AGONIST BINDING TO THE TORPEDO ACETYLCHOLINE-RECEPTOR .1. COMPLEXITIES REVEALED BY DISSOCIATION KINETICS

Examination of the kinetics of dissociation of [H-3]acetylcholine and [H-3]suberyldicholine from the membrane-bound acetylcholine receptor f rom Torpedo californica has revealed complexities in the high-affinity binding of nicotinic agonists. Each agonist binds to two high-affinit y sites per receptor with an equilibrium dissociation constant of appr oximately 15 nM. When dissociation of [H-3]acetylcholine from the rece ptor complex was triggered by dilution, dissociation occurred as a mon ophasic process with an apparent rate of 0.023 +/- 0.010 s(-1). Howeve r, when micromolar concentrations of unlabeled agonists (acetylcholine , carbamylcholine or suberyldicholine) were included in the dilution b uffer this rate increased about 5-fold. This accelerating effect occur red even when the two high-affinity sites were initially saturated wit h 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 mu M, no additional sites for [H-3]acetylc holine were detected at equilibrium. To explain these results, we prop ose that each high-affinity site is made up of two subsites, A and B, which are mutually exclusive at equilibrium. With [H-3]acetylcholine i nitially occupying site A, occupancy of site B by unlabeled ligand red uces the affinity for site A and accelerates the dissociation of the r adioligand. Studies of dissociation of [H-3]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 [H-3]acetylcholine (0.028 +/- 0.012 s(-1)), this rate was onl y marginally, if at all, affected by the presence of unlabeled ligands . These results, in addition to those presented in the accompanying ma nuscript, lead to the proposal that [H-3]suberyldicholine is able to c ross-link the two subsites or at least sterically occlude the second s ite.