Agonist binding to the Torpedo acetylcholine receptor. 1. Complexities revealed by dissociation kinetics.

Abstract

Examination of the kinetics of dissociation of [3H]acetylcholine and [3H]suberyldicholine from the membrane-bound acetylcholine receptor from Torpedo californica has revealed complexities in the high-affinity binding of nicotinic agonists. Each agonist binds to two high-affinity sites per receptor with an equilibrium dissociation constant of approximately 15 nM. When dissociation of [3H]acetylcholine from the receptor complex was triggered by dilution, dissociation occurred as a monophasic process with an apparent rate of 0.023 +/- 0.010 s(-1). However, when micromolar concentrations of unlabeled agonists (acetylcholine, carbamylcholine or suberyldicholine) were included in the dilution buffer this rate increased about 5-fold. This accelerating effect occurred even when the two high-affinity sites were initially saturated with the radioligand. This suggested the presence of an additional site (or subsite) for agonist with affinity in the micromolar range. However, at concentrations of 0-20 microM, no additional sites for [3H]acetylcholine were detected at equilibrium. To explain these results, we propose that each high-affinity site is made up of two subsites, A and B, which are mutually exclusive at equilibrium. With [3H]acetylcholine initially occupying site A, occupancy of site B by unlabeled ligand reduces the affinity for site A and accelerates the dissociation of the radioligand. Studies of dissociation of [3H]suberyldicholine, a large bis-quaternary agonist, provide some clue as to the possible physical nature of these subsites. Whereas its dissociation rate was similar to that of [3H]acetylcholine (0.028 +/- 0.012 s(-1)), this rate was only marginally, if at all, affected by the presence of unlabeled ligands. These results, in addition to those presented in the accompanying manuscript, lead to the proposal that [3H]suberyldicholine is able to cross-link the two subsites or at least sterically occlude the second site.