Mechanism of action of benzodiazepines on GABAA receptors

1 Wild-type and mutant alpha 1 beta 2 gamma 2 GABA(A) receptors were expressed in Xenopus laevis oocytes and examined using the two-electrode voltage clamp. 2 Dose - response relationships for GABA were compared in the absence and presence of 1 mu M diazepam (DZP) or methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate ( DMCM). The dose current relationships yielded EC50's ( concentration for half-maximal activation) of 41.0 +/- 73.0, 21.7 +/- 72.7, and 118.3 +/- 6.8 mu M for GABA, GABA plus DZP, and GABA plus DMCM, respectively. 3 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 EC50's of 0.078 +/- 70.005, 0.12 +/- 0.03, and 0.14 +/- 0.004 mM for GABA, GABA plus 1 mu M DZP, and GABA plus 1 mu M DMCM. 4 DZP- and DMCM-mediated modulations were examined in GABAA 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 beta L259S. In this case, the leftward and rightward shifts for DZP and DMCM were still present with EC50's 0.24 +/- 0.03, 0.14 +/- 0.02, and 1.2 +/- 0.04 mu M for GABA, GABA plus 1 mu M DZP, and GABA plus 1 mu M DMCM, respectively. 5 Oocytes expressing ultrahigh levels of wild-type GABA(A) receptors exhibited currents in response to 1 mM 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 (beta 2Y205S). The EC50's for DZP-mediated activation were 72.0 +/- 72.0 and 115 +/- 6.2 nM, respectively, similar to the EC50 for DZP-mediated enhancement of the wild-type GABA-activated current ( 64.8 +/- 73.7 nM). 6 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.