunit binding domain. The final conserved glutamate (E) is encoded by the first codon of the downstream exon. Interestingly, the c31-encoded exon has 100% conservation of the nine amino acids required for ß subunit binding, whereas the cS14a-encoded exon has only a 4/9 match, with the tyrosine (Y), tryptophan (W), isoleucine (I), and terminal glutamate (E) being conserved. If the cS14a exon is incorporated into a functional a1 subunit, this subunit might not bind ß subunits or may be involved in differential interactions with ß isoforms (Smith, 1996).

Sequencing of six cDNAs from different libraries revealed substantial heterogeneity in the IVS3-S4 loop. Some of this sequence diversity may arise from incomplete splicing, because the sequence downstream of the common region in cS26a and cSK53 contains no large ORF and begins with 5/6 or 6/6 matches to Drosophila 5' consensus splice-site sequences. Relative to cS26a, cSK53 contains six additional in-frame nucleotides before the start of the presumed unspliced exon (Smith, 1996).

Additional heterogeneity in the length of the cDNAs changes the number of amino acids in the IVS3-S4 loop from 9 to 10 or 12. Clone cS9a is the shortest (encoding the 9 amino acid IVS3-S4 loop). Clone c3p1 is slightly longer, containing an in-frame insertion of three nucleotides that are not present in cS9a but are found in both cS11 and cS26a. The latter two clones contain identical in-frame insertions of nine nucleotides; these have identical sequence to the six nucleotides in cSK53 plus the three in c3p1. The nine nucleotides found within cS11 and cS26a encode the amino acids HDD. This variable HDD segment is included as amino acids 1181-1183 in Dmca1A (Smith, 1996).

The genomic organization of a gene coding for an a 1 subunit of a voltage-gated calcium channel of Drosophila melanogaster was determined. Thirty-four exons, distributed over 45 kb of genomic sequence, have been identified and mapped, including exons in three regions involved in alternative splicing and new sites potentially involved in RNA editing. The comparison of the intron/exon boundaries of this channel with a mammalian counterpart shows that the genomic structure of these two genes has remained fairly similar during evolution, with more than half of the Drosophila intron positions being perfectly conserved compared to the human channel. Phylogenetic analysis of the mutually exclusive alternative exons reveals that they have diverged considerably. It is suggested that this divergence, rather than reflecting evolutionary age, is the likely result of accelerated rates of evolution following duplication (Peixoto, 1997).

Several alternative exons have been described in cac, including two mutually exclusive exon pairs, IS4a/IS4b and I-IIa/I-IIb, as well as a 3, 6, or 9 bp insertion adding one to three amino acids to the IVS3-IVS4 loop (Smith, 1996; Kawasaki, 2002). The latter splicing site appears to be conserved in mammalian a1 subunit genes. At the equivalent position within the a1A and a1B transcripts, inclusion of a 6 bp alternative exon adds two amino acids to the IVS3-IVS4 loop and alters functional properties of the channels (Lin, 1997; Bourinet, 1999; Hans, 1999; Krovetz, 2000). Additional mRNA splicing variants of cac have also been reported (Kawasaki, 2002 and references therein).

PROTEIN STRUCTURE

Amino Acids - 1851

Structural Domains

The Cac protein has the canonical structure of voltage-gated calcium channel a1 and sodium channel a subunits, with four internal repeats (I-IV), each containing six presumed membrane-spanning hydrophobic domains (S1-S6). Transmembrane segments S4 of each internal repeat contain positively charged amino acids every third or fourth amino acid, consistent with the postulated role of these segments in sensing and responding to transmembrane voltage changes. In addition, the conserved domains for short segments 1 and 2 (ss1, ss2) in the loop between transmembrane domains S5 and S6 of each repeat are conserved in this protein (Smith, 1996).

Comparison of both the overall protein and of these conserved domains reveals a strikingly greater similarity to calcium channel a1 subunits than to sodium channel. A conserved glutamate present in ss2 in the loop between transmembrane domains S5 and S6 of each repeat is involved in ion selectivity. Sodium channels contain this glutamate residue only in repeats I and II, whereas calcium channels have this glutamate in all four repeats. Changing the appropriate residue to glutamate in repeats III and IV of a sodium channel converts the ion selectivity of a sodium channel to that of a calcium channel. Conversely, changing the identity of these glutamate residues alters the ion selectivity and conductance of calcium channels. The glutamate residues relevant to ion selectivity are conserved in all four ss2 domains of the Dmca1A protein, consistent with identification of this protein as a calcium channel a1 subunit (Smith, 1996).

It has been proposed that a fragment of the rabbit skeletal muscle a1 subunit, including transmembrane domain IVS6 and the adjacent intra- and extra-cellular sequences, functions as a binding site for the phenylalkylamine calcium channel blockers. Combined with work suggesting that phenylalkylamines block calcium channels intracellularly, this evidence identified the intracellular portion of the IVS6 transmembrane domain and the adjacent intracellular amino acids as a binding site for phenylalkylamines. The proposed phenylalkylamine binding fragment sequence, a 17 amino acid sequence bracketing the intracellular junction of the IVS6 transmembrane domain, is conserved completely between these two proteins. This conserved region is flanked on the N-terminal side by two conservative amino acid changes (isoleucine to leucine and isoleucine to methionine), preceded by two more identical amino acids (FL). The region extends to within two amino acids of the C-terminal end of the rabbit proteolytic fragment, where there is a nonconservative tryptophan-to-serine change in the Dmca1A sequence, followed by a conserved serine (Smith, 1996).

