Identification of a GABAA receptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog

General anesthetics, including etomidate, act by binding to and enhancing the function of GABA type A receptors (GABA(A)Rs), which mediate inhibitory neurotransmission in the brain. Here, we used a radiolabeled, photoreactive etomidate analog ([H-3] azietomidate), which retains anesthetic potency in vivo and enhances GABA(A)R function in vitro, to identify directly, for the first time, amino acids that contribute to a GABA(A)R anesthetic binding site. For GABA(A)Rs purified by affinity chromatography from detergent extracts of bovine cortex, [H-3] azietomidate photoincorporation was increased by GABA and inhibited by etomidate in a concentration-dependent manner (IC50 = 30 mu M). Protein microsequencing of fragments isolated from proteolytic digests established photolabeling of two residues: one within the alpha M1 transmembrane helix at alpha 1Met-236 (and/or the homologous methionines in alpha 2,3,5), not previously implicated in etomidate function, and one within the beta M3 transmembrane helix at beta 3Met-286 ( and/or the homologous methionines in beta 1,2), an etomidate sensitivity determinant. The pharmacological specificity of labeling indicates that these methionines contribute to a single binding pocket for etomidate located in the transmembrane domain at the interface between beta and alpha subunits, in what is predicted by structural models based on homology with the nicotinic acetylcholine receptor to be a water-filled pocket similar to 50 angstrom below the GABA binding site. The localization of the etomidate binding site to an intersubunit, not an intrasubunit, binding pocket is a novel conclusion that suggests more generally that the localization of drug binding sites to subunit interfaces may be a feature not only for GABA and benzodiazepines but also for etomidate and other intravenous and volatile anesthetics.