title: Conformational mobility of immobilized alpha3beta2, alpha3beta4, alpha4beta2, and alpha4beta4 nicotinic acetylcholine receptors.
abstract: Four affinity chromatography stationary phases have been developed based upon immobilized nicotinic acetylcholine receptor (nAChR) subtypes, the alpha3beta2, alpha3beta4, alpha4beta2, and alpha4beta4 nAChRs. The stationary phases were created using membranes from cell lines expressing the subtypes and an immobilized artificial membrane stationary phase. The immobilized nAChRs were characterized using frontal chromatography with the agonist epibatidine as the marker. The observed binding affinities for the agonists epibatidine, nicotine, and cytisine were consistent with reported values, indicating that the nAChRs retained their ability to bind agonists. The noncompetitive inhibitors (NCIs) of the nAChR (R)- and (S)-mecamylamine, phencylcidine, dextromethoprphan, and levomethorphan were also chromatographed on the columns using nonlinear chromatography techniques. The studies were carried out before and after exposure of the columns to epibatidine. The NCI retention times increased after exposure to epibtatidine as did the enantioselective separation of mecamylamine and methorphan. The results indicate that the immobilized nAChRs retained their ability to undergo agonist-induced conformational change from the resting to the desensitized states. The columns provide a unique ability to study the interactions of NCIs with both of these conformational states.
authors.affiliation: Gerontology Research Center, National Institutes in Aging, National Institutes of Health, Baltimore, MD 21224-6825, USA.
authors.forename: Ruin
authors.initials: R
authors.lastname: Moaddel
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Jozwiak
authors.forename: Kevin
authors.initials: K
authors.lastname: Whittington
authors.forename: Irving W
authors.initials: IW
authors.lastname: Wainer
mesh_terms: D000470:Alkaloids / Q000737:chemistry; D001392:Azocines / Q000737:chemistry; D001667:Binding, Competitive; D015415:Biomarkers; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry; D002460:Cell Line; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid / Q000379:methods*; D003915:Dextromethorphan / Q000737:chemistry; D008464:Mecamylamine / Q000737:chemistry; D008567:Membranes, Artificial; D009538:Nicotine / Q000737:chemistry; D018722:Nicotinic Agonists / Q000737:chemistry / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000737:chemistry / Q000494:pharmacology; D010622:Phencyclidine / Q000737:chemistry; D011487:Protein Conformation; D011725:Pyridines / Q000737:chemistry; D011807:Quinolizines / Q000737:chemistry; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000187:drug effects; D000093843:Quinolizidine Alkaloids
chemical_list: D000470:Alkaloids; D001392:Azocines; D015415:Biomarkers; D019086:Bridged Bicyclo Compounds, Heterocyclic; D003915:Dextromethorphan; D008464:Mecamylamine; D008567:Membranes, Artificial; D009538:Nicotine; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D010622:Phencyclidine; D011725:Pyridines; D011807:Quinolizines; D011978:Receptors, Nicotinic; C427403:alpha(4)beta(4) nicotinic receptor; C419362:nicotinic receptor alpha3beta2; C403530:nicotinic receptor alpha3beta4; C403531:nicotinic receptor alpha4beta2; C004712:cytisine; D000093843:Quinolizidine Alkaloids; C082748:epibatidine