Trafficking and Cell Surface Stability of the Epithelial Na+ Channel Expressed in Epithelial Madin-Darby Canine Kidney Cells

The apically located epithelial Na+ channel (αβγ-ENaC) plays a key role in the regulation of salt and fluid transport in the kidney and other epithelia, yet its mode of trafficking to the plasma membrane and its cell surface stability in mammalian cells are poorly understood. Because the expression of ENaC in native tissues/cells is very low, we generated epithelial Madin-Darby canine kidney (MDCK) cells stably expressing αβγ-ENaC, where each subunit is tagged differentially at the intracellular C terminus and the β-subunit is also Myc-tagged at the ectodomain (αHAβMyc,T7γFLAG). ENaC expression in these cells was verified by immunoblotting with antibodies to the tags, and patch clamp analysis has confirmed that the tagged channel is functional. Moreover, using electron microscopy, we demonstrated apical, but not basal, membrane localization of ENaC in these cells. The glycosylation pattern of the intracellular pool of ENaC revealed peptide N-glycosidase F and endoglycosidase H sensitivity. Surprisingly, the cell surface pool of ENaC, analyzed by surface biotinylation, was also core glycosylated and lacked detectable endoglycosidase H-resistant channels. Extraction of the channel from cells in Triton X-100 demonstrated that both intracellular and cell surface pools of ENaC are largely soluble. Moreover, floatation assays to analyze the presence of ENaC in lipid rafts showed that both intracellular and cell surface pools of this channel are not associated with rafts. We have shown previously that the total cellular pool of ENaC is turned over rapidly (t 1/2 ∼ 1–2 h). Using cycloheximide treatment and surface biotinylation we now demonstrate that the cell surface pool of ENaC has a similarly short half-life (t 1/2 ∼1 h), unlike the long half-life reported recently for the Xenopus A6 cells. Collectively, these results help elucidate key aspects of ENaC trafficking and turnover rates in mammalian kidney epithelial cells. The apically located epithelial Na+ channel (αβγ-ENaC) plays a key role in the regulation of salt and fluid transport in the kidney and other epithelia, yet its mode of trafficking to the plasma membrane and its cell surface stability in mammalian cells are poorly understood. Because the expression of ENaC in native tissues/cells is very low, we generated epithelial Madin-Darby canine kidney (MDCK) cells stably expressing αβγ-ENaC, where each subunit is tagged differentially at the intracellular C terminus and the β-subunit is also Myc-tagged at the ectodomain (αHAβMyc,T7γFLAG). ENaC expression in these cells was verified by immunoblotting with antibodies to the tags, and patch clamp analysis has confirmed that the tagged channel is functional. Moreover, using electron microscopy, we demonstrated apical, but not basal, membrane localization of ENaC in these cells. The glycosylation pattern of the intracellular pool of ENaC revealed peptide N-glycosidase F and endoglycosidase H sensitivity. Surprisingly, the cell surface pool of ENaC, analyzed by surface biotinylation, was also core glycosylated and lacked detectable endoglycosidase H-resistant channels. Extraction of the channel from cells in Triton X-100 demonstrated that both intracellular and cell surface pools of ENaC are largely soluble. Moreover, floatation assays to analyze the presence of ENaC in lipid rafts showed that both intracellular and cell surface pools of this channel are not associated with rafts. We have shown previously that the total cellular pool of ENaC is turned over rapidly (t 1/2 ∼ 1–2 h). Using cycloheximide treatment and surface biotinylation we now demonstrate that the cell surface pool of ENaC has a similarly short half-life (t 1/2 ∼1 h), unlike the long half-life reported recently for the Xenopus A6 cells. Collectively, these results help elucidate key aspects of ENaC trafficking and turnover rates in mammalian kidney epithelial cells. epithelial Na+ channel endoglycosidase H peptideN-glycosidase F Madin-Darby canine kidney cells hemagglutinin N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine phosphate-buffered saline transferrin receptor The amiloride-sensitive epithelial Na+ channel (ENaC)1 is an apically located channel expressed primarily in