Mapping General Anesthetic Binding Site(s) in Human α1β3 γ-Aminobutyric Acid Type A Receptors with [³H]TDBzl-Etomidate, a Photoreactive Etomidate Analogue

The γ-aminobutyric acid type A receptor (GABA_AR) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABA_ARs purified from bovine brain, [³H]­azietomidate photolabeling of αMet-236 and βMet-286 in the αM1 and βM3 transmembrane helices identified an etomidate binding site in the GABA_AR transmembrane domain at the interface between the β and α subunits [Li, G. D., et.al. (2006) <i>J. Neurosci. 26</i>, 11599–11605]. To further define GABA_AR etomidate binding sites, we now use [³H]­TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-α1β3 GABA_ARs and more extensively identify photolabeled GABA_AR amino acids. [³H]­TDBzl-etomidate photolabeled in an etomidate-inhibitable manner β3Val-290, in the β3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [³H]­azietomidate, while no photolabeling of amino acids in the αM2 and βM2 helices that also border the etomidate binding site was detected. The location of these photolabeled amino acids in GABA_AR homology models derived from the recently determined structures of prokaryote (GLIC) or invertebrate (GluCl) homologues and the results of computational docking studies predict the orientation of [³H]­TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA_AR intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of β3Met-227 in βM1 by [³H]­TDBzl-etomidate and [³H]­azietomidate also provides evidence of a homologous etomidate binding site at the β3−β3 subunit interface in the α1β3 GABA_AR