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EMBO J. Nov 3, 1997; 16(21): 6325–6336.
PMCID: PMC1170239

Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination.

Abstract

The epithelial Na+ channel (ENaC), composed of three subunits (alpha beta gamma), plays a critical role in salt and fluid homeostasis. Abnormalities in channel opening and numbers have been linked to several genetic disorders, including cystic fibrosis, pseudohypoaldosteronism type I and Liddle syndrome. We have recently identified the ubiquitin-protein ligase Nedd4 as an interacting protein of ENaC. Here we show that ENaC is a short-lived protein (t1/2 approximately 1 h) that is ubiquitinated in vivo on the alpha and gamma (but not beta) subunits. Mutation of a cluster of Lys residues (to Arg) at the N-terminus of gamma ENaC leads to both inhibition of ubiquitination and increased channel activity, an effect augmented by N-terminal Lys to Arg mutations in alpha ENaC, but not in beta ENaC. This elevated channel activity is caused by an increase in the number of channels present at the plasma membrane; it represents increases in both cell-surface retention or recycling of ENaC and incorporation of new channels at the plasma membrane, as determined by Brefeldin A treatment. In addition, we find that the rapid turnover of the total pool of cellular ENaC is attenuated by inhibitors of both the proteasome and the lysosomal/endosomal degradation systems, and propose that whereas the unassembled subunits are degraded by the proteasome, the assembled alpha beta gamma ENaC complex is targeted for lysosomal degradation. Our results suggest that ENaC function is regulated by ubiquitination, and propose a paradigm for ubiquitination-mediated regulation of ion channels.

