Characterization of benzodiazepine binding sites after short-wave photo-affinity labeling with flunitrazepam.

Membranes prepared from rat cerebral cortex were irradiated with short-wave UV light in the presence of flunitrazepam (FZ). This photo-affinity labeling (PAL) drastically reduces the potency of FZ binding to these membranes, but the binding of 3H-beta-carboline-3-carboxylate ethyl ester (3H-BCCE) was found to be essentially unchanged. 3H-BCCE binding was therefore determined in the presence of an antagonist (BCCE itself), an agonist (FZ) and a compound reported to discriminate between multiple benzodiazepine sites (CL 218,872). The results with BCCE are consistent with a single population of sites, but FZ binds to some of the sites with a reduced affinity (KI = 30 nM) and to the remaining sites with a very low affinity (KI approximately equal to 1 microM). CL 218,872 shows a reduced affinity but appears to interact with all of the sites. Taken together, these results indicate that the binding domains for BCCE and FZ are not identical, and that CL 218,872 interacts more strongly with the antagonist domain.