title: beta-Amyloid directly inhibits human alpha4beta2-nicotinic acetylcholine receptors heterologously expressed in human SH-EP1 cells.
abstract: Amyloid-beta (Abeta) accumulation and aggregation are thought to contribute to the pathogenesis of Alzheimer's disease (AD). In AD, there is a selective decrease in the numbers of radioligand binding sites corresponding to the most abundant nicotinic acetylcholine receptor (nAChR) subtype, which contains human alpha4 and beta2 subunits (halpha4beta2-nAChR). However, the relationships between these phenomena are uncertain, and effects of Abeta on halpha4beta2-nAChR function have not been investigated in detail. We first confirmed expression of halpha4 and hbeta2 subunits as messenger RNA in transfected, human SHEP1 cells by reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses. Immunoprecipitation Western analyses confirmed alpha4 and beta2 subunit protein expression and co-assembly. Whole cell current recording demonstrated heterologous expression in SH-EP1-halpha4beta2 cells of functional halpha4beta2-nAChRs with characteristic responses to nicotinic agonists or antagonists. Nicotine-induced whole cell currents were suppressed by Abeta(1-42) in a dose-dependent manner. Functional inhibition was selective for Abeta(1-42) compared with the functionally inactive, control peptide Abeta(40-1).Abeta(1-42)-mediated inhibition of halpha4beta2-nAChR function was non-competitive, voltage-independent, and use-independent. Pre-loading of cells with guanyl-5'-yl thiophosphate failed to prevent Abeta(1-42)-induced inhibition, suggesting that down-regulation of halpha4beta2-nAChR function by Abeta(1-42) is not mediated by nAChR internalization. Sensitivity to Abeta(1-42) antagonism at 1 nm was evident for halpha4beta2-nAChRs, but not for heterologously expressed human alpha7-nAChRs, although both nAChR subtypes were functionally inhibited by 100 nm Abeta(1-42), with the magnitude of functional block being higher for 100 nm Abeta(1-42) acting on halpha7-nAChRs. These findings suggest that halpha4beta2-nAChRs are sensitive and perhaps pathophysiologically relevant targets for Abeta neurotoxicity in AD.
authors.affiliation: Division of Neurology, St. Joseph's Hospital and Medical Center, Phoenix, AZ 85013, USA. jwu2@chw.edu
authors.forename: Jie
authors.initials: J
authors.lastname: Wu
authors.forename: Yen-Ping
authors.initials: YP
authors.lastname: Kuo
authors.forename: Andrew A
authors.initials: AA
authors.lastname: George
authors.forename: Lin
authors.initials: L
authors.lastname: Xu
authors.forename: Jun
authors.initials: J
authors.lastname: Hu
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Lukas
mesh_terms: D000682:Amyloid / Q000502:physiology*; D002460:Cell Line; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D018408:Patch-Clamp Techniques; D011233:Precipitin Tests; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D000682:Amyloid; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2