Volatile anesthetics are a clinically important class of drugs whose mechanism of action is not understood. Besides the potential to rationally design safer and more effective anesthetics, elucidating anesthetic mechanisms should impact fundamental areas of neurobiology such as synaptic transmission, consciousness, and memory. Genetics is an essential tool for determining which molecular events are actually responsible for anesthetic-induced nervous system depression. The nematode Caenorhabditis elegans is a powerful genetic model that has a simple nervous system utilizing the same fundamental synaptic machinery as humans. We have found that an unusual mutation in the highly conserved gene coding for syntaxin renders C. elegans resistant to clinical concentrations of volatile anesthetics. We have also found that the anesthetic isoflurane binds to rat SNAP-25, one of the proteins that tightly interacts with syntaxin. Our proposed experiments are designed to explore this astonishing discovery by: 1) Identifying the region of mutant syntaxin necessary and sufficient to produce anesthetic resistance. 2) Identifying genes that function along with syntaxin to regulate anesthetic action and attempt to find mutants in SNAP-25 that are resistant to anesthetics 3) Determine the pathway and cells through which these genes act to mediate anesthetic effects 4) Determine whether anesthetics bind to SNAP-25 when in complex with syntaxin and define by mutagenesis the binding site for anesthetics on SNAP-25. The mechanism of action of volatile anesthetics that will be defined by the proposed work can be used to interpret and guide vertebrate experiments such as transgenic mice knockouts. The combination of invertebrate and vertebrate genetics and in vitro studies should finally allow us to reach our goal of understanding the mechanism of action of this clinically and scientifically important class of drugs.
