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[摘要]：The gamma-aminobutyric acid type A receptor (GABAAR) 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(A)Rs purified from bovine brain, [H-3]azietomidate photolabeling of alpha Met-236 and beta Met-286 in the alpha M1 and beta M3 transmembrane helices identified an etomidate binding site in the GABA(A)R transmembrane domain at the interface between the beta and alpha subunits [Li, G. D., et.al. (2006) J. Neurosci. 26, 11599-11605]. To further define GABAAR etomidate binding sites, we now use [H-3]TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietornidate, to photolabel purified human FLAG-alpha 1 beta 3 GABA(A)Rs and more extensively identify photolabeled GABA(A)R amino acids. PHYTDBzl-etomidate photolabeled in an etomidate-inhibitable manner beta 3Val-290, in the beta 3M3 transmembrane helix, as well as alpha 1Met-236 in alpha 1M1, a residue photolabeled by [H-3]azietomidate, while no photolabeling of amino acids in the alpha M2 and beta M2 helices that also border the etornidate binding site was detected. The location of these photolabeled amino acids in GABA(A)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 [H-3]TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA(A)R intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of beta Met-227 in beta M1 by [H-3]TDBzl-etomidate and [H-3]azietomidate also provides evidence of a homologous etomidate binding site at the beta 3-beta 3 subunit interface in the alpha 1 beta 3 GABA(A)R. |
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