title: Heterotropic cooperativity within and between protomers of an oligomeric M(2) muscarinic receptor.
abstract: At least four allosteric sites have been found to mediate the dose-dependent effects of gallamine on the binding of [(3)H]quinuclidinylbenzilate (QNB) and N-[(3)H]methylscopolamine (NMS) to M(2) muscarinic receptors in membranes and solubilized preparations from porcine atria, CHO cells, and Sf9 cells. The rate of dissociation of [(3)H]QNB was affected in a bell-shaped manner with at least one Hill coefficient (n(H)) greater than 1, indicating that at least three allosteric sites are involved. The level of binding of [(3)H]QNB was decreased in a biphasic manner, revealing at least two allosteric sites; binding of [(3)H]NMS was affected in a triphasic, serpentine manner, revealing at least three sites, and values of n(H) >1 pointed to at least four sites. Several lines of evidence indicate that all effects of gallamine were allosteric in nature and could be observed at equilibrium. The rates of equilibration and dissociation suggest that the receptor was predominately oligomeric, and the heterogeneity revealed by gallamine can be attributed to differences in its affinity for the constituent protomers of a tetramer. Those differences appear to arise from inter- and intramolecular cooperativity between gallamine and the radioligand.
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada M5S 3M2.
authors.forename: Rabindra V
authors.initials: RV
authors.lastname: Shivnaraine
authors.forename: Xi-Ping
authors.initials: XP
authors.lastname: Huang
authors.forename: Margaret
authors.initials: M
authors.lastname: Seidenberg
authors.forename: John
authors.initials: J
authors.lastname: Ellis
authors.forename: James W
authors.initials: JW
authors.lastname: Wells
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site; D000818:Animals; D016466:CHO Cells; D018680:Cholinergic Antagonists / Q000494:pharmacology*; D006224:Cricetinae; D005703:Gallamine Triethiodide / Q000494:pharmacology*; D007700:Kinetics; D018727:Muscarinic Antagonists / Q000494:pharmacology; D019832:N-Methylscopolamine / Q000494:pharmacology*; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011813:Quinuclidinyl Benzilate / Q000494:pharmacology*; D043585:Receptor, Muscarinic M2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D061987:Sf9 Cells; D012995:Solubility; D013552:Swine
chemical_list: D018680:Cholinergic Antagonists; D018727:Muscarinic Antagonists; D021122:Protein Subunits; D043585:Receptor, Muscarinic M2; D011813:Quinuclidinyl Benzilate; D005703:Gallamine Triethiodide; D019832:N-Methylscopolamine