Genetic Analysis of a Synaptic Calcium Channel in Drosophila: Intragenic Modifiers of a Temperature-Sensitive Paralytic Mutant of cacophony

Corresponding author: R. W. Ordway, 208 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802., rwo4{at}psu.edu (E-mail)

Communicating editor: T. C. KAUFMAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Our previous genetic analysis of synaptic mechanisms in Drosophila identified a temperature-sensitive paralytic mutant of the voltage-gated calcium channel 1 subunit gene, cacophony (cac). Electrophysiological studies in this mutant, designated cac^TS2, indicated cac encodes a primary calcium channel 1 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 cac^TS2 paralytic phenotype. The screen recovered 10 mutations that enhance or suppress cac^TS2, including second-site mutations in cac (intragenic modifiers) as well as mutations mapping to other genes (extragenic modifiers). Here we report molecular characterization of three intragenic modifiers and examine the consequences of these mutations for temperature-sensitive behavior, synaptic function, and processing of cac pre-mRNAs. These mutations may further define the structural basis of calcium channel 1 subunit function in neurotransmitter release.

TRANSMISSION of electrical impulses among neurons is a fundamental aspect of neural function. This occurs at chemical synapses when neurotransmitters are released from the presynaptic neuron and produce excitation or inhibition of the postsynaptic cell. Neurotransmitter release is evoked by calcium influx through voltage-gated calcium channels, which triggers rapid fusion of neurotransmitter-filled synaptic vesicles with the presynaptic plasma membrane.

Calcium channels implicated in neurotransmitter release are heteromultimers composed of a primary structural subunit, 1, as well as accessory ß, 2-, and possibly subunits (CATTERALL 1998 ). The 1 subunit consists of four homologous repeats, each containing six transmembrane segments and an intramembranous "P loop" (Fig 1). The four repeats are joined by three major cytoplasmic loops, which, together with the cytoplasmic N and C termini, include several protein interaction domains involved in channel regulation (IKEDA and DUNLAP 1999 ; CATTERALL 2000 ). Certain structural features of the 1 subunit have been assigned specific roles in channel function. Examples include the S4 segments, which contain positively charged residues and are thought to serve as voltage sensors (HORN 2000 ), and the P loop contributing to formation of the channel pore (Fig 1; VARADI et al. 1999 ). In vertebrates, the primary presynaptic calcium channels are of the P/Q and N types, for which the 1 subunits are encoded by the 1A (Cav 2.1) and 1B (Cav 2.2) genes, respectively. Homologous calcium channel 1 subunit genes have now been identified in a variety of organisms.

View larger version (14K): In this window In a new window Download PPT slide   Figure 1. Presynaptic voltage-gated calcium channel 1 subunit topology. The positively charged S4 voltage sensors (+) and the pore-forming P loops (P) are indicated. A calcium-dependent regulatory region within the C-terminal cytoplasmic tail contains consensus binding domains for calcium (EF hand) and calmodulin (IQ).

Several studies have utilized genetic approaches to examine the in vivo functions of specific calcium channel gene products at native synapses. In addition to work in Drosophila (see below), analysis of presynaptic calcium channels has been pursued in several other genetic model systems. The unc-2 gene of Caenorhabditis elegans, originally identified in a screen for mutants exhibiting altered adaptation to neuroactive substances, encodes a homolog of vertebrate presynaptic calcium channel 1 subunits (SCHAFER and KENYON 1995 ). In the mouse and human, mutations in the 1A subunit gene are associated with several inherited disorders characterized by ataxia and migraine as well as by altered P/Q-type channel activity (DOVE et al. 1998 ; LORENZON et al. 1998 ; WAKAMORI et al. 1998 ; LORENZON and BEAM 2000 ; MORI et al. 2000 ; PIETROBON 2002 ). In addition, knockout mice have been generated for all of the 1 subunit genes implicated in neurotransmitter release, including 1A (JUN et al. 1999 ), 1B (INO et al. 2001 ), and 1E (SAEGUSA et al. 2000 ). Despite this progress, a major remaining challenge in determining the physiological roles of these gene products is presented by long-term compensatory changes that may occur in null or hypomorphic mutants (JUN et al. 1999 ; SAEGUSA et al. 2000 ; INO et al. 2001 ; MATSUSHITA et al. 2002 ; ZHANG et al. 2002 ). Thus complementary approaches using conditional mutants, such as temperature-sensitive (TS) paralytic mutants of Drosophila, will continue to make important contributions.

