Identifying an Etomidate Binding Site in Heterologously Expressed Human Alpha1/Beta3 GABA A Receptors (GABA AR) Using Photoactive Etomidate Analogs

Abstract

The GABAAR is a target for general anesthetics, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABAARs isolated from bovine brain, [3H]azietomidate photolabeled αmMet-236 and βnMet-286 (m = 1, 2, 3 or 5; n = 1, 2 or 3) in the αM1 and βM3 transmembrane helices. To resolve uncertainties inherent in using natural sources of receptor, we used stable tetracycline-inducible HEK293 cells overexpressing α1β3 GABAARs. Furthermore, to explore the proposed site in more detail, we used two photoactivable etomidate derivatives: [3H]azietomidate, an aliphatic diazirine, and [3H]TDBzlEtomidate, an aryl diazirine with a broader amino acid side chain reactivity profile (Figure). Purified recombinant human α1β3 GABAARs, functionally reconstituted into detergent-asolectin, were photolabeled in the presence of GABA ± etomidate. [3H]Azietomidate and [3H]TDBzlEtomidate photolabeled β3Met-286 and α1Met-236, and [3H]TDBzlEtomidate photolabeled β3Val-290, one helical turn below β3Met-286. These observations prove that the photolabeled β- and α-subunits belong to a single oligomer and strengthen the hypothesis that the three photolabeled residues all belong to a single site within the β-α subunit interface. (∗S.S.J. and Z.D. contributed equally; supported by GM58448).View Large Image | View Hi-Res Image | Download PowerPoint Slide