Electrostatic interaction of ENaC, MARCKS-like protein, and PIP2

Renal Division, Emory University Medical School, Atlanta, GA 30322. Using single-cjhannel methods, we examined the interaction of a membrane-associated protein, MARCKS-like Protein-1 (MLP-1), and Epithelial Sodium Channels (ENaC), with the anionic lipid, phosphatidylinositol 4, 5-bisphosphate (PIP2). PIP2 is necessary to open ENaC. However, there is a problem with a simple model of ENaC and PIP2 association by lateral diffusion in the membrane. Given the abundance of PIP2 and ENaC and the diffusion constant of PIP2 in the apical membrane of renal cells, the mean time for PIP2 to find an ENaC channel by random diffusion would be 6.3x10 2 s or about once in 10 minutes. But, in cells expressing ENaC, the channel opens about every other second. We hypothesized that 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. We also hypothesized that MLP-1 and functional ENaC channels are associated with specific membrane domains known as lipid rafts. PIP2 within the domains stabilizes MLP-1 and ENaC while increasing the Po of individual ENaC. ENaC in these domains can be destabilized by PIP2 degradation. To investigate MLP-1-ENaC-PIP2 interactions, we (1) examined the unusual electrostatic interaction of ENaC and MLP-1 with PIP2 in the membrane; (2) investigated ENaC stability by determining if ENaC is present in PIP2-rich lipid domain and determining if MLP-1 stabilizes ENaC in these lipid domains; (3) determined the proximity of PIP2 to the N-terminal ends of β and γ ENaC using luminescence energy transfer, (4) examined the effects of making ENaC charge structure more positive on PIP2 binding and ENaC activity, and (5) examined the phenotype of renal principal cell-specific, MLP-1 knockout mice using single channel measurements in isolated, split-open collecting ducts. All of the results support a model of ENaC N-terminal interaction with MLP-1 and PIP2 to open ENaC channels. National Institutes of Digestive Diseases and Kidney R01 DK110409 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.