Previous studies in Drosophila identified cacophony (cac), also known as nightblind A (nbA), as a neurally expressed homolog of vertebrate presynaptic calcium channel 1 subunit genes (SMITH et al. 1996 ). In addition, electroretinogram recordings from cac/nbA mutant flies suggested a role for cac-encoded channels in synaptic transmission (HEISENBERG and GOTZ 1975 ; SMITH et al. 1998B ). Our laboratory subsequently isolated and characterized cac^TS2, a cac mutant exhibiting rapid paralysis at elevated temperatures (DELLINGER et al. 2000 ). This conditional mutant allowed electrophysiological analysis of synaptic transmission following acute perturbation of a specific calcium channel gene product, revealing that cac encodes a primary calcium channel functioning in neurotransmitter release (KAWASAKI et al. 2000 ).

Recent work identified the molecular lesion in cac^TS2 and demonstrated transformation rescue of cac mutants by neural expression of a specific cac-encoded 1 subunit variant (KAWASAKI et al. 2002 ). Independent characterization of the cac^TS2 mutation and its consequences for male courtship behavior have also been reported recently (CHAN et al. 2002 ). The cac^TS2 mutation maps to a calcium regulatory domain within the C-terminal cytoplasmic tail of the 1 subunit. This domain includes a binding site for calmodulin (IQ in Fig 1) as well as an adjacent EF-hand calcium-binding motif (EF hand in Fig 1). Calmodulin bound to the IQ domain has been shown to mediate calcium-dependent facilitation and inactivation (DEMARIA et al. 2001 ); the EF hand may participate in the latter process as well (PETERSON et al. 2000 ). The cac^TS2 mutation maps adjacent to the EF-hand domain, raising the possibility that altered channel inactivation may be responsible for reduced neurotransmitter release at elevated temperatures (KAWASAKI et al. 2002 and see DISCUSSION).

cac^TS2 and other TS paralytic mutants provide powerful tools for analysis of molecular mechanisms underlying synaptic function. In addition, these mutants serve as excellent starting points for broader genetic analysis (RAMASWAMI et al. 1993 ; DELLINGER et al. 2000 ). In fact, cac^TS2 was originally isolated as an enhancer of the N-ethylmaleimide-sensitive fusion protein mutant, comatose (DELLINGER et al. 2000 ), another TS paralytic mutant affecting neurotransmitter release (KAWASAKI et al. 1998 ). In this study, we conducted a genetic screen for new mutations that enhance or suppress the cac^TS2 paralytic phenotype with the goal of further defining the in vivo functions and interactions of cac-encoded 1 subunits. Among 10 mutants recovered, 4 appeared to be second-site mutations within the cac gene (intragenic modifiers) and 6 mapped to other genes (extragenic modifiers). Here we focus on behavioral, electrophysiological, and molecular characterization of the intragenic modifiers of cac^TS2. The findings are discussed in the context of previous work investigating the structural determinants of presynaptic calcium channel function.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila stocks:
cac^TS2 and C(1)RM, y w f were from our laboratory stock collection. cac^TS2 was maintained as a homozygous stock. Meiotic mapping of X-linked mutants was carried out by conventional methods using a y m wy g f chromosome constructed in our laboratory. Wild-type flies were Canton-S.

Mutagenesis and screening:
An F₂ screen was carried out essentially as described (DELLINGER et al. 2000 ). Briefly, male cac^TS2 flies were exposed for 24 hr to a solution of 25 mM ethyl methanesulfonate (EMS) in 1.6% sucrose and then mated in groups of 30 to a similar number of attached-X females [C(1)RM, y w f]. Mutagenized males were removed from these crosses after 5 days. Single F₁ male progeny were mated to attached-X females and F₂ progeny were screened at 37° for any alteration of the cac^TS2 behavioral phenotype. Flies were observed for 3 min after placing them in a vial preheated and maintained at 37° by immersion in a water bath. Crosses were maintained at room temperature and F₂ progeny were collected twice from each cross, 14 and 21 days after mating. Although only male progeny carry the cac^TS2 mutation, both males and females were examined at 37°.

