Molecular Pharmacology, Vol 12, 519-535, Copyright © 1976 by the American Society for Pharmacology and Experimental Therapeutics

Effects of Inhibitors on the Binding of Iodinated -Bungarotoxin to Acetylcholine Receptors in Rat Muscle

¹ Department of Pharmacology, Medical Sciences Building, Southampton, SO9 3TU, England

The effects of agonists and antagonists on the rate of binding of iodo--bungarotoxin to rat diaphragm have been measured. Homogenates or detergent extracts of rat diaphragm muscle (usually denervated beforehand) were used so that the time course of binding would not be affected by diffusion. Noninhibitable binding (measured in the presence of a high tubocurarine concentration) was subtracted from all binding measurements. Bungarotoxin binding was apparently a bimolecular reaction, with rate constants of 2 x 10⁶ M^-1 min^-1 (homogenate) and 5 x 10⁶ M^-1 min^-1 (solubilized). The rate constant for binding was reduced in the presence of agonists and antagonists, the relationship between retardation and inhibitor concentration being that expected for simple competitive antagonism. The equilibrium constants for antagonists, such as tubocurarine, derived from these results were close to the values found by null pharmacological methods. The apparent K_I values for agonists were similar to the concentrations needed to block neuromuscular transmission. The onset of retardation was slower with agonists than with antagonists, which suggests that desensitization may be responsible both for retardation of bungarotoxin binding and for neuromuscular blockade. Similar K_I values were found for both homogenates and detergent extracts of denervated muscle, and similar values were found in homogenates of normal and of denervated muscle, when tubocurarine or carbachol was used as inhibitor.

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ACKNOWLEDGMENT We are very grateful to Dr. Z. W. Hall for showing us his work (27, 28) before it was published.

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