Abstract

Analysis of [3H]quinuclidinyl benzilate/acetylcholine competition curves indicated that the agonist acetylcholine bound with three different affinities to chick heart muscarinic receptors. The estimated KD values for acetylcholine were 2.7, 240, and 4000 nM. Mg2+ increased and guanosine 5'-(beta, gamma-imino)triphosphate (Gpp(NH)p) decreased the proportion of the receptors in the highest affinity state without altering the KD values. Monovalent cations increased the KD values of the three affinity states and obscured the detection of the highest affinity state. The nature of the three affinity states and the sites of action of Mg2+, guanine nucleotides, and monovalent cations were probed with three experimental protocols. Treatments with N-ethylmaleimide or pertussis toxin eliminated both the highest affinity state and the sensitivity to Gpp(NH)p. In contrast, partial effects of Mg2+ were retained after either of these treatments. The effects of monovalent cations on the affinity of the receptor for agonists were unaffected by both treatments. Solubilization of the receptors with digitonin-cholate yielded preparations displaying only the low affinity state for agonist. Agonist binding to the solubilized receptors was insensitive to Mg2+ and guanine nucleotides but retained sensitivity to monovalent cations. The results indicate that chick heart muscarinic receptors can exist in vitro in three agonist affinity states and that the entire population of receptors can be interconverted from one state to another by Mg2+ and guanine nucleotides. Guanine nucleotides presumably act via the inhibitory guanine nucleotide-binding regulatory (Ni) protein, whereas there appear to be at least two distinct sites of action of Mg2+. One site is associated with Ni. Another is distinguishable from Ni but does not appear to be on the receptor itself. The effect of monovalent cations on the interaction of agonists with cardiac muscarinic receptors is qualitatively different and mediated at distinct sites from the effects of Mg2+ and guanine nucleotides.