Anionic phospholipids differentially regulate the epithelial sodium channel (ENaC) by interacting with alpha, beta, and gamma ENaC subunits.

Anionic phospholipids (APs) present a variety of lipids in the cytoplasmic leaflet of the plasma membrane, including phosphatidylinositol (PI), PI-4-phosphate (PI(4)P), phosphatidylserine (PS), PI-4,5-bisphosphate (PI(4,5)P(2)), PI-3,4,5-trisphosphate (PI(3,4,5)P(3)), and phosphatidic acid (PA). We previously showed that PI(4,5)P(2) and PI(3,4,5)P(3) upregulate the renal epithelial sodium channel (ENaC). Further studies from others suggested that PI(4,5)P(2) and PI(3,4,5)P(3) respectively target beta- and gamma-ENaC subunit. To determine whether PI(4,5)P(2) and PI(3,4,5)P(3) selectively bind to beta and gamma subunit, we performed lipid-protein overlay experiments. Surprisingly, the results reveal that most APs, including PI(4)P, PS, PI(4,5)P(2), PI(3,4,5)P(3), and PA, but not PI, non-selectively bind to not only beta and gamma but also alpha subunit. To determine how these APs regulate ENaC, we performed inside-out patch-clamp experiments and found that PS, but not PI or PI(4)P, maintained ENaC activity, that PI(4,5)P(2) and PI(3,4,5)P(3) stimulated ENaC, and that PA, however, inhibited ENaC. These data together suggest that APs differentially regulate ENaC by physically interacting with alpha-, beta-, and gamma-ENaC. Further, the data from cell-attached patch-clamp and confocal microscopy experiments indicate that PA, a product of phospholipase D, may provide one of the pathways for inhibition of ENaC by endothelin receptors.