PIP(2) Interacts Electrostatically with MARCKS-like Protein-1 and ENaC in Renal Epithelial Cells

SIMPLE SUMMARY: Epithelial Sodium Channel (ENaC) is a renal ion channel responsible for a major fraction of total body sodium balance. MARCKS-like Protein-1 (MLP-1) is a membrane protein that controls the distribution of membrane phosphatidylinositol 4, 5-bisphosphate (PIP(2)). PIP(2) strongly activates ENaC with a half-activating concentration of 21 ± 1.17 μM. Normal channel activity requires MLP-1 associated with the inner leaflet of the cell membrane. MLP-1′s strongly positively charged effector domain sequesters PIP(2) electrostatically and increases the local concentration of PIP(2) over a hundred-fold. By controlling local PIP(2) concentration, MLP-1 controls ENaC activity and, consequently, total body sodium balance. ABSTRACT: 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 (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.