Abstract
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 M1 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 ing. Only the reversible binding of xanomeline was detected purified soluble receptors, but both binding modes occurred purified receptors reconstituted into liposomes and exposed xanomeline only after reconstitution. The wash-resistant ing did not occur if the exposure of purified receptors or somes alone to xanomeline, followed by washing, reconstitution. Simultaneous presence of receptors and lipid environment is therefore essential for the binding to take place. We suggest that the binding of xanomeline involves interhelical penetration of M1 muscarinic receptor by xanomeline's O-alkyl chain and interaction with membrane lipids surrounding the receptor.
Footnotes
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This work was supported in part by Grant Agency of the Czech Republic Grants 305/02/D090 (to J.J.) and 309/02/1331 (to S.T.) in research project AVOZ 5011922 and a grant from the National Institutes of Health (NS25743) (to E.E.E.-F.).
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DOI: 10.1124/jpet.103.058594.
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ABBREVIATIONS: NMS, N-methylscopolamine; QNB, quinuclidinylbenzilate; [3H]CGP-12177, (±)-[3H]4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on hydrochloride; ABT, 3-[2′-aminobenzhydryl-oxy]tropate; CHO, Chinese hamster ovary.
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↵† Deceased.
- Received August 13, 2003.
- Accepted September 26, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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