Behavioral analysis:
TS behavior was analyzed in flies reared at 20° or as noted in the text and figure legends. Using the same apparatus described for screening, 2- to 3-day-old flies were tested in groups of six. Five groups were examined for each genotype (n = 5). Time for 50% paralysis represents the time at which three flies were no longer able to stand. In all tests exceeding 5 min in duration, the cotton plug sealing the vial was wet with water to prevent dehydration.

Synaptic electrophysiology:
Synaptic current recordings at dorsal longitudinal flight muscle (DLM) neuromuscular synapses of the adult were obtained and analyzed as described previously (KAWASAKI et al. 1998 ). Experiments were performed on 2- to 5-day-old flies reared at 20°.

Sequence analysis:
First-strand cDNA synthesis was performed by conventional methods, using total RNA isolated from heads (KAWASAKI et al. 2002 ). The resulting cDNA preparations were used as template in PCRs to amplify the entire 6-kb cac coding sequence in three overlapping fragments. cac coding sequences from modifier mutants were generated by direct sequencing of reverse transcriptase (RT)-PCR products and compared directly to those obtained from the cac^TS2 parent chromosome used in the mutagenesis. For analysis of RNA editing at nucleotide position 4106, cDNA clones including this site were generated from RT-PCR products and sequenced. Automated sequencing was carried out on an ABI Prism 377 DNA sequencer (ABI, Foster City, CA) or a CEQ 2000XL DNA analysis system (Beckman Coulter, Fullerton, CA). Sequence alignments were generated by the Clustal method, using the MegAlign feature of the Lasergene software package (DNAstar, Madison, WI).

Data analysis:
Data are presented as the mean ± SEM. All graphing and statistical analysis was carried out using Microsoft Excel (Microsoft Corp., Seattle, WA). Statistical analysis of electrophysiological and behavioral data was performed using an unpaired Student's t-test, and significance was assigned to comparisons with P values 0.05. In the figures, values significantly different from control are marked with an asterisk.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A screen for genetic modifiers of cac^TS2:
The cac^TS2 paralytic phenotype is well suited for the isolation of genetic modifiers. At 36°, cac^TS2 exhibits moderate locomotor defects including spinning, uncoordinated walking, and tumbling, but is able to stand and walk for >50 min (DELLINGER et al. 2000 ). In contrast, exposure to 38° results in rapid paralysis within 20 sec (DELLINGER et al. 2000 ). At an intermediate temperature of 37°, cac^TS2 exhibits severe motor defects approaching paralysis but can remain standing. We anticipated that mutations that enhance the cac^TS2 phenotype would cause rapid paralysis at 37°, while suppressors would alleviate the severe motor defects observed at this temperature.

Males carrying the cac^TS2 mutation were exposed to the chemical mutagen EMS and mated to attached-X (compound X) females (Fig 2). All F₁ male progeny of this cross inherit a mutagenized paternal X chromosome carrying cac^TS2 as well as mutagenized paternal autosomes. Although screens for TS paralytic mutations on the X chromosome have typically examined F₁ progeny for mutant phenotypes, we have found it advantageous to perform F₂ screens after backcrossing individual F₁ males to attached-X females (DELLINGER et al. 2000 ). In this study, F₂ progeny from 1954 lines were screened for alteration of the cac^TS2 paralytic phenotype. Of 10 modifier mutations recovered, all mapped to the X chromosome. Six were designated extragenic modifiers because they could be separated from cac^TS2 by recombination. All of these are enhancers, producing rapid paralysis at 36° in a cac^TS2 genetic background. The four remaining mutations (Table 1) were designated putative intragenic modifiers because they were not separated from cac^TS2 during recombinational mapping. Two of the intragenic modifiers are enhancers [cac^E(TS2)1 and cac^E(TS2)2] and two are suppressors [cac^su(TS2)1 and cac^su(TS2)2]. Sequence analysis of the cac open reading frame (ORF) identified missense mutations in three of these mutants (see below). In this study we focus on the behavioral, electrophysiological, and molecular characterization of the three confirmed intragenic modifiers of cac^TS2.

