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Abstract
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^sub 2^), PI-3,4,5-trisphosphate (PI(3,4,5)P^sub 3^), and phosphatidic acid (PA). We previously showed that PI(4,5)P^sub 2^ and PI(3,4,5)P^sub 3^ upregulate the renal epithelial sodium channel (ENaC). Further studies from others suggested that PI(4,5)P^sub 2^ and PI(3,4,5)P^sub 3^ respectively target β- and γ-ENaC subunit. To determine whether PI(4,5)P^sub 2^ and PI(3,4,5)P^sub 3^ selectively bind to β and γ subunit, we performed lipid-protein overlay experiments. Surprisingly, the results reveal that most APs, including PI(4)P, PS, PI(4,5)P^sub 2^, PI(3,4,5)P^sub 3^, and PA, but not PI, non-selectively bind to not only β and γ but also α 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^sub 2^ and PI(3,4,5)P^sub 3^ stimulated ENaC, and that PA, however, inhibited ENaC. These data together suggest that APs differentially regulate ENaC by physically interacting with α-, β-, and γ-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.[PUBLICATION ABSTRACT]