Volatile general anesthetics (VAs), such as isoflurane, disrupt nervous system function by undefined mechanisms. In vertebrates, VAs have multiple neurophysiological actions, including inhibition of neurotransmitter release. We have reported that
unc-64(
md130) semidominantly conferred profound VA resistance.
unc-64 encodes the ortholog of syntaxin 1A, a SNARE protein mediating neurotransmitter release.
md130 is an intron splice site mutation generating truncated products and a reduced level of full-length syntaxin. Our working model of how a truncated syntaxin antagonizes VA sensitivity is by interaction with another protein thereby preventing VA action on that protein. Recently, we completed testing of a set of transformed strains expressing various truncated UNC-64 syntaxins. These data show that the minimal syntaxin fragment producing VA resistance contains residues 1-106, which encodes the HAHB helices. In vertebrates, the HAHB fragment binds to vertebrate homologs of UNC-13; thus, we hypothesized that UNC-13 is the target of the truncated syntaxins and of VAs. This model predicts that UNC-13 overexpression in excess of truncated syntaxin should suppress VA resistance. Thus, we crossed mdIs3, which overexpresses wild type UNC-13, into a strain expressing truncated syntaxin. mdIs3 fully suppressed the VA resistance of the truncated syntaxin array without altering VA sensitivity in wild type. Next we tested
unc-13(
s69), a strong probable null mutant; in order to have adequate baseline movement of the animal, (
s69) was partially suppressed by an array carrying an open form of syntaxin and by a loss-of-function allele of tomosyn (kindly provided by Janet Richmond).
tom-1(
ok285);
unc-13(
s69);oxIs34 was insensitive to the VA isoflurane up to 6 times the N2 isoflurane EC50; both
ok285 and oxIs34 were normally isoflurane sensitive. UNC-13 has a DAG binding site. VAs have been shown to disrupt binding of DAG to a homologous site on PKC, and an anesthetic photoaffinity labeling of a fragment of PKC labeled near the DAG-binding site. Thus, we hypothesized that VAs might bind to UNC-13 and alter DAG activation. We tested
unc-13(
s69) transformed with a plasmid expressing a partially rescuing fragment of
unc-13 with a canonical myristoylation sequence (kindly provided by Janet Richmond and Josh Kaplan). This myr-UNC-13S protein is thought to bypass the requirement for DAG by direct membrane targeting. If VAs act on UNC-13 to prevent DAG binding then myr-UNC-13S should be VA resistant. Indeed,
unc-13(
s69);myr-
unc-13S was completely isoflurane resistant similar to
s69 alone. These data are consistent with a model that VAs inhibit neurotransmitter release by binding to UNC-13 and reducing its affinity for DAG.