Cloning and characterization of the human GABAA receptor α4 subunit: identification of a unique diazepam-insensitive binding site

Benzodiazepines modulate γ-aminobutyric acid (GABA)-evoked chloride currents through a specific binding site at the GABA_A receptor-chloride channel complex. The heterogeneity of diazepam-sensitive benzodiazepine binding sites (type I and type II) has been identified by pharmacological approaches both with native receptors and recombinant receptors coexpressing α, β and γ subunits. In addition, two distinguishable diazepam-insensitive benzodiazepine sites arefound, spatially distributed between cerebral cortical and cerebellar regions. Coexpression of α6 with β2 and γ2L subunits creates a pharmacologically similar benzodiazepine receptor to the diazepam-insensitive site observed in cerebellum, however, there is no evidence regarding the possible subunit combination forming the DI site in cerebral tissues. Here we report the cloning of the human α4 cDNA and its pharmacology by coexpression of this α4 subunit with β2 and γ2L subunits. This recombinant receptor complex showed a high affinity for the previously described benzodiazepine partial agonist bretazenil, the pyrazoloquinoline compounds CGS-9895 and CGS-9896, as well as the inverse agonists DMCM (methyl 6,7-dimethoxy 4-ethyl-β-carboline-3-carboxylate) and Ro15-4513 as determined by [³H]Ro15-4513 binding. However, it is insensitive to the benzodiazepine type I selective compounds CL218,872 (3-methyl-6-[3-(trifluoromethyl)phenyl]-1,2,4-triazolo[4,3-b]pyridazine) and zolpidem as well as the benzodiazepine full agonists diazepam, halazolam and midazolam. In addition, the benzodiazepine receptor ligands DMCM, β-CCE (β-carboline-3-carboxylate ethyl ester), β-CCM (β-carboline-3-carboxylate methyl ester), FG-7142, CGS-9895 and CGS-9896 showed 7 to 10 times higher affinity for α4β2γ2L than for α6β2γ2L. The pharmacology of the α4β2γ2L receptor complex appears to resemble those of the diazepam-insensitive site found in the cerebral cortex. Our study thus suggests that this subpopulation of diazepam-insensitive GABA_A receptors may be composed of α4β2γ2L subunits.