DIVERGENT MODES OF ACTION AMONG CATIONIC ALLOSTERIC MODULATORS OF MUSCARINIC M-2 RECEPTORS

We tested the hypothesis that structurally related modulators of ligan d 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 phthalimid oethyl-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. 42:63 8-641 (1992)], obidoxime was used to antagonize the actions of the tes t compounds. The allosteric delay of the dissociation of [H-3]N-methyl scopolamine ([H-3]NMS) from porcine heart muscarinic receptors was mea sured 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 mode l. The concentration-effect curves of the test compounds for the allos teric delay of [H-3]NMS dissociation were shifted to the right in the presence oi obidoxime, yet to a different extent. For WDuo3, W84, alcu ronium, and gallamine the shift induced by increasing concentrations o f obildoxime was compatible with a competitive interplay. The pK(b) va lues 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, t he ability of obidoxime to shift the concentration effect curve of Duo 3 was weak (pA(2) = 3.00) and not compatible with a competitive interp lay. 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.