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Selected References

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  • Andersson S, Davis DL, Dahlbäck H, Jörnvall H, Russell DW. Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem. 1989 May 15;264(14):8222–8229. [PubMed]
  • Botero-Velez M, Curtis JJ, Warnock DG. Brief report: Liddle's syndrome revisited--a disorder of sodium reabsorption in the distal tubule. N Engl J Med. 1994 Jan 20;330(3):178–181. [PubMed]
  • Brodsky JL, McCracken AA. ER-associated and proteasomemediated protein degradation: how two topologically restricted events came together. Trends Cell Biol. 1997 Apr;7(4):151–156. [PubMed]
  • Canessa CM, Horisberger JD, Rossier BC. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature. 1993 Feb 4;361(6411):467–470. [PubMed]
  • Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, Rossier BC. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature. 1994 Feb 3;367(6462):463–467. [PubMed]
  • Canessa CM, Merillat AM, Rossier BC. Membrane topology of the epithelial sodium channel in intact cells. Am J Physiol. 1994 Dec;267(6 Pt 1):C1682–C1690. [PubMed]
  • Cenciarelli C, Hou D, Hsu KC, Rellahan BL, Wiest DL, Smith HT, Fried VA, Weissman AM. Activation-induced ubiquitination of the T cell antigen receptor. Science. 1992 Aug 7;257(5071):795–797. [PubMed]
  • Chang SS, Grunder S, Hanukoglu A, Rösler A, Mathew PM, Hanukoglu I, Schild L, Lu Y, Shimkets RA, Nelson-Williams C, et al. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet. 1996 Mar;12(3):248–253. [PubMed]
  • Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell. 1994 Oct 7;79(1):13–21. [PubMed]
  • Deshaies RJ. Make it or break it: the role of ubiquitin-dependent proteolysis in cellular regulation. Trends Cell Biol. 1995 Nov;5(11):428–434. [PubMed]
  • Duc C, Farman N, Canessa CM, Bonvalet JP, Rossier BC. Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J Cell Biol. 1994 Dec;127(6 Pt 2):1907–1921. [PMC free article] [PubMed]
  • Egner R, Kuchler K. The yeast multidrug transporter Pdr5 of the plasma membrane is ubiquitinated prior to endocytosis and degradation in the vacuole. FEBS Lett. 1996 Jan 8;378(2):177–181. [PubMed]
  • Fenteany G, Standaert RF, Lane WS, Choi S, Corey EJ, Schreiber SL. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science. 1995 May 5;268(5211):726–731. [PubMed]
  • Firsov D, Schild L, Gautschi I, Mérillat AM, Schneeberger E, Rossier BC. Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach. Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15370–15375. [PMC free article] [PubMed]
  • Galan JM, Moreau V, Andre B, Volland C, Haguenauer-Tsapis R. Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease. J Biol Chem. 1996 May 3;271(18):10946–10952. [PubMed]
  • Galcheva-Gargova Z, Theroux SJ, Davis RJ. The epidermal growth factor receptor is covalently linked to ubiquitin. Oncogene. 1995 Dec 21;11(12):2649–2655. [PubMed]
  • Garty H, Palmer LG. Epithelial sodium channels: function, structure, and regulation. Physiol Rev. 1997 Apr;77(2):359–396. [PubMed]
  • Geering K, Beggah A, Good P, Girardet S, Roy S, Schaer D, Jaunin P. Oligomerization and maturation of Na,K-ATPase: functional interaction of the cytoplasmic NH2 terminus of the beta subunit with the alpha subunit. J Cell Biol. 1996 Jun;133(6):1193–1204. [PMC free article] [PubMed]
  • Hansson JH, Nelson-Williams C, Suzuki H, Schild L, Shimkets R, Lu Y, Canessa C, Iwasaki T, Rossier B, Lifton RP. Hypertension caused by a truncated epithelial sodium channel gamma subunit: genetic heterogeneity of Liddle syndrome. Nat Genet. 1995 Sep;11(1):76–82. [PubMed]
  • Hansson JH, Schild L, Lu Y, Wilson TA, Gautschi I, Shimkets R, Nelson-Williams C, Rossier BC, Lifton RP. A de novo missense mutation of the beta subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11495–11499. [PMC free article] [PubMed]
  • Hein C, Springael JY, Volland C, Haguenauer-Tsapis R, André B. NPl1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin-protein ligase. Mol Microbiol. 1995 Oct;18(1):77–87. [PubMed]
  • Hicke L, Riezman H. Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell. 1996 Jan 26;84(2):277–287. [PubMed]
  • Hilt W, Wolf DH. Proteasomes: destruction as a programme. Trends Biochem Sci. 1996 Mar;21(3):96–102. [PubMed]
  • Hirt RP, Poulain-Godefroy O, Billotte J, Kraehenbuhl JP, Fasel N. Highly inducible synthesis of heterologous proteins in epithelial cells carrying a glucocorticoid-responsive vector. Gene. 1992 Feb 15;111(2):199–206. [PubMed]
  • Hochstrasser M. Protein degradation or regulation: Ub the judge. Cell. 1996 Mar 22;84(6):813–815. [PubMed]
  • Hummler E, Barker P, Gatzy J, Beermann F, Verdumo C, Schmidt A, Boucher R, Rossier BC. Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice. Nat Genet. 1996 Mar;12(3):325–328. [PubMed]
  • Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, Riordan JR. Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell. 1995 Oct 6;83(1):129–135. [PubMed]
  • Jentsch S, Schlenker S. Selective protein degradation: a journey's end within the proteasome. Cell. 1995 Sep 22;82(6):881–884. [PubMed]
  • Kölling R, Hollenberg CP. The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants. EMBO J. 1994 Jul 15;13(14):3261–3271. [PMC free article] [PubMed]
  • Lingueglia E, Voilley N, Waldmann R, Lazdunski M, Barbry P. Expression cloning of an epithelial amiloride-sensitive Na+ channel. A new channel type with homologies to Caenorhabditis elegans degenerins. FEBS Lett. 1993 Feb 22;318(1):95–99. [PubMed]
