Abstract
Binding kinetics and affinities are determined for 25 antagonists interacting with the noncompetitive blocker site of the gamma-aminobutyric acidA receptor complex present in bovine brain membranes. Four radiolabeled noncompetitive antagonists are 4-tert-butylbicyclophosphoro[35S]thionate ([35S]TBPS), 4-tert-butylbicycloortho[3',4'-3H2]benzoate, 4'-cyano-4-sec-[3,4-3H2]butylbicycloorthobenzoate, and the new 4'-ethynyl-4-n-[2,3-3H2]propylbicycloorthobenzoate. The other 21 antagonists are unlabeled inhibitors of three chemical classes (other trioxabicyclooctane, dithiane, and cyclodiene insecticides). The radioligands bind to a single noninteracting site in the membranes, based on linear Scatchard plots and monophasic association and dissociation kinetics. The kinetics of unlabeled ligands are estimated by their effect on the [35S]TBPS association curve, using the theoretical model of Motulsky and Mahan [Mol. Pharmacol. 25:1-9 (1984)]. The receptor affinities of trioxabicyclooctanes and dithianes correlate with their association rates, whereas those of cyclodienes correlate with their dissociation rates. The low association rate constants for all ligands (< or = 3 x 10(7) M-1 min-1 at 25 degrees) are consistent with a slow transition to a blocked receptor conformation upon binding of these channel blockers. The association rate-controlled affinity for the trioxabicyclooctanes and dithianes is suggestive of an induced-fit model in which binding of the ligand initiates a conformational change in the receptor complex to the blocked state.
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