KINETIC AND BIOPHYSICAL ANALYSIS OF THE M2 MUSCARINIC RECEPTOR

The recombinant Pm2 muscarinic receptor expressed in Chinese hamster o vary (CHO) cells was used as a model system to examine receptor-effect or coupling and ligand binding. In CHO cells, equilibrium binding stud ies and the dependence on receptor number per cell of the maximum resp onse and EC(50) values for agonist stimulation of phosphatidylinositol metabolism and inhibition of cAMP formation were consistent with a mo dified ternary complex model of signal transduction that included a ph ysiologically noncompetent receptor state. Detailed kinetic studies of oxotremorine M (Oxo-M) binding to CHO cell membranes suggested that a gonist interactions at the high affinity class of binding sites are co mplicated and depend on receptor expression levels. At low levels of e xpression, kinetic data were consistent with a special case of a mecha nism in which Oxo-M shifts the equilibrium between two receptor confor mations while at high levels of expression, it was necessary to evoke receptor-receptor interactions to explain the kinetic data. Far ultrav iolet circular dichroism studies of the purified recombinant receptor showed a high content of alpha-helical secondary structure and small c hanges in secondary structure upon antagonist, but not agonist, bindin g.