Abstract

Previous studies have found that a mutation near the junction of the sixth transmembrane domain (TM6) and the third extracellular loop of the M₅ muscarinic receptor leads to constitutive activation and enhanced agonist affinity for the mutated receptor. These results were consistent with the extended ternary complex model, which predicts a correlation between agonist affinity and constitutive activity. We have introduced the homologous mutation into all five subtypes of the highly conserved muscarinic receptor family; SerThr→TyrPro was introduced into M₁ and M₅, and AsnThr→TyrPro was introduced into M₂, M₃, and M₄. In binding assays, these mutations produced increases in affinities toward acetylcholine and carbachol that ranged from 5-fold at the M₂ receptor to 15- to 20-fold at M₁, M₃, and M₄, to 40-fold at M₅. In functional assays, all five mutant receptors exhibited constitutive activity, at levels ranging between 30 and 80% of the maximal response elicited by carbachol. In every case, the muscarinic antagonist atropine inhibited this constitutive activity with high affinity. Thus, despite differences in effector coupling and in wild-type sequence at the mutation site, all five subtypes were activated by this mutation at the top of TM6. Previous studies of the M₅ subtype have indicated that TM6 is a ligand-dependent switch that sets the activation state of the receptor. Based on the results of the present study, it is possible that TM6 represents a general switch for the activation of the muscarinic receptor family.