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 shown previously that mutations in
unc-64 profoundly alter the volatile anesthetic (VA) sensitivity of C. elegans . Animals with the
unc-64(
md130) mutation are semidominantly resistant to isoflurane and halothane while worms carrying other hypomorphic
unc-64 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 produces only the truncated
md130 product and no wild type product conferred VA resistance similar to
unc-64(
md130) and predictably failed to rescue the lethality of
unc-64(null) . N2 transformed with syntaxin containing only the H3 and TM domains (delta-Habc 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 delta-H3 TM) are uncoordinated and slower growing compared to N2 and are not VA resistant. This plasmid also does not rescue the lethality of
unc-64 (null) . Other
unc-64 deletion constructs and amino acid substitution mutations are currently being made.