Muscarinic Acetylcholine Receptors in Torpedo Electric Organ: Effect of Guanine Nucleotides

Abstract: The effect of guanine nucleotides on the binding properties of presynaptic muscarinic receptors has been studied in a membrane preparation from the electric organ of Torpedo marmorata by measuring the competitive displacement of the radiolabelled antagonist, ³H]quinuclidinyl benzilate, by nonradioactive muscarinic ligands. The binding of the antagonists, atropine, scopolamine and pirenzepine was to a single class of sites slope factors (pseudo Hill coefficients) close to 1] and was unaffected by 0.1 m M GTP. The binding of the N -methylated antagonists, N -methylatropine and N -methyl-scopolamine was more complex (slope factors <1) but also insensitive ( N- methylatropine) to 0.1 m M GTP. Agonist binding was complex and could be resolved into two binding sites with relatively high and low affinities. The proportion of high-affinity sites varied with the nature of the agonist (15–80%). Agonist binding was depressed by 0.1 m M GTP, and the order of sensitivity was oxotremorine-M > carbamoylcholine > muscarine > acetylcholine > arecoline > oxotremorine. The binding of pilocarpine, a partial agonist, was unaffected by GTP. With carbamoylcholine as a test ligand the GTP effect on agonist binding was half-maximal at 12 μM. GDP and guanylylimidodiphosphate produced comparable inhibition of carbamoylcholine binding, but GMP and cyclic GMP were ineffective, as were various adenine nucleotides. Analysis of agonist binding in terms of a two-site model indicates that the predominant effect of guanine nucleotides is to reduce the number of sites of higher affinity.