Characterization of the Etomidate Binding Site in Expressed α1β3 GABAAR by Photoaffinity Labeling

A major site of general anesthetic action is the inhibitory γ-aminobutyric acid type A receptor (GABAAR), which responds to various anesthetics by an enhanced GABA response. Using a photoreactive analog of the general anesthetic etomidate ([3H]azietomidate), the etomidate binding site in the bovine brain GABAAR was identified by the pharmacologically specific photolabeling of two amino acids in the transmembrane domain, βMet-286 in the M3 helix and αMet-236 (α1 numbering) in the M1 helix, which both project into the interface between the β and α subunits (Li et al, J. Neurosci., 2006 26:11599-605). The GABAAR preparation used in that study was heterogeneous, as it was isolated on a benzodiazepine affinity column from a detergent extract of brain membranes. To examine anesthetic interactions with GABAAR of known subunit composition, we expressed α1β3 GABAAR in HEK cells with an N-terminal FLAG epitope tag on α1 and purified hundreds of picomoles of receptor by detergent solubilization, affinity chromatography, and reconstitution into lipid. When this GABAAR was photolabeled with [3H]azietomidate, the 3H incorporation was enhanced by GABA and inhibited by etomidate, as determined by SDS-PAGE. From a preparative scale [3H]azietomidate photolabeling of GABAAR (350 pmol of muscimol binding sites), potential photoincorporation in each of the 4 transmembrane helices from both the α1 and β3 subunits was examined by Edman degradation. In the α1β3 GABAAR, [3H]azietomidate photolabeled the two residues at the β-α interface identified previously, as well as three additional residues, two within the β3M3 helix and the third in the α1M3 helix. To determine if these new labeled residues contribute to an etomidate binding site or represent lipid-exposed labeling, we are currently characterizing the etomidate inhibition of [3H]azietomidate photolabeling at the level of individual amino acids.