Actions of two GABAA receptor benzodiazepine-site ligands that are mediated via non-γ2-dependent modulation

The potent sedative-hypnotic zolpidem and the convulsant methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) act primarily by binding to the benzodiazepine site of the main inhibitory neurotransmitter receptor, the pentameric γ-aminobutyric acid type A receptor (GABAA). This binding depends critically on the wild-type F77 residue of the GABAA receptor γ2 subunit. Mice with γ2 subunit F77I point mutation (γ2I77 mouse line) lose the high-affinity nanomolar binding of these ligands as well as their most robust behavioral actions at low doses. Interestingly, the γ2I77 mice offer a tool to study the actions of these substances mediated via other possible binding sites of the GABAA receptor. In ligand autoradiographic experiments, we discovered in γ2I77 mouse brain sections a significant amount of residual non-γ2 subunit-dependent benzodiazepine site binding enriched to the striatum and septum. Zolpidem only weakly affected this residual binding at micromolar concentrations, and only a high zolpidem dose (≥ 40 mg/kg) caused sedation and deficits in motor coordination in γ2I77 mice. DMCM had an agonistic action through a secondary, low-affinity non-benzodiazepine binding site of the GABAA receptor in the forebrain of γ2I77 mice, and this drug also fully displaced the residual benzodiazepine-site labeling. In behavioral tests, a high dose (20 mg/kg) of DMCM was sedative and modulated fear learning. DMCM, but not zolpidem, acted as an agonist in recombinant GABAA α1/6β3 receptors studied using ligand binding and electrophysiological assays. Our results highlight the less well-known actions of high doses of DMCM and zolpidem that are not mediated via the γ2 subunit-containing benzodiazepine site of the GABAA receptor.