MLP regulates ENaC Activity in Renal Distal Convoluted Tubule Cells

Abstract

The epithelial sodium channel (ENaC) plays an important role in blood pressure regulation through the fine‐tuning of sodium reabsorption in the distal nephron. We previously demonstrated that ENaC is present in distal convoluted tubule (DCT) cells, and the PIP2‐dependent regulation of ENaC is mediated by the myristoylated alanine‐rich protein kinase C substrate (MARCKS)‐like protein‐1 (MLP‐1). MLP‐1 regulation of ENaC is controlled by MLP‐1 phosphorylation of MLP‐1’s PIP2 binding domain. Here we examine the role of MLP‐1 in the function of regulating ENaC in DCT‐15 cells. We also examined whether ENaC interacts with specific domains of MLP‐1. DCT‐15 cells and Cos‐7 cells were used in our study. To further dissect the mechanism of MLP in regulating ENaC in DCT‐15 cells, we constructed various mutants of MLP. WT is the full length wild‐type protein, whereas S3A contains amino acid substitutions in the effector domain to create a molecule that could not be phosphorylated. The S3D mutant was created to mimic constitutive phosphorylation. The GA mutant had an N‐terminal glycine replaced so that it could not be myristoylated. The single‐channel patch‐clamp recordings were used to measure the channel activity of ENaC channels after transfection, and by Mammalian two‐hybrid assay we examined the interaction sites of MLP‐1 and ENaC. S3A construct of MLP has proved to be particularly effective in regulating ENaC function in DCT‐15 cells compared to other variants, given its higher channel activity. MLP‐1 regulates ENaC by changing the open probability of ENaC but not the protein expression or the number of functional channels in DCT‐15 cells. In a two‐hybrid screen, the N‐terminal (but not the C‐terminal) domain of MLP protein associated strongly with the N‐terminus of gamma ENaC and weakly with the N‐terminus of beta ENaC but not with the alpha subunit in cells.Support or Funding InformationSupported by R01 DK‐110409 to DCE and K01 DK‐115660 and ASN Gottschalk AWARD to BMW