Abstract
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 (kobsd). 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 kobsd by 4.5- and 9.1-fold, respectively; the corresponding estimates of affinity (log KL) are -2.55 ± 0.13 and -2.29 ± 0.14. Gallamine and the allosteric ligand obidoxime decreased kobsd by more than 100-fold (log KL = -4.12 ± 0.04) and by only 1.1-fold (log KL = -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.
- The American Society for Pharmacology and Experimental Therapeutics
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