Received for publication April 21, 2008.
Revised June 9, 2008.
Accepted for publication June 13, 2008.

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

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 HA- and FLAG-tagged human M₂ receptor therefore have been purified from coinfected Sf9 cells and examined for any effect of the antagonist N-methylscopolamine or the agonist oxotremorine-M on the rate at which N-[³H]methylscopolamine dissociates from the orthosteric site (k_obsd). The predominantly monomeric status was confirmed by coimmunoprecipitation and by crosslinking with bis(sulfosuccinimidyl)suberate. Both N-methylscopolamine 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-methylscopolamine, 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.

Key words: Muscarinic cholinergic, Thermodynamic and kinetic processes and modeling, Receptor binding studies