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Binding of Orthosteric Ligands to the Allosteric Site of the M₂ Muscarinic Cholinergic Receptor

Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada

The M₂ muscarinic receptor has two topographically distinct sites: the orthosteric site and an allosteric site recognized by compounds such as gallamine. It also can exhibit cooperative effects in the binding of orthosteric ligands, presumably to the orthosteric sites within an oligomer. Such effects would be difficult to interpret, however, if those ligands also bound to the allosteric site. Monomers of the hemagglutinin (HA)- and FLAG-tagged human M₂ receptor therefore have been purified from coinfected Sf9 cells and examined for any effect of the antagonist N-methyl scopolamine or the agonist oxotremorine-M on the rate at which N-[³H]methyl scopolamine dissociates from the orthosteric site (k_obsd). The predominantly monomeric status was confirmed by coimmunoprecipitation and by cross-linking with bis(sulfosuccinimidyl)suberate. Both N-methyl scopolamine and oxotremorine-M acted in a cooperative manner to decrease k_obsd by 4.5- and 9.1-fold, respectively; the corresponding estimates of affinity (log K_L) are -2.55 ± 0.13 and -2.29 ± 0.14. Gallamine and the allosteric ligand obidoxime decreased k_obsd by more than 100-fold (log K_L = -4.12 ± 0.04) and by only 1.1-fold (log K_L = -1.73 ± 0.91), respectively. Obidoxime reversed the effect of N-methyl scopolamine, oxotremorine-M, and gallamine in a manner that could be described by a model in which all four ligands compete for a common allosteric site. Ligands generally assumed to be exclusively orthosteric therefore can act at the allosteric site of the M₂ receptor, albeit at comparatively high concentrations.

Received for publication April 19, 2008.

Accepted for publication June 13, 2008.

Address correspondence to: James W. Wells, Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario, Canada M5S 3M2. E-mail: jwells{at}phm.utoronto.ca

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