View larger version (19K): In this window In a new window Download PPT slide   Figure 2. An F2 genetic screen for modifiers of cacTS2. *, mutagenized chromosome; , attached-X chromosome.

TS paralytic behavior:
Initial characterization of the intragenic modifiers involved examination of TS behavior. Enhancers and suppressors were analyzed at 36° and 38°, respectively. Note that analysis of these mutations was necessarily performed in a cac^TS2 genetic background.

The intragenic enhancer, cac^E(TS2)1, caused enhanced paralysis in double-mutant combination with cac^TS2 [cac^TS2 cac^E(TS2)1]. While cac^TS2 alone fails to paralyze at 36°, cac^TS2 cac^E(TS2)1 is paralyzed rapidly, exhibiting a time for 50% paralysis of 0.15 ± 0.01 min (n = 5; Fig 3A). Similarly, females homozygous for cac^TS2 but heterozygous for cac^E(TS2)1 exhibited a time for 50% paralysis of 0.31 ± 0.03 min (n = 5), indicating that this enhancer is dominant (Fig 3A). The suppressor mutation, cac^su(TS2)1, greatly increased the time for 50% paralysis at 38° from 0.28 ± 0.04 min (n = 5) in cac^TS2 alone to 20.43 ± 1.88 min (n = 5) in cac^TS2 cac^su(TS2)1 double mutants (Fig 3B). Females homozygous for cac^TS2 but heterozygous for cac^su(TS2)1 exhibited a time for 50% paralysis of 0.43 ± 0.06 min (n = 5), indicating that this suppressor is recessive.

View larger version (21K): In this window In a new window Download PPT slide   Figure 3. TS behavior of intragenic modifiers. (A) cacTS2 cacE(TS2)1 double mutants exhibited rapid paralysis at 36°. Note that cacTS2 behavioral tests were truncated after 50 min. (B) Both cacsu(TS2)1 and cacsu(TS2)2 markedly suppressed cacTS2 paralytic behavior at 38°. Behavioral characterization of cacsu(TS2)2 was performed on flies reared at 23°, as indicated in parentheses following the genotype designations. Asterisks mark values significantly different from control values.

The above behavioral analysis was performed on flies reared at 20°; however, characterization of the third intragenic modifier, suppressor cac^su(TS2)2, indicated no suppression of the cac^TS2 paralytic phenotype under the same conditions (data not shown). In contrast, flies reared at 23° (the approximate room temperature at which crosses were maintained for the genetic screen) showed strong suppression of cac^TS2 paralysis at 38° (Fig 3B). While cac^TS2 flies reared at 23° exhibited rapid paralysis at 38°, with a time for 50% paralysis of 0.33 ± 0.02 min (n = 5), the suppressor cac^su(TS2)2 dramatically increased this value to 29.84 ± 1.76 min (n = 5) in cac^TS2 cac^su(TS2)2 double mutants. Females homozygous for cac^TS2 and heterozygous for cac^su(TS2)2 exhibited a time for 50% paralysis of 0.39 ± 0.02 min (n = 5), indicating that this suppressor is recessive.

Synaptic physiology:
Our previous electrophysiological analysis at adult neuromuscular synapses demonstrated that cac^TS2 produces a marked reduction in neurotransmitter release at restrictive temperatures (KAWASAKI et al. 2000 ). To examine whether intragenic modifiers altered the synaptic phenotype of cac^TS2, similar experiments were performed in flies carrying both cac^TS2 and the modifier mutations. Two-electrode voltage clamp was employed at DLM synapses to record excitatory postsynaptic currents (EPSCs) evoked by DLM motor axon stimulation. Recordings from double-mutant synapses were compared to those from cac^TS2 at both 20° and 36°. As described previously, cac^TS2 exhibited a wild-type synaptic current at 20° and a marked reduction in the EPSC amplitude at 36° (Fig 4A and Fig B). Both cac^su(TS2)1 and cac^su(TS2)2 produced striking suppression of the synaptic current reduction observed in cac^TS2 (Fig 4A and Fig B). Synaptic current amplitudes recorded in cac^TS2, cac^TS2 cac^su(TS2)1, and cac^TS2 cac^su(TS2)2 at 36° were 1.16 ± 0.21 µA (n = 6), 2.26 ± 0.35 µA (n = 9), and 2.63 ± 0.31 µA (n = 6), respectively. These results indicate that each suppressor produces a second-site lesion in the cac-encoded 1 subunit that counteracts the functional consequences of the cac^TS2 mutation at neuromuscular synapses.

