Pharmacology of muscarinic receptor subtypes constitutively activated by G proteins

We have examined the effects of raising G protein concentration on the pharmacology of a series of agonist and antagonist ligands at the m1, m3, and m5 muscarinic subtypes using a functional assay. Overexpression of G(alpha q) induced constitutive activity of these receptors. The constitutive activity was reversed completely by every muscarinic antagonist tested, which indicates that they are all negative antagonists (inverse agonists). The potencies of antagonists for reversing G protein-induced activity and agonist-induced activity were identical, suggesting the same mechanism of action. Overexpression of G(alpha q) increased the potencies of every tested agonist and the efficacies of all partial agonists. The fold-gains in potency were positively correlated with ligand efficacy with the most efficacious agonists displaying the greatest potency gains. In addition, the efficacies of partial agonists approached those of full agonists. Constitutive activity of receptors has been explained by allosteric models in which receptors exist in spontaneous equilibrium between active and inactive conformations that are stabilized by agonists and antagonists, respectively. In this context, drug efficacy and potency are interrelated because they both depend on the same parameters, namely the absolute and relative affinities of a compound for receptors in active and inactive states and the ratio and concentrations of receptors in active and inactive states. All of our data are consistent with this model, in which raising G protein levels favors formation of the active conformation of receptors. Based on our findings, regulation of G protein concentration may be an important means of controlling receptor activity in vivo. These results define the functional relationship between G protein levels and muscarinic receptor pharmacology.