Background: Volatile general anesthetics (VAs) have a number of synaptic actions, one of which is to inhibit excitatory neurotransmitter release; however, no presynaptic VA binding proteins have been identified. Genetic data in Caenorhabditis elegans have led to the hypothesis that a protein that interacts with the presynaptic protein syntaxin 1A is a VA target. Motivated by this hypothesis, the authors measured the ability of syntaxin 1A and proteins that interact with syntaxin to bind to halothane and isoflurane. Methods: Recombinant rat syntaxin 1A, SNAP-25B, VAMP2, and the ternary SNARE complex that they form were tested. Binding of VAs to these proteins was detected by 19F-nuclear magnetic resonance relaxation measurements. Structural alterations in the proteins were examined by circular dichroism and ability to form complexes. Results: Volatile anesthetics did not bind to VAMP2. At concentrations in the clinical range, VAs did bind to SNAP-25B; however, binding was detected only in preparations containing SNAP-25B homomultimers. VAs also bound at clinical concentrations to both syntaxin and the SNARE complex. Addition of an N-terminal His6 tag to syntaxin abolished its ability to bind VAs despite normal secondary structure and ability to form SNARE complexes; thrombin cleavage of the tag restored VA binding. Thus, the VA binding site(s) has structural requirements and is not simply any α-helical bundle. VAs at supraclinical concentrations produced an increase in helicity of the SNARE complex; otherwise, VA binding produced no gross alteration in the stability or secondary structure of the SNARE complex. Conclusion: SNARE proteins are potential synaptic targets of volatile anesthetics.
Bibliographical noteFunding Information:
Received from the Departments of Anesthesiology and Molecular Biology/Pharmacology, Washington University School of Medicine, and the Department of Chemistry, Washington University, St. Louis, Missouri; and the Department of Anesthesiology and General Intensive Care, Medical University of Vienna, Vienna, Austria. Submitted for publication February 5, 2005. Accepted for publication May 18, 2005. Supported by the National Institutes of Health/National Institute of General Medical Sciences, Bethesda, Maryland, and the Austrian Science Fund, Vienna, Austria.