salt-transporting epithelia of the kidney (distal nephron), distal colon, lung, ducts of exocrine glands, and other organs (for review, see Ref. 1Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1036) Google Scholar). Its critical role in regulating salt and fluid transport is underscored by the findings that inactivating mutations in ENaC cause the salt-wasting nephropathy pseudohypoaldosteronism type I, and gain-of-function mutations cause Liddle syndrome, a hereditary form of hypertension (for review, see Ref. 2Lifton R.P. Gharavi A.G. Geller D.S. Cell. 2001; 104: 545-556Abstract Full Text Full Text PDF PubMed Scopus (1368) Google Scholar). Liddle syndrome is caused by mutations in one of the PY motifs (PPPxY) of ENaC, leading to increased channel numbers and opening at the plasma membrane (3Firsov D. Schild L. Gautschi I. Merillat A.M. Schneeberger E. Rossier B.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15370-15375Crossref PubMed Scopus (396) Google Scholar); the increase in channel numbers is believed to be caused by impaired binding to and suppression by the ubiquitin ligase Nedd4 (4Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (740) Google Scholar, 5Schild L. Lu Y. Gautschi I. Schneeberger E. Lifton R.P. Rossier B.C. EMBO J. 1996; 15: 2381-2387Crossref PubMed Scopus (361) Google Scholar, 6Goulet C.C. Volk K.A. Adams C.M. Prince L.S. Stokes J.B. Snyder P.M. J. Biol. Chem. 1998; 273: 30012-30017Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 7Abriel H. Loffing J. Rebhun J.F. Pratt J.H. Schild L. Horisberger J.D. Rotin D. Staub O. J. Clin. Invest. 1999; 103: 667-673Crossref PubMed Scopus (327) Google Scholar, 8Kamynina E. Debonneville C. Bens M. Vandewalle A. Staub O. FASEB J. 2001; 15: 204-214Crossref PubMed Scopus (250) Google Scholar, 9Kanelis V. Rotin D. Forman-Kay J.D. Nat. Struct. Biol. 2001; 8: 407-412Crossref PubMed Scopus (187) Google Scholar) and by impaired endocytosis of the channel (10Shimkets R.A. Lifton R.P. Canessa C.M. J. Biol. Chem. 1997; 272: 25537-25541Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). ENaC consists of three subunits, αβγ (11Canessa C.M. Horisberger J.D. Rossier B.C. Nature. 1993; 361: 467-470Crossref PubMed Scopus (827) Google Scholar, 12Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1775) Google Scholar), arranged in a stoichiometry of 2α:1β:1γ (13, 14; for another view, see Ref.15Snyder P.M. J. Biol. Chem. 1998; 273: 681-684Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Each ENaC subunit is comprised of two transmembrane domains, a large ectodomain flanking them and containing numerousN-linked glycosylation sites, and short intracellular N and C termini (16Snyder P.M. McDonald F.J. Stokes J.B. Welsh M.J. J. Biol. Chem. 1994; 269: 24379-24383Abstract Full Text PDF PubMed Google Scholar, 17Renard S. Lingueglia E. Voilley N. Lazdunski M. Barbry P. J. Biol. Chem. 1994; 269: 12981-12986Abstract Full Text PDF PubMed Google Scholar, 18Canessa C.M. Merillat A.M. Rossier B.C. Am. J. Physiol. 1994; 267: C1682-C1690Crossref PubMed Google Scholar). The N termini of and that ubiquitin O. Gautschi I. Ishikawa T. A. Schild L. Rotin D. EMBO J. 1997; PubMed Scopus Google Scholar), and the C termini of three the PY motifs (4Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (740) Google Scholar, 5Schild L. Lu Y. Gautschi I. Schneeberger E. Lifton R.P. Rossier B.C. EMBO J. 1996; 15: 2381-2387Crossref PubMed Scopus (361) Google Scholar, P.M. McDonald F.J. Adams C.M. Volk K.A. Stokes J.B. Welsh M.J. Cell. Full Text PDF PubMed Scopus Google Scholar). three ENaC are glycosylated in the role of this glycosylation is not and of glycosylation in not to channel (16Snyder P.M. McDonald F.J. Stokes J.B. Welsh M.J. J. Biol. Chem. 1994; 269: 24379-24383Abstract Full Text PDF PubMed Google Scholar, 18Canessa C.M. Merillat A.M. Rossier B.C. Am. J. Physiol. 1994; 267: C1682-C1690Crossref PubMed Google Scholar). native ENaC is a with very expression J. L. M. U. P. Rossier B.C. B. Am. J. Physiol. PubMed Google Scholar, S. C. J.B. J. Clin. Invest. 1999; 104: PubMed Scopus Google Scholar), and to be but have aspects of ENaC trafficking and stability in cell and expression primarily Xenopus A6 and mammalian expressing the ENaC for review, see Ref. D. V. Schild L. Am. J. Physiol. 