Numerous classes of general anesthetics inhibit etomidate binding to γ-aminobutyric acid type A (GABAA) receptors

Abstract

Enhancement of γ-aminobutyric acid type A receptor (GABA AR)-mediated inhibition is a property of most general anesthetics and a candidate for a molecular mechanism of anesthesia. Intravenous anesthetics, including etomidate, propofol, barbiturates, and neuroactive steroids, as well as volatile anesthetics and long-chain alcohols, all enhance GABAAR function at anesthetic concentrations. The implied existence of a receptor site for anesthetics on the GABAAR protein was supported by identification, using photoaffinity labeling, of a binding site for etomidate within the GABAAR transmembrane domain at the β-α subunit interface; the etomidate analog [3H]azietomidate photolabeled in a pharmacologically specific manner two amino acids, α1Met-236 in the M1 helix and βMet-286 in the M3 helix (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599-11605). Here, we use [3H]azietomidate photolabeling of bovine brain GABAARs to determine whether other structural classes of anesthetics interact with the etomidate binding site. Photolabeling was inhibited by anesthetic concentrations of propofol, barbiturates, and the volatile agent isoflurane, at low millimolar concentrations, but not by octanol or ethanol. Inhibition by barbiturates, which was pharmacologically specific and stereospecific, and by propofol was only partial, consistent with allosteric interactions, whereas isoflurane inhibition was nearly complete, apparently competitive. Protein sequencing showed that propofol inhibited to the same extent the photolabeling of α1Met-236 and βMet-286. These results indicate that several classes of general anesthetics modulate etomidate binding to the GABAAR: isoflurane binds directly to the site with millimolar affinity, whereas propofol and barbiturates inhibit binding but do not bind in a mutually exclusive manner with etomidate. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.