- 1Wild-type and mutant α1β2γ2 GABAA receptors were expressed in Xenopus laevis oocytes and examined using the two-electrode voltage clamp.
- 2Dose–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 EC50'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.
- 3DZP- and DMCM-mediated modulation were examined in GABAA 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 EC50'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.
- 4DZP- and DMCM-mediated modulations were examined in GABAA 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 EC50'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.
- 5Oocytes expressing ultrahigh levels of wild-type GABAA 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 EC50's for DZP-mediated activation were 72.0±2.0 and 115±6.2 nM, respectively, similar to the EC50 for DZP-mediated enhancement of the wild-type GABA-activated current (64.8±3.7 nM).
- 6Our 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.
British Journal of Pharmacology (2006) 148, 984–990. doi:10.1038/sj.bjp.0706796