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

Abstract

The γ-aminobutyric acid type A receptor (GABA<sub>A</sub>R) 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<sub>A</sub>Rs purified from bovine brain, [<sup>3</sup>H]­azietomidate photolabeling of αMet-236 and βMet-286 in the αM1 and βM3 transmembrane helices identified an etomidate binding site in the GABA<sub>A</sub>R 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<sub>A</sub>R etomidate binding sites, we now use [<sup>3</sup>H]­TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-α1β3 GABA<sub>A</sub>Rs and more extensively identify photolabeled GABA<sub>A</sub>R amino acids. [<sup>3</sup>H]­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 [<sup>3</sup>H]­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<sub>A</sub>R 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 [<sup>3</sup>H]­TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA<sub>A</sub>R intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of β3Met-227 in βM1 by [<sup>3</sup>H]­TDBzl-etomidate and [<sup>3</sup>H]­azietomidate also provides evidence of a homologous etomidate binding site at the β3−β3 subunit interface in the α1β3 GABA<sub>A</sub>R

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