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

Abstract

Background

Neurosteroids like alphaxalone are potent anxiolytics, anticonvulsants, amnestics, and sedative-hypnotics, effects linked to enhancement of GABA _A receptor gating in the central nervous system. Data locating neurosteroid binding sites on synaptic αβγ GABA _A receptors are sparse and inconsistent. Some evidence points to outer transmembrane β ⁺–α ⁻ interfacial pockets, near sites that bind the anesthetics etomidate and propofol. Other evidence suggests that steroids bind more intracellularly in β ⁺–α ⁻ interfaces.

Methods

We created 12 single-residue β3 cysteine mutations: β3T262C and β3T266C in β3-M2; and β3M283C, β3Y284C, β3M286C, β3G287C, β3F289C, β3V290C, β3F293C, β3L297C, β3E298C, and β3F301C in β3-M3 helices. We co-expressed α1 and γ2L with each mutant β3 subunit in Xenopus oocytes and electrophysiologically tested each mutant for covalent sulfhydryl modification by the water soluble reagent para-chloromercuribenzenesulfonate. We then assessed whether receptor-bound alphaxalone, etomidate, or propofol blocked cysteine modification, implying steric hindrance.

Results

Eleven mutant β3 subunits, when co-expressed with α1 and γ2L, 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 β3V290C, β3F293C, β3L297C, or β3F301C mutations. Both etomidate and propofol protected receptors with β3M286C or β3V290C mutations. Etomidate also protected β3F289C. In α1β3γ2L structural homology models, all these protected residues are located in transmembrane β ⁺–α ⁻ interfaces.

Conclusions

Alphaxalone binds in transmembrane β ⁺–α ⁻ pockets of synaptic GABA _A receptors that are adjacent and intracellular to sites for the potent anesthetics etomidate and propofol.