Allosteric modulation by persistent binding of xanomeline of the interaction of competitive ligands with the M1 muscarinic acetylcholine receptor

Xanomeline is a potent agonist that is functionally selective for muscarinic M-1 receptors. We have shown previously that a significant fraction of xanomeline binding to membranes of Chinese hamster ovary (CHO) cells expressing the M1 receptors occurs in a wash-resistant manner and speculated that this persistent binding likely does not take place at the primary binding site on the receptor. In the present work we investigated in depth the pharmacological characteristics of this unique mode of xanomeline binding and the effects of this binding on the interaction of classical competitive ligands with the receptor in CHO cells that express the M1 muscarinic receptor. Onset of persistent binding of xanomeline to the M1 muscarinic receptor was fast and was only slightly hindered by atropine. Its dissociation was extremely slow, with a half-life of over 30 h. Although persistently bound xanomeline strongly inhibited binding of the classical antagonist N-methylscopolamine (NMS) to the receptor, there are multiple indications that this is not the result of competition at the same binding domain. Namely, wash-resistant binding of xanomeline only slightly slowed the rate of NMS association, but enhanced the rate of NMS dissociation. Moreover, preincubation with xanomeline followed by extensive washing brought about an apparent decrease in the number of NMS binding sites. Our findings are best interpreted in terms of allosteric interactions between xanomeline-persistent binding to the M-1 muscarinic receptor and competitive ligands bound to the classical receptor binding site.