Ammar Hawasli

SUPPRESSORS OF SYNTAXIN PARTIAL-LOSS OF FUNCTION SHOW ANESTHETIC RESISTANCE IN CAENORHABDITUS ELEGANS

Although anesthetics were first developed and clinically introduced over a century ago, the underlying mechanisms behind anesthetics and anesthetic sensitivity are still unresolved. By taking a genetic approach to uncovering anesthetic mechanisms, scientists may be able to bridge the mysterious gap between molecular effects and behavioral effects of anesthetics. Scientists have chosen to study "anesthesia genes" in the fruit fly Drosophila melanogaster and the nematode Caenorhabditus elegans due to their powerful genetics.

Previous studies have shown that mutants of the syntaxin gene in C. elegans drastically alter anesthetic sensitivity. Syntaxin is a membrane-spanning protein located in the cellular membrane of presynaptic nerve terminals. As vesicles undergo exocytosis, syntaxin plays a fundamental role in synaptic transmitter release from the presynaptic nerve terminal. Anesthetics in C. elegans reduce transmitter release by a syntaxin mediated mechanism. In C. elegans, a partial loss or reduction of function in the syntaxin gene, unc-64, leads to viable and fertile but rather uncoordinated and abnormal adults. For example, unc-64(e246) reduction of function strain is very uncoordinated and immobile in comparison with the wild type strain.

In order to identify proteins that may function with syntaxin to regulate transmitter release and anesthetic action, Stephen Hunt mutagenized a population of unc-64(e246) mutants and screened their progeny for suppressors of the reduction of function mutation. I took two of these mutants and out-crossed them with wild-type animals several times to remove the unc-64(e246) mutation from the background. The resulting strains both have a "loopy" hyperactive phenotype. In anesthetic locamotion assays, both suppressor strains are very resistant to isoflurane; the suppressors' isoflurane EC₅₀ values (the anesthetic concentration at which the effect of locomotion is half-maximal) are 2.5 and 2.9 times greater than the wild-type value. Other behavioral anesthetic assays also confirm that both strains appear very resistant.

The loopy phenotypes in both strains are semi-dominant, with one strain being much less dominant than the other. Through a recessive mapping technique, I mapped one suppressor mutation onto the first chromosome. The other suppressor may likely be on the first or fifth chromosome; however, further mapping is necessary.

To fully map each suppressor, more fine-tuned techniques, such as three-factor mapping, will be necessary. Fine mapping will serve as a prelude to identification of the mutated gene. Furthermore, it will be essential to formally determine if the loopy phenotype corresponds to the unc-64(e246) suppression and to the anesthetic resistant phenotypes.

Ammar Hawasli¹, Stephen Hunt², Christine Liu², CM Crowder,³. ¹Howard Hughes Medical Institute Summer Undergraduate Research Fellow, Washington University; ²Department of Anesthesiology, Washington Univ. School of Medicine, St. Louis, MO; ³Department of Molecular Biology/Pharmacology, Washington Univ. School of Medicine, St. Louis, MO.