Abstract

Reducing agents cysteine, glutathione and dithiothreitol in the concentration range of 10 µM-10 mM diminished binding of [³H]3-quinuclidinyl benzilate ([³H]3-QNB) to in situ muscarinic receptor sites of membranes from rat cerebral cortex. The thiol reagents N-ethylmaleimide, pCMB, and Cd²⁺ and the oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in low concentrations (10 µM-10 mM) have also diminished muscarinic binding causing an approximately 50% inhibition at 50 µM concentration. Treatment of membranes with reducing- or thiol-reagents diminished the number of antagonist and agonist binding sites and changed the affinity toward these ligands. The apparent K_d value for [³H]3-QNB binding increased from 0.4 nM to 0.7-0.8 nM as a result of covalent modification of the membranes. The binding of carbamylcholine as determined from competition with [³H]3-QNB (0.1 mM) could be described with a two-site model with apparent K_d values 95 ± 15 µM and 52 ± 8 nM, respectively. In treated membranes these values were altered to 36-230 µM and 0.5-5 µM, respectively. The results suggested that a disulfide bond, which could be reduced and reoxidized, may play an important role in ligand binding. Furthermore, experiments with N-ethylmaleimide, pCMB, and DTNB suggested the presence of thiol groups of importance for the binding. The finding that Cd²⁺ inhibited ligand binding in low concentrations (50 µM) suggested the presence of vicinal thiol groups. Modification of thiol-disulfide state of receptor may be involved in the control of binding activity.