Volatile anesthetics bind rat synaptic snare proteins.
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 F-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 alpha-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.


CONCLUSIONS
SNARE proteins are potential synaptic targets of volatile anesthetics.
Nagele
Peter
P
Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Mendel
J Brett
JB
Placzek
William J
WJ
Scott
Barbara A
BA
D'Avignon
D André
DA
Crowder
C Michael
CM
D018685:Anesthetics, Inhalation / Q000378:metabolism*; D000818:Animals; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D050600:SNARE Proteins / Q000737:chemistry / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism*; D050827:Syntaxin 1 / Q000378:metabolism*; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism*
D018685:Anesthetics, Inhalation; D011994:Recombinant Proteins; D050600:SNARE Proteins; C494213:Stx1a protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; C494706:Vamp2 protein, rat; D050988:Vesicle-Associated Membrane Protein 2