anion-sensitive fluorophore identifies the drosophila swell-activated chloride channel in a genome-wide rna interference screenanion-sensitive荧光团标识果蝇swell-activated氯通道在全基因组rna干扰屏幕.pdfVIP

Anion-Sensitive Fluorophore Identifies the Drosophila Swell-Activated Chloride Channel in a Genome-Wide RNA Interference Screen 1 1,2,3 Stephanie C. Stotz , David E. Clapham * 1 Howard Hughes Medical Institute, Department of Cardiology, Boston Children’s Hospital, Boston, Massachusetts, United States of America, 2 Manton Center for Orphan Disease, Boston Children’s Hospital, Boston, Massachusetts, United States of America, 3 Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, United States of America Abstract When cells swell in hypo-osmotic solutions, chloride-selective ion channels (Clswell) activate to reduce intracellular osmolality and prevent catastrophic cell rupture. Despite intensive efforts to assign a molecular identity to the mammalian Clswell 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 Clswell channel. Of the 23 screen hits with mammalian homologs and predicted transmembrane domains, only RNAi specifically targeting dBest1 eliminated the Clswell current (IClswell). We further demonstrate the essential contribution of dBest1 to Drosophila IClswell with the introduction of a human Bestrophin disease-associated mutation (W94C). Overexpression of the W94C construct in Drosophila cells significantly reduced the endogenous IClswell. We confirm that exogenous expression of dBest1 alone in human embryonic kidney (HEK293) cells creates a clearly identifiable Drosophila–like IClswell. In contrast, activation of mouse Bestrophin 2 (mBest2), the closest mammalian ortholog of dBest1, is swell-insensitive. The first 64 res