View larger version (21K): In this window In a new window Download PPT slide   Figure 4. Synaptic physiology of intragenic modifiers. (A) Representative synaptic current recordings from DLM neuromuscular synapses of wild type (WT) and cacTS2, as well as cacTS2 cacsu(TS2)1 and cacTS2 cacsu(TS2)2 double mutants. Both cacsu(TS2)1 and cacsu(TS2)2 markedly suppressed the synaptic current reduction observed in cacTS2 alone at 36°. Arrows indicate stimulation of the DLM motor axon. Stimulation artifacts have been removed. The 36° traces were obtained after 7–12 min at 36°. All recordings were performed on flies reared at 20°. (B) Mean EPSC amplitudes relative to wild type at 36°. The synaptic currents in cacTS2 cacsu(TS2)1 and cacTS2 cacsu(TS2)2 were significantly different from those of cacTS2 alone.

In contrast to the suppressors, cac^E(TS2)1 did not significantly alter the cac^TS2 synaptic phenotype (Fig 4B). Synaptic current values for cac^TS2 and cac^TS2 cac^E(TS2)1 at 36° were 1.16 ± 0.21 µA (n = 6) and 1.12 ± 0.19 µA (n = 4), respectively. Thus enhancement of the cac^TS2 behavioral phenotype in cac^TS2 cac^E(TS2)1 is not reflected in DLM neuromuscular synaptic function, suggesting that enhanced paralysis may result from perturbation of central synapses. In previous studies of two TS paralytic mutants affecting synaptic transmission, comatose (KAWASAKI and ORDWAY 1999 ; SANYAL et al. 1999 ) and shibire (SALKOFF and KELLY 1978 ), these mutants exhibited increased CNS activity at elevated temperatures, suggesting that TS paralysis reflects complex interactions between central and peripheral synapses. This issue was examined in cac^TS2 cac^E(TS2)1 by performing intracellular DLM recordings under conditions in which the motor axons are not cut, but rather their connections with the CNS are left intact. In these experiments, the frequency of endogenous DLM action potentials reflects central activity. Unlike comatose and shibire, cac^TS2 at restrictive temperature exhibits a low frequency of DLM action potentials closely resembling that of wild type (Fig 5A; KAWASAKI and ORDWAY 1999 ). In contrast, the cac^TS2 cac^E(TS2)1 double mutant shows a striking increase in CNS activity at 36°. This activity occurs in bursts that reach sustained frequencies exceeding 50 Hz and reduce DLM action potential amplitude (Fig 5B). Although the precise mechanisms underlying the enhancement of cac^TS2 paralytic behavior by cac^E(TS2)1 remain to be determined, the observed increase in CNS activity is likely to play an important role.

View larger version (19K): In this window In a new window Download PPT slide   Figure 5. Intracellular DLM recordings from cacTS2 (A) and the cacTS2 cacE(TS2)1 double mutant (B) at 36°. The DLM motor axons were left intact. High-frequency bursts of action potentials driven by CNS neural activity were observed in cacTS2 cacE(TS2)1.

Molecular analysis:
To explore the mechanisms underlying the intragenic modifier phenotypes, the molecular lesions in these mutants were identified. The cac ORF from each mutant was analyzed by direct sequencing of RT-PCR products. To identify the induced mutation, the resulting sequence was compared to that of the parent cac^TS2 chromosome used in the mutagenesis. Any sequence difference between the modifier mutant and parent cac^TS2 chromosomes was confirmed through analysis of at least three independent RNA preparations. Missense mutations were identified in three of the four putative intragenic modifiers, confirming that these mutations are intragenic and defining the underlying molecular lesions.

