unc-64 encodes a homolog of vertebrate syntaxin 1A, which is expressed ubiquitously in the C. elegans nervous system. Syntaxin is a t-SNARE composed of 5 functional domains (Ha, Hb, Hc, H3, and TM) and is involved in membrane fusion of synaptic vesicles. Its H3 helical domain is known to interact with other SNARE proteins during neurotransmission. We have previously shown that mutations in unc-64 profoundly alter the volatile anesthetic (VA) sensitivity of C. elegans. Animals with the md130 mutation are resistant to isoflurane and halothane while worms carrying other hypomorphic mutations are 30 times more sensitive to these anesthetics. The md130 lesion is a G to A mutation at the splice donor site of intron 6 of unc-64. By RT-PCR, md130 produces a small amount of wild type-mRNA along with truncated forms lacking half of the H3 domain and the entire TM domain, rendering them unable to properly interact with other SNARE proteins. We want to define the structural requirements for md130’s resistance to VAs. First, we transformed N2 worms with a plasmid containing genomic unc-64 carrying the md130 lesion and found that these animals exhibit the same behavioral and VA phenotypes as unc-64(md130) homozygous animals. unc-64(null/+) animals transformed with the same plasmid are also VA resistant, and transformed unc-64(null) homozygotes are viable. Another plasmid with an added stop codon that should produce only the truncated md130 product and no wild type product not only failed to rescue unc-64 null mutants but also conferred VA resistance similar to unc-64(md130). N2 transformed with syntaxin containing only the H3 and TM domains (DHabc H3 TM) are very Unc (too Unc to test their anesthetic sensitivity), slow growing and have small brood sizes. N2 animals transformed with unc-64 lacking only the H3 domain (Habc DH3 TM) are uncoordinated and slower growing compared to N2 but are not as resistant to VAs as unc-64(md130). In addition, this plasmid does not rescue unc-64 null animals. Other unc-64 deletion constructs and amino acid substitution mutations are currently being made.
unc-64 encodes a homolog of vertebrate syntaxin 1A. Syntaxin is expressed ubiquitously in the nervous system of the nematode. unc-64 contains a high degree of homology with human and Drosophila syntaxin, suggesting that this molecule is conserved across species and performs similar functions. Syntaxin is involved in membrane fusion of synaptic vesicles and controls neurotransmitter release, in part through its H3 helical domain, which interacts with SNAP-25 and synaptobrevin. Very few viable syntaxin alleles have been identified in any animal, including C. elegans. In C. elegans, the null allele, js115, confers larval lethality: the nematode completes embryogenesis, but dies as L1 larva. Currently, only four viable syntaxin alleles have been identified in C. elegans: e246, md1259, js21, md130. js21 and e246 contain different C to T missense mutations in exon 7; md130 and md1259 contain G to A splice site mutations at the splice donors of intron 6 and intron 3, respectively. All these previously identified mutations affect the H3 domain of syntaxin or cause reduced expression. While all viable syntaxin alleles appear to reduce neurotransmitter release, large allelic differences are seen in their sensitivities to volatile general anesthetics: md130 is resistant to volatile anesthetics while js21 and md1259 are hypersensitive. In addition to the H3 domain, syntaxin also contains N-terminal HABC domains and a hinge region that are thought also to regulate membrane fusion events. However, no mutations have been identified in these regions. To better define these portions of syntaxin that regulate transmitter release and anesthetic sensitivity, a non-complementation screen using EMS as the mutagen was conducted using the unc-64 (e246) allele. Three potentially new unc-64 alleles have been isolated out of approximately 4800 F1 genomes screened. These animals exhibit phenotypes consistent with syntaxin hypomorphic alleles, including being uncoordinated and failure on re-testing to complement e246 allele. Other experiments to characterize these putative new viable syntaxin alleles are currently in progress.
We have previously shown that mutations in the neuronal syntaxin gene unc-64 profoundly alter the volatile anesthetic (VA) sensitivity of C. elegans. We found that two hypomorphic unc-64 alleles confer hypersensitivity to the VAs isoflurane and halothane but that a third unc-64 hypomorph is VA resistant. The difference between the isoflurane EC50's (the concentration where the effect on locomotion is half maximal) of the hypersensitive and resistant alleles is over 30-fold. In order to understand the molecular mechanisms of syntaxin's regulation of anesthetics, we are searching for genes that interact with the syntaxin gene. Thus, we initiated a screen for suppressors of the locomotion defect of unc-64(e246lf). The F2 progeny of EMS treated unc-64(e246) were screened for better moving animals. We have screened 14,400 F1 genomes and have established seven independent strains that clearly move better than e246. The suppressor strains moved between 6-8 times faster than e246 as measured from movies taken of them crawling on agar without food. Outcrossing of the seven strains revealed three segregating phenotypes, loopy movers (4 strains), jerky movers (1 strain), and long worms (1 strain); one strain segregated no obvious visible phenotype. We are currently determining whether these phenotypes are in fact those of the e246 suppressors. A similar screen has been performed by Owais Saifee in Mike Nonet's lab. They also isolated loopy e246 suppressors. We tested the anesthetic sensitivity of two of their loopy strains, js126 and js127;e246. Both strains were anesthetic resistant with EC50s more than twice that of N2. This confirms that this is a reasonable approach towards finding "anesthesia genes". We are currently determining the anesthetic sensitivity of the suppressors isolated in our screen.