Mechanism of action of benzodiazepines on GABA _A receptors

Wild-type and mutant α1β2γ2 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 μM 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 μM for GABA, GABA plus DZP, and GABA plus DMCM, respectively. DZP- and DMCM-mediated modulation were examined in GABA _A receptors in which the β-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 μM for GABA, GABA plus 1 μM DZP, and GABA plus 1 μM DMCM. DZP- and DMCM-mediated modulations were examined in GABA _A receptors in which the α-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 βL259S. 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 μM for GABA, GABA plus 1 μM DZP, and GABA plus 1 μM DMCM, respectively. Oocytes expressing ultrahigh levels of wild-type GABA _A receptors exhibited currents in response to 1 μM 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 (β2Y205S). 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.