Divergent modes of action among cationic allosteric modulators of muscarinic M-2 receptors

We tested the hypothesis that structurally related modulators of ligand binding to muscarinic M-2 receptors may not use a common recognition site. The applied test compounds are potent allosteric modulators [i.e., two bispyridinium model compounds substituted symmetrically either with phthalimidomethyl (WDuoS) or dichlorobenzyl (Duo3), a phthalimidoethyl-substituted hexamethonium compound (W84), alcuronium, and for sake of comparison, gallamine]. As introduced by Ellis and Seidenberg as a tool to check for a common allosteric site [Moi. Pharmacol. 42:638-641 (1992)], obidoxime was used to antagonize the actions of the test compounds. The allosteric delay of the dissociation of [H-3]N-methylscopolamine ([H-3]NMS) from porcine heart muscarinic receptors was measured in 5 mM sodium/potassium phosphate buffer (4 mM Na2HPO4 and 1 mM KH2PO4, pH 7.4) at 23 degrees (control t(1/2) approximate to 4 min). The concentration-effect curve of obidoxime: which has a weak potency and submaximal efficacy to allosterically retard [H-3]NMS dissociation, was better described with a two-site model than with a one-site model. The concentration-effect curves of the test compounds for the allosteric delay of [H-3]NMS dissociation were shifted to the right in the presence of obidoxime, yet to a different extent. For WDuo3, W84, alcuronium, and gallamine the shift induced by increasing concentrations of obildoxime was compatible with a competitive interplay. The pK(b) values of obidoxime against these modulators lay in a narrow range from pK(b) = 4.70 with gallamine to pK(b) = 4.16 with WDuo3. In contrast, the ability of obidoxime to shift the concentration effect curve of Duo3 was weak (pA(2) = 3.00) and not compatible with a competitive interplay. In conclusion, cationic allosteric modulators may stabilize [H-3]NMS binding to M-2 receptors by divergent modes of allosteric action. The finding's suggest that the M-2 receptor protein contains more than one allosteric recognition site on its extracellular face.