PIP 2 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- bis phosphate (PIP 2 ). We found that PIP 2 strongly activates ENaC in excised, inside-out patches with a half-activating concentration of 21 ± 1.17 µM. We have identified 2 PIP 2 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 PIP 2 and increase the local concentration of PIP 2 . Our previous data showed that ENaC covalently binds MLP-1 so PIP 2 bound to MLP-1 would be near PIP 2 binding sites on the cytosolic N terminal regions of ENaC. We have modified the charge structure of the PIP 2 -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.