Phosphatidylinositol 4,5-bisphosphate directly interacts with the beta and gamma subunits of the sodium channel ENaC.

The plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) regulates the activity of diverse ion channels to include the epithelial Na(+) channel ENaC. Whether PIP2 regulation of ENaC is due to a direct phospholipid-protein interaction, though, remains obscure. To date, possible interaction of PIP2 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 PIP2-ENaC interactions. We find using the CIBN-CRY2 optogenetic dimerization system that the phosphoryl group positioned at carbon 5 of PIP2 is necessary for interaction with ENaC. Previous studies have implicated conserved basic residues in the cytosolic portions of beta- and gamma-ENaC subunits as being important for PIP2-ENaC interactions. To test this, we used synthetic peptides of these regions of beta- and gamma-ENaC. Steady state intrinsic fluorescence spectroscopy demonstrated that phosphoinositides change the local conformation of the N terminus of beta-ENaC, and two sites of gamma-ENaC adjacent to the plasma membrane, suggesting direct interactions of PIP2 with these three regions. Microscale thermophoresis elaborated PIP2 interactions with the amino termini of beta- (Kd ~5.2 microM) and gamma-ENaC (Kd ~13 microM). A weaker interaction site within the carboxy terminus of gamma-ENaC (Kd ~800 microM) was also observed. These results support that PIP2 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.