Kinetic and biophysical analysis of the m2 muscarinic receptor.

The recombinant Pm2 muscarinic receptor expressed in Chinese hamster ovary (CHO) cells was used as a model system to examine receptor-effector coupling and ligand binding. In CHO cells, equilibrium binding studies and the dependence on receptor number per cell of the maximum response and EC50 values for agonist stimulation of phosphatidylinositol metabolism and inhibition of cAMP formation were consistent with a modified ternary complex model of signal transduction that included a physiologically noncompetent receptor state. Detailed kinetic studies of oxotremorine M (Oxo-M) binding to CHO cell membranes suggested that agonist interactions at the high affinity class of binding sites are complicated and depend on receptor expression levels. At low levels of expression, kinetic data were consistent with a special case of a mechanism in which Oxo-M shifts the equilibrium between two receptor conformations while at high levels of expression, it was necessary to evoke receptor-receptor interactions to explain the kinetic data. Far ultraviolet circular dichroism studies of the purified recombinant receptor showed a high content of alpha-helical secondary structure and small changes in secondary structure upon antagonist, but not agonist, binding.

A kinetic model for oxotremorine M binding to recombinant porcine m2 muscarinic receptors expressed in Chinese hamster ovary cells.

The kinetic mechanism of super high affinity [3H]oxotremorine M binding to porcine m2 muscarinic receptors expressed in Chinese hamster ovary cells was examined. In cell lines expressing low receptor numbers (10(4) binding sites/cell) and in high expression (10(6) sites/cell) cell lines treated with cholate, [3H]oxotremorine M association and dissociation kinetics were monophasic. The reciprocal relaxation time for the association reaction was independent of [3H]oxotremorine M concentration and equaled the dissociation rate constant consistent with a special case for a mechanism involving a protein conformational change followed by ligand binding. Membranes from high expression cell lines and porcine atrial membranes showed complex kinetic behavior. Two kinetic phases were observed for [3H]oxotremorine M association, and both reciprocal relaxation times were independent of ligand concentration. The number of kinetic phases and their relative amplitudes seen in dissociation experiments were dependent on whether dissociation was initiated by dilution or by addition of unlabeled ligand(s) as well as on the fractional saturation of the receptor. Computer simulations of the data led to a model consistent with the existence of asymmetric receptor dimers as well as monomers and the ligand-dependent interconversion of fully occupied dimers and monomers.