PIP2 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 (PIP2). PIP2 strongly activates ENaC with a half-activating concentration of 21 +/- 1.17 mu M. Normal channel activity requires MLP-1 associated with the inner leaflet of the cell membrane. MLP-1 ' s strongly positively charged effector domain sequesters PIP2 electrostatically and increases the local concentration of PIP2 over a hundred-fold. By controlling local PIP2 concentration, MLP-1 controls ENaC activity and, consequently, total body sodium balance. 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 mu M. We have identified 2 PIP2 binding sites in the N-terminus of ENaC beta and gamma 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.

Automated summary

The interaction between MLP-1, ENaC, and PIP2 was studied, and it was found that PIP2 activates ENaC strongly. MLP-1's positively charged effector domain is required for normal channel activity by controlling the local concentration of PIP2.