Photoaffinity Labeling of Benzodiazepine Receptors in Rat Brain with Flunitrazepam Alters the Affinity of Benzodiazepine Receptor Agonist But Not Antagonist Binding

Abstract: Recently, it was proposed that β-carbolines interact with a subset of benzodiazepine (BZD) binding sites in mouse brain. This postulate was based upon evidence showing changes in binding properties of the BZD receptor following photoaffinity labeling of membranes with flunitrazepam (FLU). Under conditions in which 80% of specific ³H]diazepam binding was lost in photolabeled membranes, specific ³H]propyl β-carboline-3-carboxylate (³H]PCC) binding was spared. In this study, the binding of the BZD antagonists ³H]PCC, ³H]Ro15 1788 and ³H]CGS 8216 was examined in rat brain membranes following photoaffinity labeling with FLU. No significant changes in the apparent K_D and small reductions in the B_max of ³H antagonist binding were observed. However, in the same membranes, up to 89% of specific ³H]FLU binding was lost. When ³H]PCC (0.05 nM) was used to label the receptors in control and photolabeled membranes, the ability of BZD receptor agonists to inhibit ³H]PCC binding was greatly diminished in the photolabeled membranes. In contrast, the potency of BZD antagonists remained the same in both control and treated membranes. Based upon PCC/³H]Ro15 1788 competition experiments, the ability of PCC to discriminate between BZD receptor subtypes was unaffected by photoaffinity labeling of cortical membranes. Overall, these findings suggest that β-carbolines do not interact with a subset of BZD binding sites per se, but may be a consequence of the differential interaction of BZD agonists and antagonists with BZD binding sites that have been photoaffinity labeled with FLU. A possible mechanism underlying this phenomenon is discussed. The ability of photolabeled membranes to differentiate between BZD agonists and antagonists provides a potential screen for agonist and antagonist activity in compounds that interact with the BZD receptor.