Anionic phospholipids differentially regulate the epithelial sodium channel (ENaC) by interacting with α, β, and γ ENaC subunits

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 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 β-and γ-ENaC subunit. To determine whether PI(4,5)P 2 and PI(3,4,5)P 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 2 , PI(3,4,5)P 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 insideout 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 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.

Depletion of Cholesterol Reduces ENaC Activity by Decreasing Phosphatidylinositol-4,5-Bisphosphate in Microvilli

“…We have shown that anionic phospholipids including PIP 2 can bind to all three ENaC subunits and directly stimulate ENaC in distal nephron A6 cells [18,19]. Recently, we have also shown that PIP 2 is predominantly located in microvilli and that PIP 2 distribution between microvilli and non-microvillar planar region depends on Cho synthesis in the CCD cells [13].…”

The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel

“…Tryptophan fluorescence anisotropy represents the ordering of the hydrophobic Trp environment. Fluorescence anisotropy of Trp 31 did not change in the presence of any lipid, indicating that the peptide is not incorporated into the membrane. 4 This result reiterates our observations that ␥NT acquires secondary structure in the presence of APs, with higher specificity for PIP 2 .…”

“…K-clusters in ␤-and ␥-ENaC Strongly Influence ENaC Proteolytic Regulation-Previous studies have reported direct interaction with APs of the N terminus of ␤-ENaC (␤NT) and ␥-ENaC (␥NT) (31). We therefore extended our computational analyses to study the possible structural changes induced by PIP 2 in ␤NT.…”

“…For example, direct phosphoinositide binding of short peptide fragments from the N terminus of different ENaC subunits has been demonstrated using lipidprotein overlay assays on PIP Strips TM (31). However, specific structural features (requirements and consequences) of interaction of ENaC N termini with APs have not been reported.…”

“…To further characterize the secondary structure gained by ␥NT in the presence of PIP 2 , we monitored the environment of the sole tryptophan (Trp 31 ) present in the peptide by measuring Trp fluorescence as a function of lipid concentration. In fluorescence spectra, ␥NT showed an emission maximum at 357 nm in phosphate buffer, indicating that Trp 31 experiences an extremely aqueous (polar) environment, which is in accordance with the unstructured nature of the peptide observed using CD and DMD simulations (Fig.…”