Alphaxalone Binds in Inner Transmembrane β+-α- Interfaces of α1β3γ2 γ-Aminobutyric Acid Type A Receptors

Background: Neurosteroids like alphaxalone are potent anxiolytics, anticonvulsants, amnestics, and sedative-hypnotics, with effects linked to enhancement of gamma-aminobutyric acid type A (GABA(A)) receptor gating in the central nervous system. Data locating neurosteroid binding sites on synaptic alpha beta gamma GABA(A) receptors are sparse and inconsistent. Some evidence points to outer transmembrane beta(+)-alpha(-) interfacial pockets, near sites that bind the anesthetics etomidate and propofol. Other evidence suggests that steroids bind more intracellularly in beta(+)-alpha(-) interfaces. Methods: The authors created 12 single-residue beta 3 cysteine mutations: beta 3T262C and beta 3T266C in beta 3-M2; and beta 3M283C, beta 3Y284C, beta 3M286C, beta 3G287C, beta 3F289C, beta 3V290C, beta 3F293C, beta 3L297C, beta 3E298C, and beta 3F301C in beta 3-M3 helices. The authors coexpressed alpha 1 and gamma 2L with each mutant beta 3 subunit in Xenopus oocytes and electrophysiologically tested each mutant for covalent sulfhydryl modification by the water-soluble reagent para-chloromercuribenzenesulfonate. Then, the authors assessed whether receptor-bound alphaxalone, etomidate, or propofol blocked cysteine modification, implying steric hindrance. Results: Eleven mutant alpha 3 subunits, when coexpressed with alpha 1 and gamma 2L, formed functional channels that displayed varied sensitivities to the three anesthetics. Exposure to para-chloromercuribenzenesulfonate produced irreversible functional changes in ten mutant receptors. Protection by alphaxalone was observed in receptors with beta 3V290C, beta 3F293C, beta 3L297C, or beta 3F301C mutations. Both etomidate and propofol protected receptors with beta 3M286C or beta 3V290C mutations. Etomidate also protected beta 3F289C. In alpha 1 beta 3 gamma 2L structural homology models, all these protected residues are located in transmembrane beta(+)-alpha(-) interfaces. Conclusions: Alphaxalone binds in transmembrane beta(+)-alpha(-) pockets of synaptic GABA(A) receptors that are adjacent and intracellular to sites for the potent anesthetics etomidate and propofol.