Wild-type and mutant alpha1beta2gamma2 GABA(A) receptors were expressed in Xenopus laevis oocytes and examined using the two-electrode voltage clamp. Dose-response relationships for GABA were compared in the absence and presence of 1 microM diazepam (DZP) or methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The dose-current relationships yielded EC(50)'s (concentration for half-maximal activation) of 41.0+/-3.0, 21.7+/-2.7, and 118.3+/-6.8 microM for GABA, GABA plus DZP, and GABA plus DMCM, respectively.DZP- and DMCM-mediated modulation were examined in GABA(A) receptors in which the beta-subunit carries the L259S mutation. This mutation has been shown to produce spontaneous opening and impart a leftward shift in the dose-response relationship. In this case, neither DZP nor DMCM produced a significant alteration in the GABA dose-response relationship with GABA EC(50)'s of 0.078+/-0.005, 0.12+/-0.03, and 0.14+/-0.004 microM for GABA, GABA plus 1 microM DZP, and GABA plus 1 microM DMCM.DZP- and DMCM-mediated modulations were examined in GABA(A) receptors in which the alpha-subunit carries the L263S mutation. This mutation also produced spontaneous opening and a leftward shift of the GABA dose-response relation, but to a lesser extent than that of betaL259S. In this case, the leftward and rightward shifts for DZP and DMCM were still present with EC(50)'s=0.24+/-0.03, 0.14+/-0.02, and 1.2+/-0.04 microM for GABA, GABA plus 1 microM DZP, and GABA plus 1 microM DMCM, respectively.Oocytes expressing ultrahigh levels of wild-type GABA(A) receptors exhibited currents in response to 1 muM DZP alone, whereas DMCM decreased the baseline current. The DZP-mediated activation currents were determined in wild-type receptors as well as receptors in which the GABA binding site was mutated (beta2Y205S). The EC(50)'s for DZP-mediated activation were 72.0+/-2.0 and 115+/-6.2 nM, respectively, similar to the EC(50) for DZP-mediated enhancement of the wild-type GABA-activated current (64.8+/-3.7 nM). Our results support a mechanism in which DZP increases the apparent affinity of the receptor, not by altering the affinity of the closed state, but rather by shifting the equilibrium towards the high-affinity open state.