An evolutionarily conserved presynaptic protein is required for isoflurane sensitivity in Caenorhabditis elegans.
BACKGROUND
Volatile general anesthetics inhibit neurotransmitter release by an unknown mechanism. A mutation in the presynaptic soluble NSF attachment protein receptor (SNARE) protein syntaxin 1A was previously shown to antagonize the anesthetic isoflurane in Caenorhabditis elegans. The mechanism underlying this antagonism may identify presynaptic anesthetic targets relevant to human anesthesia.


METHODS
Sensitivity to isoflurane concentrations in the human clinical range was measured in locomotion assays on adult C. elegans. Sensitivity to the acetylcholinesterase inhibitor aldicarb was used as an assay for the global level of C. elegans neurotransmitter release. Comparisons of isoflurane sensitivity (measured by the EC50) were made by simultaneous curve fitting and F test as described by Waud.


RESULTS
Expression of a truncated syntaxin fragment (residues 1-106) antagonized isoflurane sensitivity in C. elegans. This portion of syntaxin interacts with the presynaptic protein UNC-13, suggesting the hypothesis that truncated syntaxin binds to UNC-13 and antagonizes an inhibitory effect of isoflurane on UNC-13 function. Consistent with this hypothesis, overexpression of UNC-13 suppressed the isoflurane resistance of the truncated syntaxins, and unc-13 loss-of-function mutants were highly isoflurane resistant. Normal anesthetic sensitivity was restored by full-length UNC-13, by a shortened form of UNC-13 lacking a C2 domain, but not by a membrane-targeted UNC-13 that might bypass isoflurane inhibition of membrane translocation of UNC-13. Isoflurane was found to inhibit synaptic localization of UNC-13.


CONCLUSIONS
These data show that UNC-13, an evolutionarily conserved protein that promotes neurotransmitter release, is necessary for isoflurane sensitivity in C. elegans and suggest that its vertebrate homologs may be a component of the general anesthetic mechanism.
Metz
Laura B
LB
Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110-1010, USA.
Dasgupta
Nupur
N
Liu
Christine
C
Hunt
Stephen J
SJ
Crowder
C Michael
CM
D000818:Animals; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D002352:Carrier Proteins; D004305:Dose-Response Relationship, Drug; D019143:Evolution, Molecular; D007530:Isoflurane / Q000494:pharmacology*; D009043:Motor Activity / Q000187:drug effects / Q000502:physiology; D017729:Presynaptic Terminals / Q000187:drug effects* / Q000502:physiology*; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D017384:Sequence Deletion; D013045:Species Specificity
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D050765:Qa-SNARE Proteins; C083141:phorbol ester binding protein; D007530:Isoflurane