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.
Department of Pharmacology, Faculty of Medicine, University of Alberta, Edmonton, Canada.
L L
LL
Duncalfe
S M
SM
Dunn
D000818:Animals; D001386:Azides / Q000378:metabolism; D001569:Benzodiazepines / Q000378:metabolism*; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D005445:Flunitrazepam / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D009118:Muscimol / Q000494:pharmacology; D011869:Radioligand Assay; D011963:Receptors, GABA-A / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*
D001386:Azides; D004220:Disulfides; D011963:Receptors, GABA-A; D013438:Sulfhydryl Compounds; D001569:Benzodiazepines; D009118:Muscimol; D005445:Flunitrazepam; C042957:Ro 15-4513; D005033:Ethylmaleimide; D004229:Dithiothreitol