Isoflurane binds to presynaptic snare proteins

Abstract

GRAZ-01-15 Introduction: The molecular mechanism of action of volatile anaesthetics remains unknown. Recent results show marked effects of volatile anaesthetics on postsynaptic ligand-gated ion-channels as well as on presynaptic neurotransmitter release. In this study, we tried to elucidate the role of presynaptic SNARE proteins in the mechanism of volatile anaesthetic action, as implicated by recent genetic findings. Neuronal SNARE proteins (SNAP-25, syntaxin, and VAMP) interact with each other and are essential for synaptic vesicle exocytosis. Methods: His6-tagged recombinant rat SNARE proteins were expressed in BL21(DE3) bacteria and purified by Ni-NTA-agarose chromatography and FPLC. Binding of isoflurane to SNARE proteins was measured by 19F-NMR. Resulting T2 times are indicative of binding when significantly lower than buffer. NMR measurements were performed at various isoflurane concentrations. Results: Recombinant SNARE proteins showed distinctively different isoflurane binding characteristics. Rat SNAP-25 bound isoflurane in a dose-dependent manner at clinical concentrations (0.1-0.6 mM), whereas rat syntaxin showed non-saturable binding over a 100-fold range of isoflurane concentrations (0.06-6.0 mM). These results are suggestive of a few high-affinity binding sites on rat SNAP-25 and of many low-affinity binding sites on rat syntaxin. VAMP produced T2 values comparable to buffer alone and did therefore not show binding of isoflurane. Conclusions: In this study, we were able to identify a neuronal presynaptic protein, SNAP-25, that binds isoflurane at clinical concentrations. Binding of isoflurane to SNAP-25 might interfere with its physiological functions, particularly the interaction with other SNARE proteins. Thus, our finding might explain how volatile anaesthetics reduce presynaptic neurotransmitter release.