Multiple Sites of Action for Noncompetitive Blockers on Acetylcholine Receptor Rich Membrane Fragments from Torpedo marmorata

A vast series of compounds, different from the typical nicotinic antagonists, block in a noncompetitive manner the permeability response of several cholinergic synapses (electromotor synapse, neuromuscular junction) to acetylcholine. The interaction of several of these noncompetitive blockers was investigated in vitro with membrane fragments rich in acetylcholine receptor prepared from Torpedo marmorata electric organ. First, their effect on the conformational transitions of the membrane-bound receptor was monitored with a fluorescent cholinergic agonist and a rapid-mixing technique. All the compounds tested stabilized to various extents a “desensitized” state of the receptor exhibiting a high affinity for the agonist with minimal effect but with a high affinity in the case of the frog toxin perhydrohistrionicotoxin (H₁₂HTX). H₁₂HTX was then used to distinguish between two categories of effects of the noncompetitive blockers: (1) with some of them (phencyclidine, meproadifen, Triton X-100), the stabilization of the high-affinity state was blocked by H₁₂HTX; (2) with others (chlorpromazine, trimethisoquin), the shift of the conformational equilibrium was insensitive to H₁₂HTX. In the first instance, the stabilization of the state took place without noticeable cooperative effects, while in the second, significant positive cooperativity was systematically observed. In a second series of experiments, binding studies were carried out under equilibrium conditions with tritiated derivatives of H₁₂HTX, phencyclidine, meproadifen, Triton X-100, chlorpromazine, and trimethisoquin. All these tritiated ligands bound to “allosteric” sites distinct from the acetylcholine receptor site, but they also bound to some extent to the receptor site. Two main categories of allosteric sites for noncompetitive blockers were distinguished: (1) a “high-affinity” site which was present in one copy per acetylcholine receptor light form (250000 daltons) and blocked by H₁₂HTX and (2) a population of “low-affinity” sites present in large numbers (10–30 sites per molecule of receptor) and insensitive to H₁₂HTX. Comparison of the conformational and binding data led to the conclusion that the high-affinity site was responsible for the H₁₂HTX-sensitive effect of noncompetitive blockers on the conformational transition while the low-affinity sites were engaged in the H₁₂HTX-insensitive ones. In the cases of chlorpromazine and trimethisoquin, low-affinity H₁₂HTX-insensitive binding was significant at concentrations at which the effects on conformational transitions occurred. Reconstitution experiments indicated that the number of these low-affinity sites depended on the lipid to protein ratio, suggesting that these sites are located at the interface of the receptor protein with membrane lipids. It was also shown that the unique high-affinity site for noncompetitive blockers is in contact with all four types of subunits of the receptor molecule. The possibility is considered that this site lies within the central depression of the molecule which potentially may serve as an ion channel.