title: Molecular mechanisms of methoctramine binding and selectivity at muscarinic acetylcholine receptors.
abstract: Methoctramine (N,N'-bis[6-[[(2-methoxyphenyl)-methyl]hexyl]-1,8-octane] diamine) is an M(2)-selective competitive antagonist of muscarinic acetylcholine receptors and exhibits allosteric properties at high concentrations. To reveal the molecular mechanisms of methoctramine binding and selectivity we took advantage of reciprocal mutations of the M(2) and M(3) receptors in the second and third extracellular loops that are involved in the binding of allosteric ligands. To this end we performed measurements of kinetics of the radiolabeled antagonists N-methylscopolamine (NMS) in the presence of methoctramine and its precursors, fluorescence energy transfer between green fluorescent protein-fused receptors and an Alexa-555-conjugated precursor of methoctramine, and simulation of molecular dynamics of methoctramine association with the receptor. We confirm the hypothesis that methoctramine high-affinity binding to the M(2) receptors involves simultaneous interaction with both the orthosteric binding site and the allosteric binding site located between the second and third extracellular loops. Methoctramine can bind solely with low affinity to the allosteric binding site on the extracellular domain of NMS-occupied M(2) receptors by interacting primarily with glutamate 175 in the second extracellular loop. In this mode, methoctramine physically prevents dissociation of NMS from the orthosteric binding site. Our results also demonstrate that lysine 523 in the third extracellular loop of the M(3) receptors forms a hydrogen bond with glutamate 219 of the second extracellular loop that hinders methoctramine binding to the allosteric site at this receptor subtype. Impaired interaction with the allosteric binding site manifests as low-affinity binding of methoctramine at the M(3) receptor.
authors.affiliation: Institute of Physiology, v.v.i. (J.J., P.Z., A.R., V.D.) and Institute of Experimental Botany, v.v.i. (K.F.), Academy of Sciences of the Czech Republic, Prague, Czech Republic; and Department of Experimental and Clinical Pharmacology, University of Minnesota College of Pharmacy, Minneapolis, Minnesota (E.E.E.) jakubik@biomed.cas.cz.
authors.forename: Jan
authors.initials: J
authors.lastname: Jakubík
authors.affiliation: Institute of Physiology, v.v.i. (J.J., P.Z., A.R., V.D.) and Institute of Experimental Botany, v.v.i. (K.F.), Academy of Sciences of the Czech Republic, Prague, Czech Republic; and Department of Experimental and Clinical Pharmacology, University of Minnesota College of Pharmacy, Minneapolis, Minnesota (E.E.E.).
authors.forename: Pavel
authors.initials: P
authors.lastname: Zimčík
authors.affiliation: Institute of Physiology, v.v.i. (J.J., P.Z., A.R., V.D.) and Institute of Experimental Botany, v.v.i. (K.F.), Academy of Sciences of the Czech Republic, Prague, Czech Republic; and Department of Experimental and Clinical Pharmacology, University of Minnesota College of Pharmacy, Minneapolis, Minnesota (E.E.E.).
authors.forename: Alena
authors.initials: A
authors.lastname: Randáková
authors.affiliation: Institute of Physiology, v.v.i. (J.J., P.Z., A.R., V.D.) and Institute of Experimental Botany, v.v.i. (K.F.), Academy of Sciences of the Czech Republic, Prague, Czech Republic; and Department of Experimental and Clinical Pharmacology, University of Minnesota College of Pharmacy, Minneapolis, Minnesota (E.E.E.).
authors.forename: Květoslava
authors.initials: K
authors.lastname: Fuksová
authors.affiliation: Institute of Physiology, v.v.i. (J.J., P.Z., A.R., V.D.) and Institute of Experimental Botany, v.v.i. (K.F.), Academy of Sciences of the Czech Republic, Prague, Czech Republic; and Department of Experimental and Clinical Pharmacology, University of Minnesota College of Pharmacy, Minneapolis, Minnesota (E.E.E.).
authors.forename: Esam E
authors.initials: EE
authors.lastname: El-Fakahany
authors.affiliation: Institute of Physiology, v.v.i. (J.J., P.Z., A.R., V.D.) and Institute of Experimental Botany, v.v.i. (K.F.), Academy of Sciences of the Czech Republic, Prague, Czech Republic; and Department of Experimental and Clinical Pharmacology, University of Minnesota College of Pharmacy, Minneapolis, Minnesota (E.E.E.).
authors.forename: Vladimír
authors.initials: V
authors.lastname: Doležal
mesh_terms: D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D003374:Coumarins / Q000494:pharmacology; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics; D003959:Diamines / Q000378:metabolism*; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*
chemical_list: D003374:Coumarins; D004272:DNA, Mitochondrial; D003959:Diamines; D006576:Heterocyclic Compounds, 4 or More Rings; D007546:Isoquinolines; D011976:Receptors, Muscarinic; C455554:lamellarin D; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human; C054938:methoctramine