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

Clapham DE (2012) Anion-Sensitive Fluorophore Identifies the Drosophila Swell-Activated Chloride Channel in a Genome-Wide RNA Interference Screen. PLoS ONE 7(10): e46865. doi:10.1371/journal.pone.0046865 Anion-Sensitive Fluorophore Identifies the Drosophila Swell-Activated Chloride Channel in a Genome-Wide RNA Interference Screen Stephanie C. Stotz 0 David E. Clapham 0 Alexander A. Mongin, Albany Medical College, United States of America 0 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 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 residues of dBest1 conferred swell activation to mBest2. The chimera, however, maintains mBest2-like pore properties, strongly indicating that the Bestrophin protein forms the Clswell 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. - Funding: SCS is supported by the Tommy Kaplan Fellowship, Boston Childrens Hospital, Boston. Howard Hughes Medical Institute provided funding for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. All mammalian cells express chloride channels activated by decreases in extracellular osmolality, albeit with different biophysical properties [1]. The ubiquitous expression of Clswell suggests its essential cellular function. Tightly regulated Clswell channels participate in volume regulation, motility, cell survival, and cell division [1]. In contrast, de-regulated constitutively active Clswell channels exacerbate several cardiac diseases, including myocardial hypertrophy and heart failure [2]. The mammalian Clswell channel- encoding gene has yet to be identified despite the wealth of proteins nominated by candidate approaches [3]. These proteins include ClC-2 [4], ClC-3 [5], P-glycoprotein [6,7], pICln [8,9], p64 [10], phospholemman [11], Best1 and 2 [12], TMEM16A [13], and TMEM16F [14]. The research community has yet to agree on any of these candidates as a bona fide Clswell channel. In Drosophila, however, accumulating evidence indicates that dBest1 encodes for a Clswell channel. RNAi targeting dBest1 eliminates Drosophila Schneider (S2) cell IClswell, an effect rescued by re-introduction of dBest1 [15]. Further, swell activated dBest1 mutants have altered biophysical properties and reactivity to sulfhydryl reagents [16]. dBest1 likely forms the chloride conducting pore, but it may be an obligate auxillary subunit of Drosophila IClswell that modifies channel properties akin to CaV b subunits [17]. Assigning chloride channel function to any protein is difficult. The known chloride channel families (e.g., ClC, Anoctamin/ TMEM16, CFTR, and ionotropic GABAA and GlyR) lack structural pore or gating motifs that might form the basis for in silico identification. Expression cloning approaches have also failed due to widespread Clswell channel expression that precludes the separation of endogenous and over-expressed protein activities. Moreover, known chloride channels blockers are non-specific and their affinities are far too low to encourage affinity purification. Finally, previous chloride indicators are poor tools for screening due to loading and retention issues, inconsistent results, and poor reproducibility [18]. Here we present an unbiased genome-wide, high-throughput RNAi screen designed to identify the Drosop (...truncated)