Gamma-aminobutyric acid (GABA) and pentobarbital induce different conformational rearrangements in the GABA A receptor alpha1 and beta2 pre-M1 regions.

Gamma-aminobutyric acid (GABA) binding to GABA(A) receptors (GABA(A)Rs) triggers conformational movements in the alpha(1) and beta(2) pre-M1 regions that are associated with channel gating. At high concentrations, the barbiturate pentobarbital opens GABA(A)R channels with similar conductances as GABA, suggesting that their open state structures are alike. Little, however, is known about the structural rearrangements induced by barbiturates. Here, we examined whether pentobarbital activation triggers movements in the GABA(A)R pre-M1 regions. Alpha(1)beta(2) GABA(A)Rs containing cysteine substitutions in the pre-M1 alpha(1) (K219C, K221C) and beta(2) (K213C, K215C) subunits were expressed in Xenopus oocytes and analyzed using two-electrode voltage clamp. The cysteine substitutions had little to no effect on GABA and pentobarbital EC(50) values. Tethering chemically diverse thiol-reactive methanethiosulfonate reagents onto alpha(1)K219C and alpha(1)K221C affected GABA- and pentobarbital-activated currents differently, suggesting that the pre-M1 structural elements important for GABA and pentobarbital current activation are distinct. Moreover, pentobarbital altered the rates of cysteine modification by methanethiosulfonate reagents differently than GABA. For alpha(1)K221Cbeta(2) receptors, pentobarbital decreased the rate of cysteine modification whereas GABA had no effect. For alpha(1)beta(2)K215C receptors, pentobarbital had no effect whereas GABA increased the modification rate. The competitive GABA antagonist SR-95531 and a low, non-activating concentration of pentobarbital did not alter their modification rates, suggesting that the GABA- and pentobarbital-mediated changes in rates reflect gating movements. Overall, the data indicate that the pre-M1 region is involved in both GABA- and pentobarbital-mediated gating transitions. Pentobarbital, however, triggers different movements in this region than GABA, suggesting their activation mechanisms differ.