Molecular lesions in three intragenic modifiers:
The cac^E(TS2)1 mutation is an a t transversion at position 2407, resulting in substitution of serine for a conserved threonine (T619S) in the P-loop region of the second repeat (Fig 6, bottom left). A mutation at the analogous position in the human 1A gene (T666M) is associated with familial hemiplegic migraine (OPHOFF et al. 1996 ). cac^su(TS2)1 is a t c transition at position 4112, substituting threonine for a conserved isoleucine (I1187T). This residue lies within the short extracellular loop between IVS3 and IVS4 (Fig 6, top right) in proximity to the S4 voltage sensor. As described below, cac^su(TS2)1 also alters RNA processing at an adjacent site. Finally, the molecular lesion in cac^su(TS2)2 is a t a transversion at position 3300, substituting leucine for a conserved phenylalanine (F916L) in IIIS5 (Fig 6, top left). As noted in the DISCUSSION, phenylalanine residues within S5 have been proposed to interact with the S4 voltage sensor and play an important role in voltage-dependent gating.

View larger version (47K): In this window In a new window Download PPT slide   Figure 6. Molecular lesions in the intragenic modifiers of cacTS2. Alignment of CAC with related calcium channel 1 subunit polypeptide sequences is shown. Amino acid identities with CAC are shaded. Characteristic domains are indicated with horizontal bars below the alignments. The aligned sequences correspond to CAC (U55776), Mouse 1A (NM007578), and C. elegans UNC-2 (U25119).

A fourth putative intragenic modifier, cac^E(TS2)2, exhibited a wild-type cac ORF sequence and thus was not confirmed to be an allele of cac. In meiotic mapping experiments examining >1000 recombinant chromosomes, cac^E(TS2)2 was not separated from cac^TS2, indicating that this modifier is either intragenic or within a closely linked gene.

cac^su(TS2)1 alters pre-mRNA editing:
Despite the short length of the IVS3-IVS4 extracellular loop, the corresponding cac mRNA sequence is subject to both alternative splicing and A-to-I RNA editing (SMITH et al. 1996 , SMITH et al. 1998A ; PEIXOTO et al. 1997 ). As reported previously, the IVS3-IVS4 loop varies from 9 to 12 amino acids in length depending upon the inclusion or exclusion of a 3-, 6-, or 9-bp alternative exon (SMITH et al. 1996 ; PEIXOTO et al. 1997 ; KAWASAKI et al. 2002 ). The cac^su(TS2)1 mutation (t4112a) is not within any of these alternative exons, and RT-PCR analysis of cac cDNAs suggests that alternative splicing in this region is not perturbed in this mutant (data not shown). However, the cac^su(TS2)1 mutation does alter A-to-I RNA editing at an adjacent site.

A-to-I editing is a form of pre-mRNA processing in which a genomically encoded adenosine is deaminated to inosine (reviewed in REENAN 2001 ). Inosine is read as guanosine during translation and may alter the amino acid sequence of the encoded protein. Editing of adenosine 4106, 1 of 12 previously characterized RNA editing sites in cac (SMITH et al. 1998A ; KAWASAKI et al. 2002 ), results in substitution of serine for a genomically encoded asparagine (Fig 7A). Consistent with previous work, our direct sequencing of RT-PCR products derived from wild-type or cac^TS2 head RNA indicates that the majority of cac transcripts are edited at this position. Sequence chromatographs (Fig 7B) show that this editing site exhibits major and minor peaks corresponding to the edited and unedited forms, respectively. Interestingly, the cac^su(TS2)1 mutation at nucleotide 4112 appears to suppress editing at position 4106, resulting in similar and often superimposed peaks corresponding to the edited and unedited sequences (Fig 7C). However, these sequence chromatograph peaks may not provide a quantitative measure of the relative abundance of specific cDNA variants, and thus editing at this site was further examined by generating and sequencing cloned cDNAs. RT-PCR was utilized to generate cDNA clones including the region of interest from wild type, cac^TS2, and cac^TS2 cac^su(TS2)1 double mutants. Sequences from >20 clones for each genotype confirmed that editing at position 4106 is reduced by the cac^su(TS2)1 mutation. The percentages of clones edited at this site in WT, cac^TS2, and cac^TS2 cac^su(TS2)1 were 91.7% (n = 24), 80.0% (n = 25), and 59.1% (n = 22), respectively, in agreement with the sequence chromatograph data. These results indicate that the cac^su(TS2)1 mutation acts on an adjacent editing site to increase the fraction of 1 subunits containing a genomically encoded asparagine and raise the possibility that altered RNA editing may contribute to the suppressor phenotype.

