Role of Receptor Protein and Membrane Lipids in Xanomeline Wash-Resistant Binding to Muscarinic M₁ Receptors

Xanomeline is a novel agonist functionally selective for muscarinic receptors of the M₁ subtype. It binds to this receptor in two modes, reversible and quasi-irreversible (wash-resistant). We investigated the unknown mechanism of the wash-resistant binding in experiments with muscarinic M ₁ receptors expressed in transfected Chinese hamster ovary cells. Xanomeline's structure consists of two heterocycles and O-hexyl side chain. We compared the wash-resistant binding of xanomeline and its analogs with shorter O-alkyl side chains. For the wash-resistant binding to occur, the O-alkyl chain had to be at least O-butyl or longer. Accumulation of inositol phosphates was enhanced in washed cells that had been preexposed to xanomeline or its pentyl analog, whereas the agonistic effects of the methyl, propyl, and butyl analogs were abolished by washing. Only the reversible binding of xanomeline was detected on purified soluble receptors, but both binding modes occurred on purified receptors reconstituted into liposomes and exposed to xanomeline only after reconstitution. The wash-resistant binding did not occur if the exposure of purified receptors or liposomes alone to xanomeline, followed by washing, preceded reconstitution. Simultaneous presence of receptors and their lipid environment is therefore essential for the wash-resistant binding to take place. We suggest that the wash-resistant binding of xanomeline involves interhelical penetration of the M₁ muscarinic receptor by xanomeline's O-alkyl chain and its interaction with membrane lipids surrounding the receptor.