PIP2 Binds Multiple Cationic Sites of ENaC

Abstract

Phosphatidylinositol 4,5‐bisphosphate (PIP2) regulates the activity and gating of many different ion channels to include the epithelial Na+ channel (ENaC). The negatively charged phosphoryl groups of the PIP2 headgroup are expected to interact with cationic residues of the cytoplasmic tails of ENaC. To date, understanding of the biophysical interaction between ENaC and PIP2 has been limited to indirect functional assays. Consequently, the precise binding sites and their affinities have not been defined. We began this study using secondary structure prediction tools to show that helical conformation that may cluster the cationic residues of ENaC cytoplasmic tails. Such clustering would form an electropositive region attractive to the PIP2 headgroup. We used a CIBN/CRY2‐5‐ptase optogenetic dimerization system in HEK cells to show that the C5 phosphoryl group of PIP2 plays a role in regulating ENaC activity. To more precisely define the PIP2 binding sites of ENaC, we designed short peptides corresponding to the cationic clusters of ENaC. Steady state intrinsic fluorescence ligand binding data showed that PIP2 interacted with the extreme amino terminus of the β‐ENaC subunit (βN1 peptide) and the amino and carboxy termini of γ‐ENaC nearest the transmembrane domains (γN2 and γC peptides, respectively), but no significant changes were observed with the other peptides or mutant controls. Microscale thermophoresis data revealed that the βN1 and γN2 sites has the strongest interaction with the PIP2 headgroup with Kd ~5.2 and 15 μM, respectively, whereas the γC site interacts with much weaker affinity. Again, no interaction was observed between PIP2 and the other peptides and mutant controls. These results are consistent with PIP2 binding ENaC to stabilize the open conformation of channel via moderate and weak electrostatic interactions within the cytoplasmic termini.Support or Funding InformationThis work is supported by NIH/HHLBI T32 HL07446 to CRA and JDS, and NIH/NIDDK R01 DK987460 and DK117909 to JDS.