title: Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms.
abstract: We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished the functional responses to both classical and atypical agonists. Our data show that both classical and atypical agonists activate hM1 receptors by the same molecular switch that involves D71 in the second transmembrane helix. The principal difference among the studied agonists is rather in the way they interact with D105 in the orthosteric binding site. Furthermore, our data demonstrate a key role of D105 in xanomeline wash-resistant binding and persistent activation of hM1 by wash-resistant xanomeline.
authors.affiliation: Institute of Physiology Czech Academy of Sciences, v.v.i., 142 20 Prague, Czech Republic.
authors.forename: Alena
authors.initials: A
authors.lastname: Randáková
authors.affiliation: Institute of Physiology Czech Academy of Sciences, v.v.i., 142 20 Prague, Czech Republic.
authors.forename: Eva
authors.initials: E
authors.lastname: Dolejší
authors.affiliation: Institute of Physiology Czech Academy of Sciences, v.v.i., 142 20 Prague, Czech Republic.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Rudajev
authors.affiliation: Institute of Physiology Czech Academy of Sciences, v.v.i., 142 20 Prague, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Zimčík
authors.affiliation: Institute of Physiology Czech Academy of Sciences, v.v.i., 142 20 Prague, Czech Republic.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Doležal
authors.affiliation: Department of Experimental and Clinical Pharmacology, University of Minnesota College of Pharmacy, Minneapolis, MN 55455, USA.
authors.forename: Esam E
authors.initials: EE
authors.lastname: El-Fakahany
authors.affiliation: Institute of Physiology Czech Academy of Sciences, v.v.i., 142 20 Prague, Czech Republic. Electronic address: jakubik@biomed.cas.cz.
authors.forename: Jan
authors.initials: J
authors.lastname: Jakubík
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D007295:Inositol Phosphates / Q000378:metabolism; D008958:Models, Molecular; D018721:Muscarinic Agonists / Q000494:pharmacology*; D009154:Mutation; D010095:Oxotremorine / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D043643:Receptor, Muscarinic M1 / Q000819:agonists* / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects; D013830:Thiadiazoles / Q000494:pharmacology
chemical_list: D007295:Inositol Phosphates; D018721:Muscarinic Agonists; D011725:Pyridines; D043643:Receptor, Muscarinic M1; D043562:Receptors, G-Protein-Coupled; D013830:Thiadiazoles; D010095:Oxotremorine; D002217:Carbachol; C075257:xanomeline; D002118:Calcium
keywords: Activation mechanism; Atypical agonists; Carbachol (PubChem CID: 5831); Muscarinic acetylcholine receptors; N-desmethylclozapine (PubChem CID: 2820); Oxotremorine (PubChem CID: 4630); Xanomeline; Xanomeline (PubChem CID: 60809)