Photoaffinity labeling of [ 3H] flunitrazepam- and [ 3H] RO15-4513-bound pellets in rat cerebral cortex and cerebellum

Irreversible incorporation of [ 3H] flunitrazepam and [ 3H] Ro15-4513 into GABA/benzodiazepine receptor subunits was studied by UV irradiation using ligand-bound membrane pellets from rat cerebral cortical and cerebellar synaptic membranes. Specific incorporation for [ 3H] flunitrazepam was greater in the pellet than in the suspension. The incorporation was identical for [ 3H] Ro15-4513 in both pellet and suspension. With the ligand-bound pellets, 50% of the available binding sites were photolabeled by both ligands in cortex and cerebellum. SDS polyacrylamide gel electrophoresis and fluorography of [ 3H] flunitrazepam photo-labeled receptor revealed the same number of major sites in both brain regions. In contrast, [ 3H] Ro15-4513 appears to label fewer sites in cortex and cerebellum. Photoaffinity labeling with [ 3H] flunitrazepam in ligand-bound membrane pellet provides a more selective and reliable method for studying the subunit structure of GABA/benzodiazepine receptor complex.