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Photoaffinity Labeling of Benzodiazepine Receptor Proteins with the Partial Inverse Agonist [ 3 H]Ro 15–4513: A Biochemical and Autoradiographic Study
Author(s) -
Sieghart W.,
Eichinger A.,
Richards J. G.,
Möhler H.
Publication year - 1987
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1987.tb13125.x
Subject(s) - inverse agonist , flunitrazepam , chemistry , benzodiazepine , photoaffinity labeling , agonist , receptor , gabaa receptor , partial agonist , biochemistry
Photolabeling of the benzodiazepine receptor, which to date has been done with benzodiazepine agonists such as flunitrazepam, can also be achieved with Ro 15‐4513, a partial inverse agonist of the benzodiazepine receptor. [ 3 H]Ro 15‐4513 specifically and irreversibly labeled a protein with an apparent molecular weight of 51, 000 (P 51 ) in cerebellum and at least two proteins with apparent molecular weights of 51, 000 (P 51 ) and 55, 000 (P 55 ) in hippocampus. Photolabeling was inhibited by 10 μM diazepam but not by 10 μM Ro 5‐4864. The BZ 1 receptor‐selective ligands CL 218872 and ß‐carboline‐3‐carboxylate ethyl ester preferentially inhibited irreversible binding of [ 3 H]Ro 15‐4513 to protein P 51 . Not only these biochemical results but also the distribution and density of [ 3 H]Ro 15–4513 binding sites in rat brain sections were similar to the findings with [ 3 H]flunitrazepam. Thus, the binding sites for agonists and inverse agonists appear to be located on the same pro‐teins. In contrast, whereas [ 3 H]flunitrazepam is known to label only 25% of the benzodiazepine binding sites in brain membranes, all binding sites are photolabeled by [ 3 H]Ro 15‐4513. Thus, all benzodiazepine receptor sites are associated with photolabeled proteins with apparent molecular weights of 51, 000 and/or 55, 000. In cerebellum, an additional protein (MW 57, 000) unrelated to the benzodiazepine receptor was labeled by [ 3 H]Ro 15‐4513 but not by [ 3 H]flunitrazepam. In brain sections, this component contributed to higher labeling by [ 3 H]Ro 15–4513 in the granular than the molecular layer.