Desensitization of membrane-bound Torpedo acetylcholine receptor by amine noncompetitive antagonists and aliphatic alcohols: studies of [3H]acetylcholine binding and sodium-22 ion fluxes

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Abstract
Measurements of the kinetics of binding of [3H]acetylcholine ([3H]AcCh) to membrane-bound nicotinic AcCh receptors from Torpedo electric tissue were used to characterize the effects of a series of amine and alcohol noncompetitive antagonists on receptor conformational equilibria. The receptor exists in multiple, interconvertible conformations distinguished by agonist binding affinity. In the absence of cholinergic ligands, certain aromatic amines including proadifen, dimethisoquin and lidocaine, as well as propanol and butanol, produce a dose-dependent increase in the fraction of receptors (f) in a high-affinity conformation from a value of fmax .apprx. 0.17 in the absence of drug of fmax .apprx. 0.9. Not all noncompetitive antagonists produce that same value of fmax. For histrionicotoxin (HTX), fmax .apprx. 0.3, and the aromatic amine adiphenine did not alter f while tetracaine actually decreased f to 0.1. The high-affinity receptor conformation stabilized by noncompetitive antagonists was characterized by the rate constant (krec) for receptor reisomerization upon removal of stabilizing ligand and the rate constant (kdis) for dissociation of [3H]AcCh-receptor complexes. On the basis of these criteria, the high-affinity receptor conformation stabilized by amine and alcohol noncompetitive blockers is the same as that stabilized by agonist. A 4.degree. C, krec = (2.2 .+-. 0.2) .times. 10-3 s-1 and kdis = 4 .times. 10-2 s-1. Since HTX and adiphenine produced only a small conformational perturbation, their effects on the actions of proadifen and 2-propanol were examined. HTX and adiphenine antagonized the conformational perturbation caused by proadifen, while mixtures of HTX and 2-propanol produced additive effects. Effects of noncompetitive blockers were also assayed in terms of the inhibition of agonist-induced efflux of 22Na+ from Torpedo vesicles. Exposure to proadifen in the absence of agonist produced a reversible inhibition (desensitization) of the flux response, and recovery from desensitization occurred at the same rate as the reisomerization from the high-affinity receptor state. HTX, which did not cause desensitization of the flux response, reduced the desensitization by proadifen. These results are compatible with the hypothesis that certain noncompetitive antagonists modify receptor function by stabilizing the same high-affinity (desensitized) conformation that is stabilized by agonist. This is accomplished either as a consequence of binding to the distinct allosteric site or by an alternate, presumably nonspecific mechanism. However, not all noncompetitive antagonists that bind to that specific site stabilize the same high-affinity conformation.

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