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

Abstract

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 GABA_A agonist [³H]muscimol and for the chloride ion channel blocking agent [³⁵S]t-butylbicyclophosphoro-thionate (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 53000 and Mr 57000. 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 53000 band, as shown by fluorography. Proteolytic degradation by trypsin of the isolated photo-affinity labeled receptor in detergent solution proceeds via a labeled Mr 48000 polypeptide. Proteolytic destruction of the reversible [3H]flunitrazepam and [3H]muscimol binding activities requires > 100 fold higher concentrations of trypsin than the decomposition of the receptor polypeptides into fragments < M_r 10000.