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    Mapping General Anesthetic Binding Site(s) in Human ╬▒1╬▓3 ╬│-Aminobutyric Acid Type A Receptors with [<sup>3</sup>H]TDBzl-Etomidate, a Photoreactive Etomidate Analogue

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    The ╬│-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]┬şazietomidate photolabeling of ╬▒Met-236 and ╬▓Met-286 in the ╬▒M1 and ╬▓M3 transmembrane helices identified an etomidate binding site in the GABA<sub>A</sub>R transmembrane domain at the interface between the ╬▓ and ╬▒ subunits [Li, G. D., et.al. (2006) <i>J. Neurosci. 26</i>, 11599ÔÇô11605]. To further define GABA<sub>A</sub>R etomidate binding sites, we now use [<sup>3</sup>H]┬şTDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-╬▒1╬▓3 GABA<sub>A</sub>Rs and more extensively identify photolabeled GABA<sub>A</sub>R amino acids. [<sup>3</sup>H]┬şTDBzl-etomidate photolabeled in an etomidate-inhibitable manner ╬▓3Val-290, in the ╬▓3M3 transmembrane helix, as well as ╬▒1Met-236 in ╬▒1M1, a residue photolabeled by [<sup>3</sup>H]┬şazietomidate, while no photolabeling of amino acids in the ╬▒M2 and ╬▓M2 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]┬şTDBzl-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 ╬▓3Met-227 in ╬▓M1 by [<sup>3</sup>H]┬şTDBzl-etomidate and [<sup>3</sup>H]┬şazietomidate also provides evidence of a homologous etomidate binding site at the ╬▓3Ôłĺ╬▓3 subunit interface in the ╬▒1╬▓3 GABA<sub>A</sub>R