Cooperativity has been investigated as the mechanistic basis for effects observed with cardiac muscarinic receptors in washed membranes from Syrian hamsters. Specifically, N-[3H]methylscopolamine labeled only 66-75% of the sites labeled by [3H]quinuclidinylbenzilate at apparently saturating concentrations of each radioligand. Also, receptors labeled by N-[3H]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 preparations, guanylylimidodiphosphate (GMP-PNP) effected an apparent interconversion of sites from higher to lower affinity for agonists and from lower to higher affinity for the antagonist. Excellent and mechanistically consistent descriptions of the data were obtained in terms of a model comprising cooperative and noncooperative forms of the receptor; the former was described by a variant of the Adair equation, and the latter was included to account for low-affinity sites that survived treatment with the mustard. If differences in apparent capacity derive from negative cooperativity in the binding of N-[3H]methylscopolamine, the cooperative form of the receptor was at least trivalent in native membranes; otherwise, constraints imposed by the effects of GMP-PNP at the concentrations of radioligand used in the assays dictate that the cooperative form of the receptor was at least tetravalent. In contrast, a divalent receptor is sufficient with the data from alkylated membranes, in accord with the reduced likelihood of interactions between 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-PNP can be rationalized as a shift in the equilibrium between the different states.

Muscarinic cholinergic receptors can appear to be more numerous when labeled by [(3)H]quinuclidinylbenzilate (QNB) than by N-[(3)H]methylscopolamine (NMS). The nature of the implied heterogeneity has been studied with M(2) receptors in detergent-solubilized extracts of porcine atria. The relative capacity for [(3)H]NMS and [(3)H]QNB was about 1 in digitonin-cholate, 0.56 in cholate-NaCl, and 0.44 in Lubrol-PX. Adding digitonin to extracts in cholate-NaCl increased the absolute capacity for both radioligands, and the relative capacity increased to near 1. The latency cannot be attributed to a chemically impure radioligand, instability of the receptor, an irreversible effect of NMS, or a failure to reach equilibrium. Binding at near-saturating concentrations of [(3)H]QNB in cholate-NaCl or Lubrol-PX was blocked fully by unlabeled NMS, which therefore appeared to inhibit noncompetitively at sites inaccessible to radiolabeled NMS. Such an effect is inconsistent with the notion of functionally distinct, noninterconverting, and mutually independent sites. Both the noncompetitive effect of NMS on [(3)H]QNB and the shortfall in capacity for [(3)H]NMS can be described quantitatively in terms of cooperative interactions within a receptor that is at least tetravalent; no comparable agreement is possible with a receptor that is only di- or trivalent. The M(2) muscarinic receptor therefore appears to comprise at least four interacting sites, presumably within a tetramer or larger array, and ligands appear to bind in a cooperative manner under at least some conditions.