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The γ-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 GABAARs 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 GABAAR transmembrane domain at the interface between the β and α subunits [Li, G. D., et.al. (2006) J. Neurosci. 26, 11599–11605]. To further define GABAAR 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 GABAARs and more extensively identify photolabeled GABAAR 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 GABAAR 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 GABAAR 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 GABAAR. [ABSTRACT FROM AUTHOR]

We synthesized 2,6-diisopropyl-4-[3-(3-methyl-3H-diazirin-3-yl)propyl]phenol (p-(4-azipentyl)propofol),or p-4-AziC5-Pro, a novel photoactivable derivativeof the general anesthetic propofol. p-4-AziC5-Prohas an anesthetic potency similar to that of propofol. Like propofol,the compound potentiates inhibitory GABAAreceptor currentresponses and allosterically modulates binding to both agonist andbenzodiazepine sites, assayed on heterologously expressed GABAAreceptors. p-4-AziC5-Pro inhibits excitatorycurrent responses of nACh receptors expressed in Xenopusoocytes and photoincorporates into native nACh receptor-enriched Torpedomembranes. Thus, p-4-AziC5-Prois a functional general anesthetic that both modulates and photoincorporatesinto Cys-loop ligand-gated ion channels, making it an excellent candidatefor use in identifying propofol binding sites. [ABSTRACT FROM AUTHOR]