Abstract
As a pathway for Na(+) reabsorption, the epithelial Na(+) channel ENaC is critical for Na(+) homeostasis and blood pressure control. Na(+) transport is regulated by Nedd4-2, an E3 ubiquitin ligase that decreases ENaC expression at the cell surface. To investigate the underlying mechanisms, we proteolytically cleaved/activated ENaC at the cell surface and then quantitated the rate of disappearance of cleaved channels using electrophysiological and biochemical assays. We found that cleaved ENaC channels were rapidly removed from the cell surface. Deletion or mutation of the Nedd4-2 binding motifs in alpha, beta, and gammaENaC dramatically reduced endocytosis, whereas a mutation that disrupts a YXXØ endocytosis motif had no effect. ENaC endocytosis was also decreased by silencing of Nedd4-2 and by expression of a dominant negative Nedd4-2 construct. Conversely, Nedd4-2 overexpression increased ENaC endocytosis in human embryonic kidney 293 cells but had no effect in Fischer rat thyroid epithelia. In addition to its effect on endocytosis, Nedd4-2 also increased the rate of degradation of the cell surface pool of cleaved alphaENaC. Together the data indicate that Nedd4-2 reduces ENaC surface expression by altering its trafficking at two distinct sites in the endocytic pathway, inducing endocytosis of cleaved channels and targeting them for degradation.
Highlights
The epithelial Naϩ channel ENaC forms a pathway for Naϩ reabsorption across epithelia, including the kidney, lung, and colon
Proteolytic Cleavage of Cell Surface ENaC—In Fig. 1A, we transfected FRT epithelia with ␣, , and ␥ENaC and measured the short circuit current blocked by amiloride as an assay of ENaC activity
Together with the results of the electrophysiological assay, we conclude that proteolytically cleaved/active ENaC is rapidly removed from the cell surface and that Liddle syndrome mutations dramatically slow the rate of removal
Summary
CDNA Constructs—Human ␣, , ␥ENaC [24, 25] and Nedd4-2 [12] in pMT3 were cloned as described previously. For RNA interference experiments, cells were cotransfected with 0.27 g of each ENaC subunit and siRNA against GFP (control) or Nedd (0.2 g) [26]. ENaC subunits contained the following mutations: R566X, Y620A, P616 – 618A, or ␥T629A. 48 h after transfection, the cells were washed with phosphatebuffered saline (PBS)-CM (PBS with 1 mM MgCl2 and CaCl2) and incubated with trypsin (5 g/ml) for 5 min at 37 °C. The cells were washed three times with PBS-CM to remove trypsin, incubated at 37 °C for times between 0 and 60 min to allow endocytosis of cleaved channels, and placed on ice. Cleaved channels remaining at the cell surface were labeled with biotin, as previously described [7].
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