  • Lingueglia E, Renard S, Waldmann R, Voilley N, Champigny G, Plass H, Lazdunski M, Barbry P. Different homologous subunits of the amiloride-sensitive Na+ channel are differently regulated by aldosterone. J Biol Chem. 1994 May 13;269(19):13736–13739. [PubMed]
  • McDonald FJ, Snyder PM, McCray PB, Jr, Welsh MJ. Cloning, expression, and tissue distribution of a human amiloride-sensitive Na+ channel. Am J Physiol. 1994 Jun;266(6 Pt 1):L728–L734. [PubMed]
  • McDonald FJ, Price MP, Snyder PM, Welsh MJ. Cloning and expression of the beta- and gamma-subunits of the human epithelial sodium channel. Am J Physiol. 1995 May;268(5 Pt 1):C1157–C1163. [PubMed]
  • Miyazawa K, Toyama K, Gotoh A, Hendrie PC, Mantel C, Broxmeyer HE. Ligand-dependent polyubiquitination of c-kit gene product: a possible mechanism of receptor down modulation in M07e cells. Blood. 1994 Jan 1;83(1):137–145. [PubMed]
  • Mori S, Heldin CH, Claesson-Welsh L. Ligand-induced polyubiquitination of the platelet-derived growth factor beta-receptor. J Biol Chem. 1992 Mar 25;267(9):6429–6434. [PubMed]
  • Nelson RM, Long GL. A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Anal Biochem. 1989 Jul;180(1):147–151. [PubMed]
  • O'Brodovich HM. The role of active Na+ transport by lung epithelium in the clearance of airspace fluid. New Horiz. 1995 May;3(2):240–247. [PubMed]
  • Palombella VJ, Rando OJ, Goldberg AL, Maniatis T. The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B. Cell. 1994 Sep 9;78(5):773–785. [PubMed]
  • Paolini R, Kinet JP. Cell surface control of the multiubiquitination and deubiquitination of high-affinity immunoglobulin E receptors. EMBO J. 1993 Feb;12(2):779–786. [PMC free article] [PubMed]
  • Puoti A, May A, Canessa CM, Horisberger JD, Schild L, Rossier BC. The highly selective low-conductance epithelial Na channel of Xenopus laevis A6 kidney cells. Am J Physiol. 1995 Jul;269(1 Pt 1):C188–C197. [PubMed]
  • Renard S, Lingueglia E, Voilley N, Lazdunski M, Barbry P. Biochemical analysis of the membrane topology of the amiloride-sensitive Na+ channel. J Biol Chem. 1994 Apr 29;269(17):12981–12986. [PubMed]
  • Rotin D, Bar-Sagi D, O'Brodovich H, Merilainen J, Lehto VP, Canessa CM, Rossier BC, Downey GP. An SH3 binding region in the epithelial Na+ channel (alpha rENaC) mediates its localization at the apical membrane. EMBO J. 1994 Oct 3;13(19):4440–4450. [PMC free article] [PubMed]
  • Saumon G, Basset G. Electrolyte and fluid transport across the mature alveolar epithelium. J Appl Physiol (1985) 1993 Jan;74(1):1–15. [PubMed]
  • Scheffner M, Huibregtse JM, Vierstra RD, Howley PM. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell. 1993 Nov 5;75(3):495–505. [PubMed]
  • Schild L, Canessa CM, Shimkets RA, Gautschi I, Lifton RP, Rossier BC. A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5699–5703. [PMC free article] [PubMed]
  • Schild L, Lu Y, Gautschi I, Schneeberger E, Lifton RP, Rossier BC. Identification of a PY motif in the epithelial Na channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome. EMBO J. 1996 May 15;15(10):2381–2387. [PMC free article] [PubMed]
  • Schild L, Schneeberger E, Gautschi I, Firsov D. Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation. J Gen Physiol. 1997 Jan;109(1):15–26. [PMC free article] [PubMed]
  • Shimkets RA, Warnock DG, Bositis CM, Nelson-Williams C, Hansson JH, Schambelan M, Gill JR, Jr, Ulick S, Milora RV, Findling JW, et al. Liddle's syndrome: heritable human hypertension caused by mutations in the beta subunit of the epithelial sodium channel. Cell. 1994 Nov 4;79(3):407–414. [PubMed]
  • Snyder PM, McDonald FJ, Stokes JB, Welsh MJ. Membrane topology of the amiloride-sensitive epithelial sodium channel. J Biol Chem. 1994 Sep 30;269(39):24379–24383. [PubMed]
  • Snyder PM, Price MP, McDonald FJ, Adams CM, Volk KA, Zeiher BG, Stokes JB, Welsh MJ. Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na+ channel. Cell. 1995 Dec 15;83(6):969–978. [PubMed]
  • Staub O, Dho S, Henry P, Correa J, Ishikawa T, McGlade J, Rotin D. WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome. EMBO J. 1996 May 15;15(10):2371–2380. [PMC free article] [PubMed]
  • Strautnieks SS, Thompson RJ, Gardiner RM, Chung E. A novel splice-site mutation in the gamma subunit of the epithelial sodium channel gene in three pseudohypoaldosteronism type 1 families. Nat Genet. 1996 Jun;13(2):248–250. [PubMed]
  • Strous GJ, van Kerkhof P, Govers R, Ciechanover A, Schwartz AL. The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor. EMBO J. 1996 Aug 1;15(15):3806–3812. [PMC free article] [PubMed]
  • Stutts MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier BC, Boucher RC. CFTR as a cAMP-dependent regulator of sodium channels. Science. 1995 Aug 11;269(5225):847–850. [PubMed]
  • Tamura H, Schild L, Enomoto N, Matsui N, Marumo F, Rossier BC. Liddle disease caused by a missense mutation of beta subunit of the epithelial sodium channel gene. J Clin Invest. 1996 Apr 1;97(7):1780–1784. [PMC free article] [PubMed]
  • Treier M, Staszewski LM, Bohmann D. Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain. Cell. 1994 Sep 9;78(5):787–798. [PubMed]
  • Voilley N, Lingueglia E, Champigny G, Mattéi MG, Waldmann R, Lazdunski M, Barbry P. The lung amiloride-sensitive Na+ channel: biophysical properties, pharmacology, ontogenesis, and molecular cloning. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):247–251. [PMC free article] [PubMed]
  • Ward CL, Omura S, Kopito RR. Degradation of CFTR by the ubiquitin-proteasome pathway. Cell. 1995 Oct 6;83(1):121–127. [PubMed]

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