Requirement for sulfhydryl groups in the differential effects of magnesium ion and GTP on agonist binding of muscarinic cholinergic receptor sites in rat atrial membrane fraction

Abstract

The binding of agonist (carbachol) and antagonist (atropine) to the rat heart atrial muscarinic cholinergic receptor sites was investigated. Divalent cations (Mg²⁺ or Ca²⁺), in low concentrations, modestly increased the agonist binding affinity of the receptor site without any effect on the antagonist binding affinity. 3. Guanine nucleotides (e. g. GTP), on the other hand, decreased the agonist binding affinity (but not the antagonist binding affinity), and the extent of GTP effect depended on the absence or presence of divalent cation (Mg²⁺) in the binding assay. 4. Pretreatment of atrial membranes with N-ethylmaleimide (NEM) altered the agonist binding curve (obtained with varying concentrations of carbachol) such that the Hill coefficient (nH) became very close to 1.0, whereas the corresponding nH values for control (untreated) or dithiothreitol (DTT)-treated membranes were much less than 1.0; NEM or DTT treatments failed to show any effect of antagonist binding curve. 5. NEM treatment abolished both divalent cation-induced and guanine nucleotide-induced alterations in the agonist binding affinity of the receptor site. 6. Monovalent cations in low concentrations did not mimic the effects of Mg²⁺ or Ca²⁺ on agonist binding. Instead, concentration dependent decreases in both agonist and antagonist binding affinities and abilities were observed. Neither NEM nor DTT treatments failed to alter the monovalent cation effects on carbachol and atropine binding. 7. These observations indicate a likely involvement of-SH groups in the opposing effects of Mg²⁺ and guanine nucleotides (GTP) on cardiac muscarinic receptor-agonist interaction. The results further suggest some subtle in vitro differences in the brain and heart muscarinic receptor sites with regard to the influence by divalent cations and guanine nucleotides on the receptor-agonist interaction.