Divergent modes of action among cationic allosteric modulators of muscarinic M2 receptors
Tränkle, C.; Mohr, K.
Molecular Pharmacology 51(4): 674-682
1997
ISSN/ISBN: 0026-895X PMID: 9106634 Document Number: 479952
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 (WDuo3) 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 (Mol. Pharmacol. 42:638-641 (1992)), obidoxime was used to antagonize the actions of the test compounds. The allosteric delay of the dissociation of (3H)N-methylscopolamine (3H)NMS) from porcine heart muscarinic receptors was measured in 5 mm sodium/potassium phosphate buffer (4 mm Na-2HPO-4 and 1 mm KH-2PO-4, pH 7.4) at 23 degree (control t-1/2 apprxeq 4 min). The concentration-effect curve of obidoxime, which has a weak potency and submaximal efficacy to allosterically retard (3H)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 (3H)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 obidoxime 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 (3H)NMS binding to M-2 receptors by divergent modes of allosteric action. The findings suggest that the M-2 receptor protein contains more than one allosteric recognition site on its extracellular face.