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.

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor

“…[ 3 H]-ACh (specific activity = 46.9 mCi/mmol) was from DuPont-NEN Canada and [ 3 H]-SbCh (74 mCi/mmol) was synthesized and its specific activity determined as described [10]. [ 3 H]-(±)-epibatidine (two batches of 33.8 Ci/mmol and 66.6 Ci/mmol) was obtained from New England Nuclear Life Sciences (Boston, MA) and was isotopically diluted to make stock solutions of 1 mM with a specific activity of 48 -50 mCi/mmol.…”

“…Prior to binding experiments, acetylcholinesterase activity was inhibited by incubating concentrated membrane preparations (approximately 10 µM in α-BTx binding sites) with 0.5% volume of a stock solution of 0.3 M DNPP in isopropanol for 3 minutes at room temperature [10]. Following treatment, the membranes were diluted approximately 50-fold in Torpedo Ringers containing 4 mM CaCl 2 and stored on ice until use.…”

“…The DNPP-treated membranes (0.4 ml per sample) were dialysed against 0.4 ml radiolabelled ligand for 6 hours with rocking at 4° C [10]. Total and free radioligand concentrations were estimated by taking duplicate 50 µl samples from each compartment and counting for radioactivity.…”

“…We have previously proposed that each high affinity site in the nAChR is made up of two subsites that could be bridged by SbCh [10,11]. In order to investigate whether the "extra" binding sites for [ 3 H]-(±)-epibatidine may be one of the subsites, we studied the ability of Ach and (±)-epibatidine to displace [ 3 H]-SbCh from its receptor sites.…”

“…Additionally, the ability of various monoclonal antibodies to differentially block the binding of different classes of cholinergic ligands suggested the presence of multiple, possibly three, distinct subsites within each high affinity binding domain [9]. Later, studies of the kinetics of association and dissociation of radiolabelled ACh and the bisquaternary agonist, suberyldicholine (SbCh), again suggested that each high affinity site may be made up of two subsites [10,11].…”