title: Flunitrazepam photoaffinity labeling of the GABA(A) receptor reduces inhibition of [3H]Ro15-4513 binding by GABA.
abstract: The benzodiazepine drugs modulate gamma-aminobutyric acid (GABA)-mediated synaptic transmission via a high-affinity binding site that is part of the GABA(A) receptor complex, but which is distinct from the GABA binding site. Ro15-4513 is a benzodiazepine negative modulator of GABA action that displays unique anti-ethanol properties both in vivo and in vitro. Ro15-4513 has been reported to photoaffinity label nearly 100% of the benzodiazepine binding sites in rat brain homogenates. In contrast, the benzodiazepine positive modulator flunitrazepam photoaffinity labels only 25% of the sites. Here, we have examined the reversible binding of [3H]Ro15-4513, [3H]flumazenil (Ro15-1788), and [3H]flunitrazepam to embryonic chick brain membranes, and to membranes that have been photoaffinity labeled with nonradioactive flunitrazepam. Photoaffinity labeling with flunitrazepam decreased the subsequent reversible binding of [3H]flunitrazepam and [3H]flumazenil, but increased the binding of [3H]Ro15-4513. The increase in [3H]Ro15-4513 binding after flunitrazepam photoaffinity labeling was due to a decrease in the apparent Kd, with no change in Bmax. Following photoaffinity labeling, negative modulation of [3H]Ro15-4513 binding by GABA was lost, whereas positive modulation of residual [3H]flunitrazepam binding was retained. We conclude that the site photoaffinity labeled by flunitrazepam is distinct from the site responsible for reversible binding of [3H]Ro15-4513.
authors.affiliation: Department of Anatomy and Cell Biology, State University of New York, Brooklyn 11203.
authors.forename: L A
authors.initials: LA
authors.lastname: Borden
authors.forename: T T
authors.initials: TT
authors.lastname: Gibbs
mesh_terms: D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001569:Benzodiazepines / Q000378:metabolism*; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002642:Chick Embryo; D005442:Flumazenil / Q000378:metabolism; D005445:Flunitrazepam / Q000378:metabolism*; D007700:Kinetics; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D010777:Photochemistry; D011963:Receptors, GABA-A / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D001386:Azides; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D005442:Flumazenil; D005680:gamma-Aminobutyric Acid; D005445:Flunitrazepam; C042957:Ro 15-4513