PIP2 Interacts with MLP and ENaC Electrostatically 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 phosphatidylinositol 4, 5-bisphosphate (PIP2). PIP2 activates ENaC in inside-out patches (half activating concentration of 21 ± 1.17). There are several PIP2 binding sites on ENaC, but 2 binding sites with a large number of basic residues at the N-terminus of ENaC b and g are critical sites for PIP2 regulation of ENaC. PIP2 is necessary to open ENaC, but there is a problem with a simple model of ENaC and PIP2 associating by membrane lateral diffusion. ENaC is a relatively rare protein (a few functional apical channels/m2). PIP2 is also a rare molecule: less than 1 in 1000 membrane lipids. Given the known abundance of PIP2 and ENaC and the diffusion constant of PIP2 in the apical membrane, the mean time it would take a PIP2 molecule to find an ENaC channel by random lateral diffusion is 6.3x102s or approximately 10 minutes. But, in normal epithelial cells expressing ENaC, the channel opens about every 2 seconds. 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 without direct covalent binding. This implies differences between PIP2 association with MLP-1 and the binding of a ligand to a specific receptor. First, the effect of MLP-1 charge acts at a distance. The coulombic force exerted by MLP-1 on PIP2 does decrease as the square of the distance between MLP-1 effector domain and PIP2, but the distances within the membrane are relatively small and the membrane dielectric constant is also very small so the forces can be significant over a large membrane area. In the absence of MLP-1, PIP2 moves around the membrane lipid in a two-dimensional random walk, but the presence of the charged effector domain biases the walk so that PIP2 always moves towards MLP-1, at first slowly and then rapidly until it is in MLP-1's potential well. This implies that MLP-1 increases the local concentration of PIP2 exponentially as electrostatic theory predicts. MLP-1 membrane concentration is near 10mM which is comparable to the concentration of PIP2. Given PIP2's lateral mobility in the membrane of about 10-8cm2s-1, MLP-1 would rapidly sequester most membrane PIP2. This would increase the local PIP2 concentration over a hundred-fold. Our data shows that ENaC covalently binds MLP-1, so this increased PIP2 concentration would be in close proximity to PIP2 binding sites on the cytosolic N terminus of ENaC. This binding is also electrostatic since the ENaC binding sites contain multiple basic residues. The advantage of electrostatic interaction is that PIP2 can remain in close proximity to both ENaC and MLP-1. 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 consistent with electrostatic theory. Using STED FRAP, we also measured the diffusion rates of MLP-1, ENaC, and PIP2 and ENaC and PIP2 in the absence of MLP-1 that also support an electrostatic model.