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dc.contributor.authorStotz, Stephanie Christine
dc.contributor.authorClapham, David Eldon
dc.date.accessioned2013-04-10T15:31:11Z
dc.date.issued2012
dc.identifier.citationStotz, Stephanie C., and David E. Clapham. 2012. Anion-sensitive fluorophore identifies the drosophila swell-activated chloride channel in a genome-wide RNA interference screen. PLoS ONE 7(10): e46865.en_US
dc.identifier.issn1932-6203en_US
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:10522863
dc.description.abstractWhen cells swell in hypo-osmotic solutions, chloride-selective ion channels (\(Cl_{swell}\)) activate to reduce intracellular osmolality and prevent catastrophic cell rupture. Despite intensive efforts to assign a molecular identity to the mammalian \(Cl_{swell}\) channel, it remains unknown. In an unbiased genome-wide RNA interference (RNAi) screen of Drosophila cells stably expressing an anion-sensitive fluorescent indicator, we identify Bestrophin 1 (dBest1) as the Drosophila \(Cl_{swell}\) channel. Of the 23 screen hits with mammalian homologs and predicted transmembrane domains, only RNAi specifically targeting dBest1 eliminated the \(Cl_{swell}\) current (\(I_{Clswell}\)). We further demonstrate the essential contribution of dBest1 to Drosophila \(I_{Clswell}\) with the introduction of a human Bestrophin disease-associated mutation (W94C). Overexpression of the W94C construct in Drosophila cells significantly reduced the endogenous \(I_{Clswell}\). We confirm that exogenous expression of dBest1 alone in human embryonic kidney (HEK293) cells creates a clearly identifiable Drosophila–like \(I_{Clswell}\). In contrast, activation of mouse Bestrophin 2 (mBest2), the closest mammalian ortholog of dBest1, is swell-insensitive. The first 64 residues of dBest1 conferred swell activation to mBest2. The chimera, however, maintains mBest2-like pore properties, strongly indicating that the Bestrophin protein forms the \(Cl_{swell}\) channel itself rather than functioning as an essential auxiliary subunit. dBest1 is an anion channel clearly responsive to swell; this activation depends upon its N-terminus.en_US
dc.language.isoen_USen_US
dc.publisherPublic Library of Scienceen_US
dc.relation.isversionofdoi:10.1371/journal.pone.0046865en_US
dc.relation.hasversionhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464265/pdf/en_US
dash.licenseLAA
dc.subjectBiologyen_US
dc.subjectAnatomy and Physiologyen_US
dc.subjectPhysiological Processesen_US
dc.subjectHomeostasisen_US
dc.subjectCell Physiologyen_US
dc.subjectElectrophysiologyen_US
dc.subjectBiotechnologyen_US
dc.subjectBioengineeringen_US
dc.subjectBiological Systems Engineeringen_US
dc.subjectModel Organismsen_US
dc.subjectAnimal Modelsen_US
dc.subjectDrosophila Melanogasteren_US
dc.subjectMolecular Cell Biologyen_US
dc.subjectGene Expressionen_US
dc.subjectRNA interferenceen_US
dc.subjectCellular Stress Responsesen_US
dc.subjectSignal Transductionen_US
dc.subjectProteomicsen_US
dc.subjectProteomic Databasesen_US
dc.titleAnion-Sensitive Fluorophore Identifies the Drosophila Swell-Activated Chloride Channel in a Genome-Wide RNA Interference Screenen_US
dc.typeJournal Articleen_US
dc.description.versionVersion of Recorden_US
dc.relation.journalPLoS ONEen_US
dash.depositing.authorClapham, David Eldon
dc.date.available2013-04-10T15:31:11Z
dc.identifier.doi10.1371/journal.pone.0046865*
dash.contributor.affiliatedStotz, Stephanie Christine
dash.contributor.affiliatedClapham, David


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