Received for publication May 19, 2008.
Revised August 2, 2008.
Accepted for publication August 4, 2008.

Use of acetylcholine mustard to study allosteric interactions at the M₂ muscarinic receptor

Abstract

We explored the interaction of a nitrogen mustard derivative of acetylcholine with the human M₂ muscarinic receptor expressed in CHO cells using the muscarinic radioligand, [³H]N-methylscopolamine. Acetylcholine mustard caused a concentration-dependent, first order loss of [³H]N-methylscopolamine binding at 37°C, with the half maximal rate constant occurring at 24 µM and a maximal rate constant of 0.16 min^-1. We examined the effects of various ligands on the rate of alkylation of M₂ receptors by acetylcholine mustard. N-methylscopolamine and McN-A-343 (4-(trimethylamino)-2-butynyl-(3-chlorophenyl)carbamate) competitively slowed the rate of alkylation, whereas the inhibition by gallamine reached a plateau at high concentrations, indicating allosteric inhibition. In contrast, WIN 51708 (17--hydroxy-17--ethynyl-5--androstano[3,2-b]pyrimido[1,2-a]benzimidazole) had no effect. We also measured the inhibition of [³H]NMS binding by acetylcholine mustard at 0°C, conditions under which there is little or no detectable covalent binding. In these experiments, the dissociation constant of the aziridinium ion of acetylcholine mustard was estimated to be 12.3 µM. In contrast, the parent mustard and alcoholic hydrolysis product of acetylcholine mustard were without effect. Our results show that measurement of the effects of ligands on the rate of inactivation of the orthosteric site by a small site-directed electrophile is a powerful method for discriminating competitive inhibition from allosterism.

Key words: M2 muscarinic receptor, acetylcholine mustard, allosterism, competitive inhibition, gallamine, muscarinic receptor