A Transmembrane Amino Acid in the GABAA Receptor β2 Subunit Critical for the Actions of Alcohols and Anesthetics

Alcohols and inhaled anesthetics enhance the function of GABA(A) receptors containing alpha, beta, and gamma subunits. Molecular analysis has focused on the role of the alpha subunits; however, there is evidence that the beta subunits may also be important. The goal of our study was to determine whether Asn265, which is homologous to the site implicated in the alpha subunit (Ser270), contributes to an alcohol and volatile anesthetic binding site in the GABA A receptor beta(2) subunit. We substituted cysteine for Asn265 and exposed the mutant to the sulfhydryl-specific reagent octyl methanethiosulfonate (OMTS). We used two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes and found that, after OMTS application, GABA-induced currents were irreversibly potentiated in mutant alpha(1)beta(2)(N265C)gamma(2S) receptors [but not alpha(1)beta(2)(I264C)gamma(2S)], presumably because of the covalent linking of octanethiol to the thiol group in the substituted cysteine. It is noteworthy that this effect was blocked when OMTS was applied in the presence of octanol. We found that potentiation by butanol, octanol, or isoflurane in the N265C mutant was nearly abolished after the application of OMTS, suggesting that an alcohol and volatile anesthetic binding site at position 265 of the beta(2) subunit was irreversibly occupied by octanethiol and consequently prevented butanol or isoflurane from binding and producing their effects. OMTS did not affect modulation or direct activation by pentobarbital, but there was a partial reduction of allosteric modulation by flunitrazepam and alphaxalone in mutant alpha(1)beta(2)(N265C)gamma(2S) receptors after OMTS was applied. Our findings provide evidence that Asn265 may contribute to an alcohol and anesthetic binding site.