View larger version (43K): In this window In a new window Download PPT slide   Figure 7. The cacsu(TS2)1 mutation alters RNA editing at an adjacent site. (A) A-to-I RNA editing of the cac transcript at position 4106 in effect produces a codon change from aac to agc, resulting in substitution of serine (S) for a genomically encoded asparagine (N). (B) Sequence from cacTS2 shows major and minor peaks at position 4106, corresponding to the edited (G) and unedited (a) sequences, respectively. (C) The same region from cacTS2 cacsu(TS2)1 mutant cDNA shows that edited (G) and unedited (A) peaks are of similar size. The adjacent cacsu(TS2)1 molecular lesion is indicated at position 4112.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have carried out a screen for genetic modifiers of cac^TS2, a calcium channel 1 subunit mutant exhibiting rapid paralysis and a marked reduction in neurotransmitter release at elevated temperatures. Here we report isolation and characterization of three intragenic modifier mutations, including one enhancer and two suppressors. Analysis of these mutants has identified second-site molecular lesions in the cac coding sequence and defined the resulting behavioral and electrophysiological phenotypes. These findings extend our knowledge of the structural determinants governing the function of a specific presynaptic calcium channel 1 subunit at native synapses.

Intragenic modifiers of cac^TS2:
Prior to the isolation of cac^TS2 (DELLINGER et al. 2000 ), viable mutant alleles of cac were recovered in screens for mutations altering male courtship (VON SCHILCHER 1976 , VON SCHILCHER 1977 ) and the optomotor response (HEISENBERG and GOTZ 1975 ). In contrast, no cac mutants were identified in previous screens for X-linked TS paralytic mutants (SUZUKI et al. 1971 ; GRIGLIATTI et al. 1973 ; SIDDIQI and BENZER 1976 ). In this study, three confirmed alleles of cac were recovered in a screen of 1954 X chromosomes. This relative abundance of cac mutations may have resulted from the sensitized background provided by the cac^TS2 behavioral phenotype at 37°.

The molecular lesion in cac^TS2 maps to the second proline of a highly conserved proline pair located in a calcium regulatory domain within the C-terminal cytoplasmic tail of the 1 subunit (KAWASAKI et al. 2002 ). Interestingly, this proline pair is adjacent to an EF-hand calcium-binding motif, which may participate in calcium-dependent regulation of channel gating (PETERSON et al. 2000 ). This study has begun to define the molecular basis of functional interactions between cac^TS2 and the intragenic modifiers through molecular characterization of the modifier mutations.

cac^E(TS2)1:
The cac^E(TS2)1 mutation enhanced the cac^TS2 phenotype, producing rapid paralysis at 36°. This behavioral phenotype was not associated with enhancement of the cac^TS2 neuromuscular synaptic phenotype at the same temperature. However, the marked increase in CNS activity observed by recording endogenous DLM action potential frequencies at 36° (Fig 5) likely enhances TS paralysis through fatigue or general disruption of neural function.

The cac^E(TS2)1 mutation results in substitution of serine for a highly conserved threonine within the P loop of repeat II (T619S). A mutation at the analogous position within the human 1A gene (T666M) is one of several 1A mutations associated with the human autosomal dominant disorder, familial hemiplegic migraine (OPHOFF et al. 1996 ). Consistent with the position of T666M within the P loop, a reduction in ion permeation has been observed (HANS et al. 1999 ); this mutation has also been shown to enhance channel inactivation (KRAUS et al. 1998 ). At present the lack of a cac^E(TS2)1 phenotype at neuromuscular synapses remains unexplained. Although one possibility is that neuromuscular synapses express an 1 subunit variant lacking the mutant form of the P loop, to date no sequence variation has been observed within the P loop of repeat II.

