Flunitrazepam photoaffinity labeling of the GABAA receptor reduces inhibition of [3H]Ro15-4513 binding by GABA (original) (raw)

The benzodiazepine drugs modulate 7-aminobutyric acid (GABA)-mediated synaptic transmission via a high-affinity binding site that is part of the GABA^ receptor complex, but which is distinct from the GABA binding site. Ro! 5-4513 is a b~.~z_~-li~_~pkne neg~.five modulator of GABA action Lh~t ~sphys ~nique ~ati-ethanol properties both in vivo and in vitro. Ro15-4513 has been reported to photoaffimity label nearly 100% of the benzodi~zepine b~ding sites in rat brain homogeaat~. In contrast, the ~azepine positive modulator fluaitrazepam photoaffirtity labels only 25% of the sites. Here, we have examined the reverszbie biLding of [3H]Ro15-4513, [3l-I]flumazenil (Ro15-1788), mad [3H]fltmitrazepam to embryonic chick brain membranes, and to membranes that have been photoaffinity labeled with nonraclioaetive fltmitrazepam. Photoaffinity labeling with flunitrazep~n decreased the subsequent revergible binding of [~H]flunitrazepam and [3H]flumazenil, but increased the binding of [3H]RoI5-4513. The increase in [3H]Ro15..4513 binding after flunitrazepam photoaffinity labeling was due to a decrease in the apparent Ko, with no change in B~,. Following photoaffmity labeling, negative modulation of [3HIRo15-4513 bhading 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 rever~ible bmd;~ng of [~H]Ro!5-45t3. GABA (7-aminobutyric acid); Benzodiazepine; Ro15-4513; Photoaffinity labeling; Flumazeni|; Ro15-1788 I. iniroduction The GABA A receptor is a multimefic protein complex that mediates many of the effects of y-aminobutyde acid (GABA), the major inhibitory neurotransmitter in the vertebrate brain.