Muscarinic cholinergic receptors in the embryonic chick heart: interaction of agonist, receptor, and guanine nucleotides studied by an improved assay for direct binding of the muscarinic agonist [3H]cismethyldioxolane

JB Galper, LS Haigh, AC Hart, DS O'Hara and DJ Livingston

Muscarinic agonist binding has been studied by the indirect method of competition between binding of agonist and 3H-antagonist. Studies of 3H- agonist binding have either been complicated by high levels of nonspecific binding or have been carried out at low concentrations of agonist, which bind only to high affinity sites. A new assay for binding of the muscarinic agonist, [3H]cismethyldioxolane, allows measurement of binding at concentrations up to 1000 nM with a high degree of specificity (92% at 18 nM, 50% at 1000 microM). Using this new binding method, it is possible not only to directly establish the validity of those observations made using competition binding studies, but also to obtain new insight into the mechanism of interaction of receptor agonist and guanine nucleotide. Specifically, data have been presented which demonstrate: 1) that binding of agonist to the high and low affinity forms of the muscarinic receptor involves two independent parallel reactions, and, in the absence of guanine nucleotides, agonist binding alone does not mediate the interconversion of the receptor from one affinity state to another, thus suggesting that high affinity receptors are present in chick heart membranes and do not require agonist binding for their formation; 2) that the interaction of agonist and guanine nucleotide with the receptor-guanine nucleotide-binding protein complex involves formation of an intermediate state in which both agonist and guanine nucleotide are bound to the receptor-guanine nucleotide-binding protein complex; and, finally, 3) that the order of binding of agonist and guanine nucleotide during formation of this intermediate state is random.

Volume 32, Issue 1, pp. 230-240, 08/01/1987
Copyright © 1987 by American Society for Pharmacology and Experimental Therapeutics