Abstract
Dysfunction of ENaC, the epithelial sodium channel that regulates salt and water reabsorption in epithelia, causes several human diseases, including cystic fibrosis (CF). To develop a global understanding of molecular regulators of ENaC traffic/function and to identify of candidate CF drug targets, we performed a large-scale screen combining high-content live-cell microscopy and siRNAs in human airway epithelial cells. Screening over 6,000 genes identified over 1,500 candidates, evenly divided between channel inhibitors and activators. Genes in the phosphatidylinositol pathway were enriched on the primary candidate list, and these, along with other ENaC activators, were examined further with secondary siRNA validation. Subsequent detailed investigation revealed ciliary neurotrophic factor receptor (CNTFR) as an ENaC modulator and showed that inhibition of (diacylglycerol kinase, iota) DGKι, a protein involved in PiP2 metabolism, downgrades ENaC activity, leading to normalization of both Na+ and fluid absorption in CF airways to non-CF levels in primary human lung cells from CF patients.
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•High-content siRNA screens reveal cystic-fibrosis-linked sodium channel modulators•Phosphatidylinositol pathway genes are enriched in the primary screen data set•Ciliary neurotrophic factor regulator (CNFTR) implicated in channel control•DGKι inhibitor normalizes Na+ and fluid absorption in human CF primary airway cells
Screening of live human cells reveals both inhibitors and activators of the epithelial Na+ channel, including an extensive network of phosphatidylinositol pathway genes and DGKι, which can be inhibited to revert ion flow and fluid absorption in cells from CF patient airways.