cac^su(TS2)1:
The cac^su(TS2)1 mutation strongly suppressed both the TS paralytic and the neuromuscular synaptic phenotypes of cac^TS2. cac^su(TS2)1 results in substitution of threonine for a conserved isoleucine (I1187T) within the short IVS3-S4 extracellular loop and also reduces A-to-I RNA editing at an adjacent site. Editing at a single site within the region encoding the IVS3-S4 loop (a4106) leads to substitution of serine for a genomically encoded asparagine (N1185S; SMITH et al. 1996 , SMITH et al. 1998A ). This editing event is disrupted by the nearby cac^su(TS2)1 mutation, resulting in a lower percentage of edited transcripts. During A-to-I editing, specific secondary structures are thought to form between the edited region and an editing site complementary sequence (ECS) often located in a 3' intron (SMITH et al. 1998A ). The close proximity of the cac^su(TS2)1 mutation to the a4106 editing site raises the possibility that the mutation disrupts pairing with the ECS and thus the secondary structure required for efficient editing. Future studies will determine the relative contributions of the cac^su(TS2)1 mutation and altered RNA editing to the observed suppression of cac^TS2.

It is noteworthy that the cac^su(TS2)1 lesion(s) within an extracellular loop of the 1 subunit can suppress the phenotype produced by the altered cytoplasmic tail in cac^TS2, despite the apparent inability of these domains to interact directly. The close proximity of the cac^su(TS2)1 mutation to the IVS4 segment raises the possibility that a change in the position or mobility of the voltage sensor may be involved. Such intramolecular functional interactions have been observed, for example, in voltage-gated sodium channels where fast inactivation produces immobilization of the IIIS4 and IVS4 voltage sensors (CHA et al. 1999 ; KUHN and GREEFF 1999 ). Interestingly, suppressors cac^su(TS2)1 and cac^su(TS2)2 (see below) map to repeat IV and III residues that may interact with the respective S4 voltage sensors.

cac^su(TS2)2:
The cac^su(TS2)2 mutation produces strong suppression of both the TS paralytic and neuromuscular synaptic phenotypes of cac^TS2. Surprisingly, suppression of the paralytic phenotype required rearing flies at 23°, whereas suppression of the neuromuscular synaptic phenotype was observed in flies reared at 20°. The mechanism underlying this effect is not clear; however, it is likely that central and neuromuscular synapses differ in their sensitivity to culture temperature. The suppressor phenotype suggests important intramolecular interactions between the respective channel domains affected by the two mutations. cac^su(TS2)2 maps to one of several phenylalanine residues within the IIIS5 segment (F916L). Previously, a systematic analysis of S5 segment phenylalanines was initiated on the basis of another Drosophila ion channel mutation, Shaker⁵, which maps to an S5 phenylalanine (F401I) of the Shaker-encoded potassium channel (GAUTAM and TANOUYE 1990 ; KANEVSKY and ALDRICH 1999 ). Substituting phenylalanine 401 with leucine (F401L), but not isoleucine, valine, or alanine, appeared to dramatically stabilize the open state of the channel, resulting in slowed deactivation and increased open probability over a wide range of membrane potentials (KANEVSKY and ALDRICH 1999 ). These effects were modeled in terms of stabilizing interactions between S5 phenylalanines and positively charged residues within the S4 voltage sensor. We speculate that such an interaction operating in the cac^su(TS2)2 mutant (F916L) might counteract inhibition of channel activity in cac^TS2.

The results reported here raise interesting questions about the interactions of calcium channel 1 subunit domains in channel gating mechanisms underlying neurotransmitter release. It will be of great interest to examine the TS behavior of cac^TS2 mutant channels, as well as the interactions of cac^TS2 and the modifier mutations, through direct functional analysis of wild-type and mutant channels. Together with in vivo analysis examining the function of mutant channels at native synapses, these studies may provide new insights into the mechanisms underlying presynaptic calcium channel function.

	ACKNOWLEDGMENTS

We thank S. Schaeffer (Penn State University) as well as the Penn State Nucleic Acids Facility for assistance with automated sequencing. This work was supported by a predoctoral fellowship from the American Heart Association (I.M.B.) and grants from the National Institutes of Health (NIH R01-NS38064), the National Science Foundation (NSF IBN-9986990), and the Penn State President's Fund for Undergraduate Research.

Manuscript received February 1, 2002; Accepted for publication January 3, 2003.

	LITERATURE CITED

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