Nedd4-2 induces endocytosis and degradation of proteolytically cleaved epithelial Na+ channels.
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.
Kabra
Rajesh
R
Departments of Internal Medicine and Molecular Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242, USA.
Knight
Kristin K
KK
Zhou
Ruifeng
R
Snyder
Peter M
PM
D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D002460:Cell Line; D004705:Endocytosis / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D000075702:Nedd4 Ubiquitin Protein Ligases; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D051381:Rats; D011916:Rats, Inbred F344; D012964:Sodium / Q000378:metabolism; D013961:Thyroid Gland / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D012964:Sodium; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases