THE INTERACTION OF 4-DAMP MUSTARD WITH SUBTYPES OF THE MUSCARINIC RECEPTOR

The compound 4-DAMP mustard (N-2-chloroethyl-4-piperidinyl diphenylace tate) is a 2-chloroethylamine derivative of the selective muscarinic a ntagonist 4-DAMP (N,N-dimethyl-4-piperidinyl diphenylacetate). At neut ral pH, 4-DAMP mustard cyclizes spontaneously into an aziridinium ion that binds covalently with muscarinic receptors. Analysis of the kinet ics of receptor alkylation showed that the interaction of 4-DAMP musta rd with M(2) and M(3) receptors was consistent with a model in which t he aziridinium ion rapidly forms a reversible complex with the recepto r which converts to a covalent complex at a relatively slower rate. Th e rate constant (k(2)) for alkylation of M(2) and M(3) receptors was a pproximately the same (k(2) = 0.1 min(-1)): however, the affinity of t he aziridinium ion for the M(3) receptor (K-D = 7.2 nM) was approximat ely 6.3-fold greater than that for the M(2) receptor (K-D = 43 nM). Th e results of competitive binding experiments on Chinese hamster ovary cells transfected with the M(1) - M(5) subtypes of the muscarinic rece ptor 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 t ool for selectively inactivating all non-M(2) muscarinic receptors, pa rticularly when it is used in the presence of a reversible M(2) select ive antagonist to protect the M(1) receptor from alkylation. The resul ts of studies on isolated smooth muscle preparations that have had the ir M(3) receptors alkylated with 4-DAMP mustard are consistent with th e postulate that the M(2) receptor can elicit contraction by inhibitin g the relaxant effect of isoproterenol and forskolin on histamine indu ced contractions.