Allosteric interactions at the M3 muscarinic acetylcholine receptor

摘要: The extracellular loops of muscarinic acetylcholine receptors are predicted to play a role in the binding and regulation allosteric modulators. Furthermore, sequence five subtypes shows large degree diversity this region. M3 receptor mutants, K523E, D518K N132G, which substituted residues were those corresponding M1 subtype, studied. As amino acids positions 518 523 charged, uncharged K523Q D518N, also created order observe any possible effect charge. One question examined is whether these mutations changed orthosteric ligands, generating phenotype.Radioligand experiments revealed that one mutant, had profound potentiating on prototypical modulators like gallamine, strychnine, brucine N-chloromethylbrucine, but minimal effects number including [3H]N-methylscopolamine ([3H]NMS) (ACh). increase affinity was found at both unoccupied [3H]NMS-occupied receptors, with up 70 fold increases being observed. Switches from negative positive cooperativity for some strychnine-related compounds found.At affinities ligands increased 160 receptor-ACh complex, 35 Positive magnitude highest has been reported receptors.The dramatic changes cooperativities K523E did not result generation phenotype. data suggest introduction negatively charged glutamate residue loss positively lysine. seems be solely transmission their site.For acting gallamine site, all ACh have reproduced functional studies, indicating modulation, seen binding, transmitted cellular response. A novel unexpected finding WIN62,577 an agonist N132G. study nanomolar concentrations may present assays function give misleading interpretations data. These artefacts removed by preincubation acetylcholinesterase, control previously used studies sensitivity composition assay buffer investigated detail. In phosphate low ionic strength (PB) constants studied, allosteric, increased, relative Hepes higher strength, except WIN 62,577, ligand binds different site modulators, SVT-40776 new selective antagonist, modes binding. Cooperativities switched changing buffer.The two factors affecting parameters PB mutation mutually potentiate each others effects. We able obtain 10,000 6400 occupied receptor.The location K523, other external shown important explored using models based X-ray structures rhodopsin â2 adrenergic receptor.