Differential effects of 4'-chlorodiazepam on expressed human GABAA receptors

The interactions of the atypical benzodiazepine 4'-chlorodiazepam (Ro 5-4864) with functionally expressed human GABA A receptor cDNAs were determined. Cotransfection of human α 2 , β 1 , and γ 2 subunits was capable of reconstituting a 4'-chlorodiazepam recognition site as revealed by a dose-dependent potentiation of t-[ 35 S] butylbicyclophosphorothionate ([ 35 S]-TBPS) binding to the GABA-activated chloride channel. This site is found on GABA A receptor complexes containing sites for GABA agonist-like benzodiazepines and neuroactive steroids. The importance of the α subunit was further demonstrated as substitution of either α 1 , or α 3 for the α 2 subunit did not reconstitute a 4'-chlorodiazepam recognition site that was capable of modulating [ 35 S]TBPS binding under the same experimental conditions. The 4'-chlorodiazepam modulatory site was shown to be distinct from the benzodiazepine site, but the phenylquinolines PK 8165 and PK 9084 produced effects similar to 4'-chlorodiazepam, consistent with the previous analysis of the 4'-chlorodiazepam site in brain homogenates. Further analysis of the subunit requirements revealed that coexpression of α 2 and β 1 , alone reconstituted a 4'-chlorodiazepam recognition site. It is interesting, however, that the 4'-chlorodiazepam site was found to inhibit [ 35 S]TBPS binding to the GABA-activated chloride channel. Thus, the 4'-chlorodiazepam site may be reconstituted with only the α and β polypeptides