Abstract
The plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulates the activity of diverse ion channels to include the epithelial Na(+) channel ENaC. Whether PIP(2) regulation of ENaC is due to a direct phospholipid-protein interaction, remains obscure. To date, possible interaction of PIP(2) with ENaC primarily has been tested indirectly through assays of channel function. A fragment-based biochemical analysis approach is used here to directly quantify possible PIP(2)-ENaC interactions. We find using the CIBN-CRY2 optogenetic dimerization system that the phosphoryl group positioned at carbon 5 of PIP(2) is necessary for interaction with ENaC. Previous studies have implicated conserved basic residues in the cytosolic portions of β- and γ-ENaC subunits as being important for PIP(2)-ENaC interactions. To test this, we used synthetic peptides of these regions of β- and γ-ENaC. Steady-state intrinsic fluorescence spectroscopy demonstrated that phosphoinositides change the local conformation of the N terminus of β-ENaC, and two sites of γ-ENaC adjacent to the plasma membrane, suggesting direct interactions of PIP(2) with these three regions. Microscale thermophoresis elaborated PIP(2) interactions with the N termini of β- (K(d) ∼5.2 μm) and γ-ENaC (K(d) ∼13 μm). A weaker interaction site within the carboxyl terminus of γ-ENaC (K(d) ∼800 μm) was also observed. These results support that PIP(2) regulates ENaC activity by directly interacting with at least three distinct regions within the cytoplasmic domains of the channel that contain conserved basic residues. These interactions are probably electrostatic in nature, and are likely to bear a key structural role in support of channel activity.