Differences in kinetics of xanomeline binding and selectivity of activation of G proteins at M₁ and M₂ muscarinic acetylcholine receptors

Xanomeline is a functionally selective M₁/M₄ 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 M₁ receptors characterized by persistent binding and receptor activation after extensive washout has been shown previously. In the present study, using human M ₁ and M₂ receptors expressed in Chinese hamster ovary cells and [³H]N-methylscopolamine as a tracer, we show that persistent binding of xanomeline also occurs at the M₂ receptor with similar affinity as at the M₁ receptor (K_I = 294 and 296 nM, respectively). However, kinetics of formation of xanomeline wash-resistant binding to M₂ receptors was markedly slower than to M₁ receptors. Xanomeline was a potent fast-acting full agonist in stimulating guanosine 5′-O-(3-[³⁵S]thio)triphosphate binding at M ₁ receptors, whereas at M₂ receptors it behaved as a potent partial agonist (40% of carbachol maximal response) only upon preincubation 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 M₁ than at M₂ 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 M₁ and the M₂ receptor.