Enac degradation in A6 cells by the ubiquitin-proteosome proteolytic pathway.
Amiloride-sensitive epithelial Na(+) channels (ENaC) are responsible for trans-epithelial Na(+) transport in the kidney, lung, and colon. The channel consists of three subunits (alpha, beta, gamma) each containing a proline rich region (PPXY) in their carboxyl-terminal end. Mutations in this PPXY domain cause Liddle's syndrome, an autosomal dominant, salt-sensitive hypertension, by preventing the channel's interactions with the ubiquitin ligase Neural precursor cell-expressed developmentally down-regulated protein (Nedd4). It is postulated that this results in defective endocytosis and lysosomal degradation of ENaC leading to an increase in ENaC activity. To show the pathway that degrades ENaC in epithelial cells that express functioning ENaC channels, we used inhibitors of the proteosome and measured sodium channel activity. We found that the inhibitor, MG-132, increases amiloride-sensitive trans-epithelial current in Xenopus distal nephron A6 cells. There also is an increase of total cellular as well as membrane-associated ENaC subunit molecules by Western blotting. MG-132-treated cells also have increased channel density in patch clamp experiments. Inhibitors of lysosomal function did not reproduce these findings. Our results suggest that in native renal cells the proteosomal pathway is an important regulator of ENaC function.
Department of Physiology and Renal Division, Emory University, Atlanta, Georgia 30322, USA. bmalik@ccms-renal.physio.emory.edu
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B
Malik
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Schlanger
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Al-Khalili
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Bao
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Yue
S R
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Price
W E
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Mitch
D C
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Eaton
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053503:Epithelial Sodium Channels; D006801:Humans; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008954:Models, Biological; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D009399:Nephrons; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*; D019459:Urothelium / Q000166:cytology / Q000502:physiology; D014982:Xenopus laevis
D015853:Cysteine Proteinase Inhibitors; D053503:Epithelial Sodium Channels; D007976:Leupeptins; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D021122:Protein Subunits; D015222:Sodium Channels; D014452:Ubiquitins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde