CARDIAC MUSCARINIC RECEPTORS - COOPERATIVITY AS THE BASIS FOR MULTIPLE STATES OF AFFINITY

Cooperativity has been investigated as the mechanistic basis for effec ts observed with cardiac muscarinic receptors in washed membranes from Syrian hamsters, Specifically, N-[H-3]methylscopolamine labeled only 66-75% of the sites labeled by [H-3]quinuclidinylbenzilate at apparent ly saturating concentrations of each radioligand. Also, receptors labe led by N-[H-3]methylscopolamine revealed three states of affinity for agonists, both in native membranes and following irreversible blockade of about 80% of the sites by propylbenzilylcholine mustard; in both p reparations, guanylylimidodiphosphate (GMP-PNP) effected an apparent i nterconversion of sites from higher to lower affinity for agonists and from lower to higher affinity for the antagonist. Excellent and mecha nistically consistent descriptions of the data were obtained in terms of a model comprising cooperative and noncooperative forms of the rece ptor; the former was described by a variant of the Adair equation, and the latter was included to account for low-affinity sites that surviv ed treatment with the mustard, If differences in apparent capacity der ive from negative cooperativity in the binding of N-[H-3]methylscopola mine, the cooperative form of the receptor was at least trivalent in n ative membranes; otherwise, constraints imposed by the effects of GMP- PNP at the concentrations of radioligand used in the assays dictate th at the cooperative form of the receptor was at least tetravalent, In c ontrast, a divalent receptor is sufficient with the data from alkylate d membranes, in accord with the reduced likelihood of interactions bet ween functional sites within an oligomeric array, A model is presented wherein the receptor interconverts spontaneously between two or more states differing in their cooperative properties. The effects of GMP-P NP can be rationalized as a shift in the equilibrium between the diffe rent states.