Molecular Mechanisms of Methoctramine Binding and Selectivity at Muscarinic Acetylcholine Receptors
Autor: | Vladimír Doležal, Alena Randáková, Esam E. El-Fakahany, Jan Jakubík, K. Fuksová, Pavel Zimčík |
---|---|
Rok vydání: | 2014 |
Předmět: |
Pharmacology
Binding Sites Stereochemistry Allosteric regulation Muscarinic acetylcholine receptor M3 Muscarinic acetylcholine receptor M2 Diamines Isoquinolines Binding Competitive DNA Mitochondrial Heterocyclic Compounds 4 or More Rings Receptors Muscarinic Mitochondria chemistry.chemical_compound DNA Topoisomerases Type I chemistry Coumarins Competitive antagonist Cell Line Tumor Muscarinic acetylcholine receptor Methoctramine Humans Molecular Medicine Binding site Receptor |
Zdroj: | Molecular Pharmacology. 86:180-192 |
ISSN: | 1521-0111 0026-895X |
Popis: | 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. |
Databáze: | OpenAIRE |
Externí odkaz: |