A transmembrane amino acid in the GABAA receptor β2 subunit critical for the actions of alcohols and anesthetics

Abstract

Alcohols and inhaled anesthetics enhance the function of GABA(A) receptors containing α, β, and γ subunits. Molecular analysis has focused on the role of the α subunits; however, there is evidence that the β subunits may also be important. The goal of our study was to determine whether Asn265, which is homologous to the site implicated in the α subunit (Ser270), contributes to an alcohol and volatile anesthetic binding site in the GABA(A) receptor β(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 α(1)β(2)(N265C)γ(2S) receptors [but not α(1)β(2)(I264C)γ(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 β(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 α(1)β(2)(N265C)γ(2S) receptors after OMTS was applied. Our findings provide evidence that Asn265 may contribute to an alcohol and anesthetic binding site.

Fig. 1.

The five subunits that make up the TM domain of an α₁β₂γ_2S GABA_A receptor are rendered as ribbon structures that trace the α-helical backbone. For clarity, the ligand-binding and intracellular domains are not shown. The view is from the extracellular surface, directly down the chloride ion pore. In one subunit, residues Ile264 and Asn265 are rendered with space-filling surfaces. The model was built by replacing the corresponding residues in the prokaryotic proton-gated ion channel GLIC, as described under Materials and Methods.

Fig. 2.

GABA concentration response curves for wild-type α₁β₂γ_2S and mutant GABA_A receptors expressed in X. laevis oocytes. All values are presented as mean ± S.E.M. from four to five oocytes.

Fig. 3.

The β₂(N265C) point mutation reduced alcohol modulation of GABA_A receptor function. A, representative tracings showing the potentiation of EC₅ GABA-induced current by 11 mM butanol in wild-type α₁β₂γ_2S and mutant α₁β₂(N265C)γ_2S GABA_A receptors expressed in X. laevis oocytes. B, wild-type and mutant α₁β₂(I264C)γ_2S receptors were potentiated by butanol (11, 22, and 32 mM) in a concentration-dependent manner, unlike mutant α₁β₂(N265C)γ_2S receptors, which were significantly less sensitive to the potentiating effects of butanol, compared with the wild type at all concentrations tested. C, the enhancement of GABA_A receptor function by octanol was also less in the α₁β₂(N265C)γ_2S mutant than the wild type, and this effect was statistically significant at 114 and 171 μM octanol concentrations. Octanol modulation of mutant α₁β₂(I264C)γ_2S receptors did not differ significantly from the wild type. All values are presented as mean ± S.E.M. from four to five oocytes. *, p < 0.05; **, p < 0.01; ***, p < 0.001 denotes values significantly different from the wild type, as tested by two-way analysis of variance with Bonferroni's post-test.

Fig. 4.

Effect of OMTS on the EC₅ GABA responses of wild-type and mutant GABA_A receptors expressed in X. laevis oocytes. A, representative tracings showing EC₅ GABA-induced current from wild-type α₁β₂γ_2S and mutant α₁β₂(N265C)γ_2S GABA_A receptors, before and after 1-min application of 50 μM OMTS. B, OMTS irreversibly enhanced the GABA responses of mutant α₁β₂(N265C)γ_2S GABA_A receptors. The GABA responses of wild-type and mutant α₁β₂(I264C)γ_2S GABA_A receptors were not affected by OMTS. C, the effect of 50 μM OMTS on the α₁β₂(N265C)γ_2S mutant was blocked in the presence of 114 μM octanol. D, the GABA responses of α₁β₂(N265C)γ_2S receptors were irreversibly enhanced in the presence of 0.4 μM flunitrazepam. All values are presented as mean ± S.E.M. from three to six oocytes. *, p < 0.05 denotes values significantly different from the pre-OMTS condition, as tested by one-way analysis of variance with Bonferroni's post-test.

Fig. 5.

Allosteric modulation of wild-type α₁β₂γ_2S and mutant α₁β₂(N265C)γ_2S GABA_A receptors expressed in X. laevis oocytes before and after the application of 50 μM OMTS. A, in wild-type α₁β₂γ_2S receptors, there was no change in the potentiation of EC₅ GABA responses by 114 μM octanol, 0.4 μM flunitrazepam, 0.6 mM isoflurane, 50 μM pentobarbital, or 3 μM alphaxalone after a l-min application of OMTS. B, in mutant α₁β₂(N265C)γ_2S receptors, OMTS abolished the effects of 11 mM butanol, 114 μM octanol, and 0.6 mM isoflurane. The potentiating effects of 0.4 μM flunitrazepam and 3 μM alphaxalone were also reduced after OMTS was applied. However, OMTS did not affect the enhancement of EC₅ GABA responses by 50 μM pentobarbital. All values are presented as mean ± S.E.M. from four to seven oocytes. *, p < 0.05; **, p < 0.01 denotes values significantly different from the pre-OMTS condition, as tested by Student's t test.