Differences in Kinetics of Xanomeline Binding and Selectivity of Activation of G Proteins at M_1 and M_2 Muscarinic Acetylcholine Receptors

摘要

Xanomeline is a functionally selective M_1,/M_4 muscarinic acetylcholine receptor agonist that nevertheless binds with high affinity to all five subtypes of muscarinic receptors. A novel mode of interaction of this ligand with the muscarinic M_1 receptors characterized by persistent binding and receptor activation after extensive washout has been shown previously. In the present study, using human M_1, and M_2 receptors expressed in Chinese hamster ovary cells and [~3H]A/-methylsco-polamine as a tracer, we show that persistent binding of xanomeline also occurs at the M_2 receptor with similar affinity as at the M_1, receptor (K_1 = 294 and 296 nM, respectively). However, kinetics of formation of xanomeline wash-resistant binding to M_2 receptors was markedly slower than to M_1 receptors. Xanomeline was a potent fast-acting full agonist in stimulating guanosine 5'-O-(3-[~(35)S]thio)triphosphate binding at M_1 receptors, whereas at M_2 receptors it behaved as a potent partial agonist (40% of carbachol maximal response) only upon pre-incubation for 1 h. Development of xanomeline agonistic effects at the M_2 receptor was slower than its ability to attenuate carbachol responses. We also demonstrate that xanomeline discriminates better between G protein subtypes at M_1 than at M_2 receptors. Our data support the notion that xanomeline interacts with multiple sites on the muscarinic receptor, resulting in divergent conformations that exhibit differential effects on ligand binding and receptor activation. These conformations are both time- and concentration-dependent and vary between the M_1 and the M_2 receptor