The interaction of 4-DAMP mustard with subtypes of the muscarinic receptor

The compound 4-DAMP mustard (N-2-chloroethyl-4-piperidinyl diphenylacetate) is a 2-chloroethylamine derivative of the selective muscarinic antagonist 4-DAMP (N,N-dimethyl-4-piperidinyl diphenylacetate). At neutral pH, 4-DAMP mustard cyclizes spontaneously into an aziridinium ion that binds covalently with muscarinic receptors. Analysis of the kinetics of receptor alkylation showed that the interaction of 4-DAMP mustard with M(2) and M(3) receptors was consistent with a model in which the aziridinium ion rapidly forms a reversible complex with the receptor which converts to a covalent complex at a relatively slower rate. The rate constant (k(2)) for alkylation of M(2) and M(3) receptors was approximately the same (k(2) = 0.1 min(-1)): however, the affinity of the aziridinium ion for the M(3) receptor (K-D = 7.2 nM) was approximately 6.3-fold greater than that for the M(2) receptor (K-D = 43 nM). The results of competitive binding experiments on Chinese hamster ovary cells transfected with the M(1) - M(5) subtypes of the muscarinic receptor showed that the affinity of the aziridinium ion for the M(1), M(3), M(4) and M(5) subtypes was approximately the same and about 11-fold greater than that for the M(2) receptor. 4-DAMP mustard is a useful tool for selectively inactivating all non-M(2) muscarinic receptors, particularly when it is used in the presence of a reversible M(2) selective antagonist to protect the M(1) receptor from alkylation. The results of studies on isolated smooth muscle preparations that have had their M(3) receptors alkylated with 4-DAMP mustard are consistent with the postulate that the M(2) receptor can elicit contraction by inhibiting the relaxant effect of isoproterenol and forskolin on histamine induced contractions.