Defining anesthetic mechanism: the role of SNARE proteins

In contrast to most drugs, the molecular mechanism of general anesthesia is unknown. Recently a C. elegans mutant that is resistant to isoflurane and halothane has been identified by Mike Crowder`s lab. This mutation, called unc-64(mdl30), was located in the C. elegans syntaxin gene, which codes for the presynaptic syntaxin protein, a member of the large, highly conserevd SNARE protein family. SNARE proteins are essential for exocytosis and, in particular, for synaptic neurotransmitter release. Syntaxin, along with two other SNARE proteins, forms a tight ternary complex. Based on genetic data and the structure of the SNARE complex (four-alphahelix bundle with hydrophobic core), syntaxin or a syntaxin binding protein represent plausible targets for general anesthetics. In my project I will focus on generating recombinant SNARE proteins and on testing their affinity for volatile anesthetics. I will use 19F-NMR spectroscopy, a technique partially developed at Washington University, to test binding of isoflurane to SNARE proteins and complex. Preliminary results - I have started my research fellowship in Aug 2000 show that rat SNAP-25, a syntaxin-binding SNARE protein, is binding isoflurane saturably at clinical concentrations. This result is very promising as it identifies anesthetic binding to a highly conserved vertebrate synaptic protein. To further elucidate the structural requirements for isoflurane binding I will generate mutant SNARE proteins with various deletions and test for changes in anesthetic binding. The general goal during my research fellowship is to identify the molecular mechanism of presynaptic volatile anesthetic action and the role of SNARE proteins therein.