Anion-Sensitive Fluorophore Identifies the Drosophila Swell-Activated Chloride Channel in a Genome-Wide RNA Interference Screen

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Anion-Sensitive Fluorophore Identifies the Drosophila Swell-Activated Chloride Channel in a Genome-Wide RNA Interference Screen

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Title: Anion-Sensitive Fluorophore Identifies the Drosophila Swell-Activated Chloride Channel in a Genome-Wide RNA Interference Screen
Author: Stotz, Stephanie Christine; Clapham, David Eldon

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Citation: Stotz, 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.
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Abstract: When 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.
Published Version: doi:10.1371/journal.pone.0046865
Other Sources: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464265/pdf/
Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA
Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:10522863
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