Differences in Kinetics of Xanomeline Binding and Selectivity of Activation of G Proteins at M₁ and M₂ Muscarinic Acetylcholine Receptors

Abstract

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 [3 H]N-methylscopolamine 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 I ϭ 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 5Ј-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 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 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.

Involvement of a Subpopulation of Neuronal M4Muscarinic Acetylcholine Receptors in the Antipsychotic-like Effects of the M1/M4Preferring Muscarinic Receptor Agonist Xanomeline

“…Since xanomeline is not entirely M 1 /M 4 -specific (Jakubík et al, 2006;Noetzel et al, 2009), classical cholinomimetic side effects, e.g., gastrointestinal side effects, were observed in humans (Bodick et al, 1997;Shekhar et al, 2008). Previous efforts to develop highly selective orthosteric agonists of the individual muscarinic subtypes have not been successful.…”

Long-term wash-resistant effects of brief interaction of xanomeline at the M1 muscarinic receptor

“…In addition to its reversible interaction with the classical orthosteric binding site of the M 1 mAChR, xanomeline also binds persistently to a secondary site(s) on the receptor [2,3]. This wash-resistant binding occurs almost instantaneously at the M 1 , but not the M 2 mAChR [7], which suggests induction of receptor subtype-specific conformations by xanomeline. This receptor subtype selectivity and the lack of similar effects when liposomes are treated with xanomeline prior to reconstitution of M1 receptors [8] support a receptor specific mechanism.…”

“…These findings also strongly argue against the possibility of membrane lipids serving as a nonspecific depot of xanomeline where xanomeline leeches out to rebind to the receptor causing persistent activation. It has been shown that persistent xanomeline binding to the M 1 mAChR acutely modulates binding of ligands to the orthosteric site in an allosteric manner [2,7,9]. It also causes wash-resistant receptor activation that is silenced by atropine [2].…”

Wash-Resistantly Bound Xanomeline Inhibits Acetylcholine Release by Persistent Activation of Presynaptic M2and M4Muscarinic Receptors in Rat Brain

“…Wash-resistantly bound xanomeline activates guanosine 5Ј-O-(3-thio)triphosphate binding at the M 1 receptor, and, although with lower efficacy but similar affinity, also at the M 2 receptor (Jakubík et al, 2006). Likewise, wash-resistantly bound xanomeline induces durable antagonism of M 5 receptor activation (Grant and El-Fakahany, 2005).…”

“…3-[3-Hexyloxy-1,2,5-thiadiazo-4-yl]-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline) is an atypical agonist that binds to all muscarinic receptor subtypes with high affinity (Bymaster et al, 1997;Watson et al, 1998;Wood et al, 1999;Jakubík et al, 2006), but it displays functional selectivity for the M 1 and M 4 receptors (Shannon et al, 1994;Ward et al, 1995;Bymaster et al, 1997Bymaster et al, , 1998. Xanomeline was supposed to be developed as an M 1 receptor-selective drug for treatment of Alzheimer's disease.…”