Mapping of GABA A receptor sites that are photoaffinity-labelled by [ 3H]flunitrazepam and [ 3H]Ro 15-4513

Abstract

The GABA A receptor in brain membranes prepared from bovine cerebral cortex and cerebellum has been photoaffinity-labelled by the classical benzodiazepine agonist, [ 3H]flunitrazepam, or by the partial inverse agonist [ 3H]Ro 15-4513. Following solubilization and precipitation with trichloroacetic acid, the photoaffinity-labelled receptor preparations were subjected to specific chemical cleavage using hydroxylamine, a reagent which cleaves specifically at a relatively rare Asn-Gly bond. The resulting peptides were resolved by denaturing polyacrylamide gel electrophoresis and mapping of these peptides to the known amino acid sequences of the GABA A receptor subunits has localized the photoaffinity-labelling sites for these two ligands to distinct portions of the α subunits. It is shown that the site for [ 3H]flunitrazepam photoaffinity-labelling in the receptor populations of both the cerebral cortex and cerebellum occurs within residues 1–103 of the bovine α 1 subunit sequence (or within analogous segments of homologous α subunits). In contrast, the site of photoaffinity-labelling by [ 3H]Ro 15-4513 in the cerebral cortex and in the diazepam-sensitive GABA A receptor population of the cerebellum lies between residues 104 and the carboxy-terminus of the bovine α 1 or homologous α subunits. However, the [ 3H]Ro 15-4513 photoaffinity-labelling site for the diazepam-insensitive receptors of the cerebellum is shown to occur within residues 1–101 ( α 6 subunit numbering). These results demonstrate that the photoaffinity-labelling sites for [ 3H]flunitrazepam and [ 3H]Ro 15-4513 on the GABA A receptor are localized to distinct domains of the α 1 subunit and that [ 3H]Ro 15-4513 photoaffinity labels a site on the α 6 subunit that is unique from its site of labelling on the α 1 subunit.