Identification of a GABA A Receptor Anesthetic Binding Site at Subunit Interfaces by Photolabeling with an Etomidate Analog

Abstract

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 ([ 3 H]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, [ 3 H]azietomidate photoincorporation was increased by GABA and inhibited by etomidate in a concentration-dependent manner (IC 50 = 30 μ m ). Protein microsequencing of fragments isolated from proteolytic digests established photolabeling of two residues: one within the αM1 transmembrane helix at α1Met-236 (and/or the homologous methionines in α2,3,5), not previously implicated in etomidate function, and one within the βM3 transmembrane helix at β3Met-286 (and/or the homologous methionines in β1,2), 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 β and α subunits, in what is predicted by structural models based on homology with the nicotinic acetylcholine receptor to be a water-filled pocket ∼50 Å 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.