Constitutive activity and inverse agonism at the M2 muscarinic acetylcholine receptor

Introduction of a single-point mutation (Asn to Tyr) at position 410 at the junction between transmembrane domain 6 and the third extracellular loop of the human M 2 muscarinic acetylcholine (mACh) receptor generated a mutant receptor (N410Y) that possesses many of the hallmark features of a constitutively active mutant receptor. These included enhanced agonist binding affinity and potency, in addition to agonist-independent accumulation of [H-3] inositol phosphates in cells coexpressing the chimeric G alpha(qi5) protein and the N410Y mutant M-2 mACh receptor. Constitutive activity was sensitive to inhibition by a range of muscarinic ligands, including those used clinically in the management of overactive bladder (oxybutynin, tolterodine, and darifenacin), indicating that these ligands behave as inverse agonists at the M-2 mACh receptor. Long-term (24- h) treatment of Chinese hamster ovary cells expressing the N410Y mutant M 2 mACh receptor with certain mACh receptor inverse agonists (atropine, darifenacin, and pirenzepine) elicited a concentration-dependent up-regulation of cell surface receptor expression. However, not all ligands possessing negative efficacy in the [H-3] inositol phosphate accumulation assays were capable of significantly up-regulating receptor expression, perhaps indicating a spectrum of negative efficacies among ligands traditionally defined as mACh receptor antagonists. Finally, structurally distinct agonists exhibited differences in their relative potencies for the activation of G alpha(i/o) versus G alpha(s), consistent with agonist-directed trafficking of signaling at the N410Y mutant, but not at the wild-type M-2 mACh receptor. This indicates that the N410Y mutation of the M-2 mACh receptor alters receptor-G-protein coupling in an agonist-dependent manner, in addition to generating a constitutively active receptor phenotype.