Martin_1984_J.Pharmacol.Exp.Ther_230_424

Modulation of the affinity of agonists and antagonists at muscarinic cholinergic receptors in rat heart membranes was investigated using the radiolabeled antagonist, [3H]quinuclidinyl benzilate ([3H]QNB), and the radiolabeled agonist, [methyl-3H]oxotremorine acetate ([3H]OXO). Receptor affinity for oxotremorine measured in competition binding assays with [3H]QNB or by equilibrium binding of [3H]OXO was increased when the incubation temperature was reduced to 4 degrees C. In contrast, the receptor affinity for [3H]QNB was decreased at lower incubation temperatures and a marked effect of guanine nucleotides on the affinity for [3H]QNB was revealed. Guanine nucleotides increased receptor affinity for [3H]QNB without changing the total number of binding sites. The GTP-induced increase in the affinity for [3H]QNB was reflected by an increase in the rate constant for association of [3H]QNB. At subsaturating ligand concentrations, guanine nucleotides increased [3H]QNB binding and decreased [3H]OXO binding with the same order of potency: GppNHp = GTP gamma S greater than GTP greater than guanosine 5'-diphosphate greater than GMP. Free Mg++ ion was required to observe guanine nucleotide effects on antagonist binding. Pretreatment of heart membranes with N-ethyl-maleimide increased [3H]QNB affinity and blocked the effects of guanine nucleotides. N-Ethylmaleimide also decreased [3H]OXO binding and increased [3H]QNB binding with a similar concentration-effect relationship. Thus, antagonist and agonist binding to muscarinic cholinergic receptors is modulated in a reciprocal manner by a number of factors; this modulation appears to reflect interaction of agonist and antagonist-occupied receptors with a guanine nucleotide regulatory protein, Ni.