Proteolytic fragments containing the IIIS6 and IVS6 transmembrane domains and regions immediately adjacent to them have been shown to bind dihydropyridines. Dihydropyridine sensitivity of an L-type channel is abolished when a portion of the polypeptide overlapping the extracellular end of IVS6 is replaced with non-L-type sequence. Conversely, dihydropyridine sensitivity can be conferred upon a non-L-type channel by replacing the IIIS5-S6 and IVS5-S6 regions with sequences from L-type (carp or rabbit) skeletal muscle subunits. Because dihydropyridines bind to the channel from the outside, the portions of these fragments that begin in the extracellular domain and enter into the transmembrane segments from the outside are, most likely, involved in dihydropyridine binding (Smith, 1996).

The sequences from a dihydropyridine-sensitive carp skeletal muscle a1 subunit (sequences that confer dihydropyridine sensitivity to chimeric channels) align with Dmca1A in the IIIS5-S6 and IVS5-S6 regions. Certain amino acids in the dihydropyridine-sensitive carp skeletal muscle subunit are potentially relevant to dihydropyridine sensitivity. In the region of IIIS5-S6 and IVS5-S6, there are 102 such amino acids. Of these, Dmca1A is identical to dihydropyridine-sensitive channels at only 18 sites. By comparing all known dihydropyridine-sensitive and -resistant channels, 23 positions within these regions have been identified where the amino acid is different between dihydropyridine-resistant and -sensitive vertebrate a1 subunit, but 100% identical within the resistant and sensitive subgroups. Of these 23 amino acids, Dmca1A is identical to the dihydropyridine-resistant channels at 20 sites and shows identity to the sensitive channels at only three sites. The lack of correspondence between Dmca1A and dihydropyridine-sensitive channels at these positions suggests that the Drosophila Dmca1A a1 subunit may be insensitive to dihydropyridines (Smith, 1996 and references therein).

Calcium and sodium channels often contain in their C-terminal intracellular regions an EF-hand motif, which forms a structure of two a helices flanking a calcium-binding loop. This motif has been correlated functionally with Ca²⁺-sensitive inactivation of calcium channels. A potential EF-hand in Dmca1A is immediately C terminal to transmembrane domain IVS6. The Dmca1A sequence has 12 matches of 16 for residues important for calcium binding. Allowing conservative substitutions increases this match to 14 of 16 positions (Smith, 1996).

There are several sites of possible post-translational modification of the Dmca1A protein. A single extracellular site matching the consensus sequence [N]-[~P]-[S/T]-[~P] for N-linked glycosylation is found at N865 near the N terminus of the IIIS4 transmembrane domain. Nine intracellular consensus sites for cAMP-dependent protein kinase phosphorylation [R/K]-[X]-[X]-[S/T] are found: in the N terminus at T31; in the I/II loop at S386 and S392, and in the C terminus at S1348, S1519, S1559, S1616, S1650, and S1836. Fifteen intracellular sites matching the consensus PKC phosphorylation site [S/T]-[X]-[R/K] are found: in the I/II loop at T437; in the IIS4-S5 loop at S552 and S563; in the IIIS4-S5 loop at S900; in the III/IV loop at T1083; in the IVS4-S5 loop at T1209 and S1220, and in the C terminus at T1370, S1432, S1493, S1496, S1559, S1683, S1748, and T1820. The clustering of 13 potential phosphorylation sites in the C terminus suggests that this region may be involved in phosphorylation-dependent modification of calcium channel function (Smith, 1996).

Of particular interest because of their possible functional significance are five conserved PKC sites that are found in the S4-S5 loops of all non-L-type channels. These sites are not found in any L-type channels and thus may mediate a property that distinguishes these channels functionally. In Dmca1A these sites are S552, S563, S900, T1209, and S1220. Their proximity to the voltage-sensing S4 transmembrane domain is intriguing. There is, in addition, a cAMP-dependent protein kinase phosphorylation site conserved in the segment between IVS6 and the EF-hand in all calcium channels sequenced to date. This site is likely to modulate a function common to all calcium channels (Smith, 1996).

Calcium channel ß subunits interact with a1 subunits to stimulate peak current amplitude, to increase the rate of activation, and to modify the voltage dependence of activation and inactivation in Drosophila as well as in other species. A conserved 18 amino acid sequence (QQ-E-L-GY-WI--E) has been identified in the I-II cytoplasmic linker that bindsß subunits. Mutations in this conserved domain inhibit ß subunit binding. Analysis of the cac cDNA clones shows that alternative splicing in the region encoding this I/II linker in Dmca1A generates a1 subunits with major differences in the ß subunit binding domain (Smith, 1996).

date revised: 22 December 2002

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