The purified GABA/benzodiazepine/barbiturate receptor complex: four types of ligand-binding sites, and the interactions between them, are preserved in a single isolated protein complex.

A GABA/benzodiazepine/barbiturate receptor complex has been purified from bovine cerebral cortex by affinity chromatography on a benzodiazepine column. Depending on the detergent present during the isolation of the receptor (deoxycholate/Triton X-100 or CHAPS/Asolectin), and during the binding assays (Triton X-100 or CHAPS), the receptor displays different binding properties for the GABAA agonist [3H]muscimol and for the chloride ion channel blocking agent [35S]t-butylbicyclophosphorothionate (TBPS), whereas the binding properties for the benzodiazepine [3H]flunitrazepam are independent of isolation and assay conditions. Both methods of isolation yield a protein complex consisting of the same two subunits of Mr 53 000 and Mr 57 000. Therefore the different binding properties reflect different conformations of the isolated receptor protein. [3H]flunitrazepam binding to the CHAPS-purified receptor is stimulated by GABA and the barbiturate pentobarbital in a dose-dependent manner. Photo-affinity labeling of the purified receptor with [3H]flunitrazepam leads to incorporation of radioactivity into both subunits, but predominantly into the Mr 53 000 band, as shown by fluorography. Proteolytic degradation by trypsin of the isolated photo-affinity labeled receptor in detergent solution proceeds via a labeled Mr 48 000 polypeptide. Proteolytic destruction of the reversible [3H]flunitrazepam and [3H]muscimol binding activities requires greater than 100 fold higher concentrations of trypsin than the decomposition of the receptor polypeptides into fragments less than Mr 10 000.