Benzodiazepine binding to GABAA receptors: differential effects of sulfhydryl modification

The effects of sulfhydryl modification on benzodiazepine binding to gamma-aminobutyric acid type A (GABAA) receptors have been studied in membrane preparations from bovine cerebral cortex. After reduction of membranes with increasing concentrations of dithiothreitol, the binding of the partial inverse agonist [3H]Ro15-4513 (2.5 nM) was inhibited with an IC50 of 4.6 mM, while the binding of the classical agonist [3H]flunitrazepam was affected only at much higher concentrations (> 30 mM). Prior desensitization of the GABAA receptor by incubation with 10 microM muscimol had no effect on dithiothreitol inhibition of [3H]Ro15-4513 binding. In equilibrium assays, 10 mM dithiothreitol increased the KD for [3H]Ro15-4513 binding by more than three-fold and reduced the density of binding sites by more than one-third. The binding sites for [3H]Ro15-4513 were protected from the effects of dithiothreitol by carrying out the reduction step in the presence of 10 microM Ro15-4513 or 10 microM flunitrazepam. Alkylation of brain membranes by N-ethylmaleimide inhibited the binding of both [3H]Ro15-4513 and [3H]flunitrazepam with a similar concentration dependence. Equilibrium binding assays in the presence of 10 mM N-ethylmaleimide showed that both the affinity and number of binding sites for [3H]Ro15-4513 were reduced, whereas only the KD for [3H]flunitrazepam binding was affected. These results demonstrate that sulfhydryl modification of GABAA receptors has different effects on the binding of a classical benzodiazepine agonist and a partial inverse agonist, suggesting differences in the modes of binding of the two ligands.