Separate subunits for agonist and benzodiazepine binding in the gamma-aminobutyric acidA receptor oligomer

• Published in: The Journal of biological chemistry Vol. 261; no. 32; pp. 15013 - 15016
• Main Authors: Casalotti, S O, Stephenson, F A, Barnard, E A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 15.11.1986; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Binding Sites; Biological and medical sciences; Brain - metabolism; Cattle; Cell Membrane - metabolism; Cell receptors; Cell structures and functions; Cerebral Cortex - metabolism; Flunitrazepam - metabolism; Fundamental and applied biological sciences. Psychology; Kinetics; Molecular and cellular biology; Molecular Weight; Muscimol - metabolism; Receptors, GABA-A - isolation & purification; Receptors, GABA-A - metabolism; Gabaergic receptor; Vertebrata; Mammalia; Purification; Benzodiazepine derivatives; Ox; Brain (Vertebrata); Molecular interaction; Artiodactyla; Peptide chain; Photoaffinity labelling; Ungulata
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)66821-8

The gamma-aminobutyric acidA (GABAA) agonist muscimol can be photoactivated by 254 nm illumination to affinity label its binding site in the GABAA receptor. We have conducted this reaction on the pure receptor from bovine cerebral cortex in detergent solution, showing that [3H]muscimol can produce then a specific saturable labeling. In the detergent solution, the receptor alone is sensitive to 254 nm irradiation; this reduces the efficiency of incorporation to below that in the membranes, but the competing photoreaction with [3H]muscimol is sufficient and occurs at a representative set of the muscimol-binding sites, such that it can be employed for the photolabeling of those sites. The affinity of [3H]muscimol displayed in this irreversible reaction is indistinguishable from that of its reversible binding. gamma-Aminobutyric acid and bicuculline compete in the photolabeling reaction according to their known affinities at the gamma-aminobutyric acid-binding site. The labeling is shown to occur at the beta-subunit (apparent Mr 57,000) in the pure receptor. The binding sites for gamma-aminobutyric acid agonists, on the beta-subunits, and the benzodiazepine binding sites, on the alpha-subunits, are linked allosterically so that a strongly cooperative hetero-oligomeric structure of this receptor is deduced.