ENaC Activity and Regulation in Renal Distal Convoluted Tubule Cells

Abstract

Our previous work has shown that epithelial sodium channel (ENaC) proteins are present in distal convoluted tubule (DCT) cells of the distal nephron. The purpose of these experiments was to show whether these proteins were functional. We also wish to show whether the channels were stimulated by aldosterone, inhibited by amiloride, and regulated by the ENaC chaperone protein, MLP‐1, as ENaC is regulated in collecting duct principal cells. We used DCT‐15 cells in monolayer culture on permeable polyester supports to measure transepithelial voltage, resistance, and current. The properties of DCT‐15 cells are quite similar to those of in situ cells of the DCT2 segment of the nephron. Our results showed that DCT‐15 cells had relatively low resistances (206 ± 5.71 Ω‐cm2, n=36) and small, but measurable amiloride‐sensitive currents (2.87 ± 0.237 μA, n=36), showing that these cells contain functional ENaC. 100 nM aldosterone significantly increased mean DCT‐15 cell current (from 2.87 ± 0.237, n=36, to 5.33 ± 0.395μA, n=8). Amiloride significantly lowered the current across DCT cells to very low levels (from 2.74 ± 0.219, n=42, to 0.825 ± 0.186μA, n=15). MLP‐1 is a myristoylated protein that when associated with the membrane promotes ENaC activity. When three critical serines are phosphorylated by PKC, MLP‐1 dissociates from the membrane and ENaC activity decreases. We transfected the cells with four MLP FLAG‐tagged mutants: wild type (WT), constitutively active (S3A), constitutively inactive (S3D), and myristoylation negative (GA) and measured DCT‐15 current. For comparison, we examined a principal cell line, mpkCCD cells. Transfection with MLP‐1 modified current in both DCT and CCD cells: median current was highest in S3A mutant and lowest in S3D mutant; the current after transfection with either construct was significantly different from WT. ENaC and MLP construct expression was also studied in lysates of both cell types by Western blotting with FLAG and anti‐ENaC subunit antibodies. In DCT‐15 cells with the highest current, expression of ENaC was greatest. Lastly, we calculated based on an average current of 2 μA/cm2 and ENaC single channel current of 0.4 pA that DCT‐15 cells must have approximately 5 million functional channels per square centimeter.Support or Funding InformationSupported by R01 DK‐110409 to DCE and K01 DK‐115660 and ASN Gottschalk AWARD to BMWThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.