Cloning and characterization of the human GABAA receptor alpha 4 subunit: identification of a unique diazepam-insensitive binding site.

Benzodiazepines modulate gamma-aminobutyric acid (GABA)-evoked chloride currents through a specific binding site at the GABAA 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 alpha, beta and gamma subunits. In addition, two distinguishable diazepam-insensitive benzodiazepine sites are found, spatially distributed between cerebral cortical and cerebellar regions. Coexpression of alpha 6 with beta 2 and gamma 2L 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 alpha 4 cDNA and its pharmacology by coexpression of this alpha 4 subunit with beta 2 and gamma 2L subunits. This recombinant receptor complex showed a high affinity for the previously described benzodiazepine partial agonist bretazenill, the pyrazoloquinoline compounds CGS-9895 and CGS-9896, as well as the inverse agonists DMCM (methyl 6,7-dimethoxy 4-ethyl-beta-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, beta-CCE (beta-carboline-3-carboxylate ethyl ester), Beta-CCM (beta-carboline-3-carboxylate methyl ester), FG-7142, CGS-9895 and CGS-9896 showed 7 to 10 times higher affinity for alpha 4 beta 2 gamma 2L. The pharmacology of the alpha 4 beta 2 gamma 2L 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 GABAA receptors may be composed of alpha 4 beta 2 gamma 2L subunits.