PIP₂ Interacts Electrostatically with MARCKS-like Protein-1 and ENaC in Renal Epithelial Cells

We examined the interaction of a membrane-associated protein, MARCKS-like Protein-1 (MLP-1), and an ion channel, Epithelial Sodium Channel (ENaC), with the anionic lipid, phosphatidylinositol 4, 5-bisphosphate (PIP₂). We found that PIP₂ strongly activates ENaC in excised, inside-out patches with a half-activating concentration of 21 ± 1.17 µM. We have identified 2 PIP₂ binding sites in the N-terminus of ENaC β and γ with a high concentration of basic residues. Normal channel activity requires MLP-1's strongly positively charged effector domain to electrostatically sequester most of the membrane PIP₂ and increase the local concentration of PIP₂. Our previous data showed that ENaC covalently binds MLP-1 so PIP₂ bound to MLP-1 would be near PIP₂ binding sites on the cytosolic N terminal regions of ENaC. We have modified the charge structure of the PIP₂ -binding domains of MLP-1 and ENaC and showed that the changes affect membrane localization and ENaC activity in a way consistent with electrostatic theory.