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Divergent Modes of Action among Cationic Allosteric Modulators of Muscarinic M 2 Receptors

1997; American Society for Pharmacology and Experimental Therapeutics; Volume: 51; Issue: 4 Linguagem: Inglês

10.1124/mol.51.4.674

1521-0111

Christian Tränkle, Klaus Mohr,

Ion channel regulation and function

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 [ 3 H] N -methylscopolamine ([ 3 H]NMS) from porcine heart muscarinic receptors was measured in 5 mm sodium/potassium phosphate buffer (4 mm Na 2 HPO 4 and 1 mmKH 2 PO 4 , pH 7.4) at 23° (control t 1/2 ≈ 4 min). The concentration-effect curve of obidoxime, which has a weak potency and submaximal efficacy to allosterically retard [ 3 H]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 [ 3 H]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 p K b values of obidoxime against these modulators lay in a narrow range from p K b = 4.70 with gallamine to p K b = 4.16 with WDuo3. In contrast, the ability of obidoxime to shift the concentration-effect curve of Duo3 was weak (p A 2 = 3.00) and not compatible with a competitive interplay. In conclusion, cationic allosteric modulators may stabilize [ 3 H]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.