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

PROJECT ABSTRACT This application investigates the role of electrostatic forces in the interaction of membrane proteins and charged membrane lipids. Specifically, we will examine the interaction of a membrane-associated protein, MARCKS-like Protein-1 (MLP-1), and the Epithelial Sodium Channel (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, the mean time it would take PIP2 to find an ENaC channel by random diffusion would be 6.3x10 2s or about once in 10 minutes. But, in cells expressing ENaC, the channel opens about every other second. We hypothesize 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 hypothesize that MLP-1 and functional ENaC channels are associated with specific membrane domains known as lipid rafts. PIP2 within these domains stabilizes MLP-1 and ENaC while increasing opening (Po) of individual ENaC. ENaC in these domains can be destabilized by PIP2 degradation. To investigate MLP-1-ENaC-PIP2 interactions, we will (1) examine the unusual electrostatic interaction of ENaC and MLP-1 with PIP2 in the membrane. (2) Investigate ENaC stability by determining if ENaC is present in PIP2- rich lipid domains and determining if MLP-1 stabilizes ENaC in these lipid domains. (3) Determine if cytoskeletal interactions maintain MLP-1 and ENaC in the PIP2-rich lipid domains by using FRAP and STED FCS before and after latrunculin or cytochalasin E disruption of the cytoskeleton. 4) Examine the phenotype of renal principal cell-specific, MLP-1 knockout mice using single channel measurements in isolated, split-open collecting ducts.