2001; PubMed Google Scholar). ENaC trafficking in mammalian kidney epithelial cells stably expressing has not this was the of The ENaC to in the C.M. Snyder P.M. Welsh M.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), but of the of trafficking to the cell surface are in ENaC has the of an endoglycosidase H transmembrane that have glycosylation at the is unlike the ENaC channel in an H-resistant pool is detectable its S. Lingueglia E. Lazdunski M. Barbry P. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). an to be is ENaC not but the H-resistant pool is has that in the ENaC is of its core glycosylation and in at the plasma membrane L.S. Welsh M.J. J. 1998; PubMed Scopus Google Scholar, L.S. Welsh M.J. Am. J. Physiol. 1999; PubMed Google Scholar). O. Gautschi I. Ishikawa T. A. Schild L. Rotin D. EMBO J. 1997; PubMed Scopus Google Scholar) has demonstrated that the total cellular pool of ENaC expressed in mammalian cells has a short half-life (t 1/2 h), also shown for A6 ENaC J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. Horisberger J.D. Rossier B.C. J. Am. 1997; 8: PubMed Google Scholar). expressing ENaC, are at a this half-life is Canessa C.M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). the short half-life of the intracellular pool of ENaC in has recently that the half-life of the surface pool of ENaC in A6 cells is long for and for the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J.B. P. K.A. B. B. Am. J. Physiol. 2001; Scholar). in that the cell surface pool of ENaC is short (10Shimkets R.A. Lifton R.P. Canessa C.M. J. Biol. Chem. 1997; 272: 25537-25541Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, O. Gautschi I. Ishikawa T. A. Schild L. Rotin D. EMBO J. 1997; PubMed Scopus Google Scholar), this was not The stability of ENaC at the cell surface of mammalian epithelial for ENaC has not this we the of kidney epithelial cells stably expressing an Each subunit was tagged with a at the intracellular C and an was to the of cells ENaC at the Using this cell we that the cell ENaC is to intracellular that the channel not glycosylation trafficking to the cell We also that ENaC is not associated with lipid and its intracellular and cell surface pools are primarily soluble. Moreover, we demonstrate that unlike A6 the cell surface ENaC has a very short are for of the regulation of channel numbers at the plasma a key role in regulating ENaC and Liddle expressing ENaC generated from cells a was of the and the was a and R.P. O. J. N. PubMed Scopus Google Scholar). in the stably expressing cells a for the of was generated by an intracellular of the and a in the presence of the cells to a a was in the ectodomain and in a previously to have channel (3Firsov D. Schild L. Gautschi I. Merillat A.M. Schneeberger E. Rossier B.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15370-15375Crossref PubMed Scopus (396) Google and an intracellular was of the The tagged was the and the was the cells. the presence of and the cells The cells have previously O. Gautschi I. Ishikawa T. A. Schild L. Rotin D. EMBO J. 1997; PubMed Scopus Google Scholar). cells in containing and at in The cells in the with and and and in Triton X-100 and by at for at a (for was in and an Because the expression of the was very low, a of was and by D. PubMed Scopus Google Scholar) to a of from the cells was also the with antibodies to the and by from from cells in to and assays to and of in the of cells. assays using both with results cell from cells using the cell of the patch clamp reported previously T. Y. Rotin D. J. Physiol. 1998; PubMed Scopus Google Scholar). patch clamp was to cell The was by to the of with of the cell The patch clamp from using a of with a The was an was to the an with a using The was from to over a of amiloride-sensitive by of for the of The and The of the was with to The cells in a containing and the of cell the was to the one containing and The of the was with at by from The results are reported the of and to the of cells in the a a and the three with with and with in biotinylation with with in and for with with with in and at at in and at to and the of of of for at by and of the to the intracellular three with by in with and and cells surface by with for in the with in with for cell surface expression using the for surface biotinylation, to the of of the cell the large intracellular pool of ENaC the an was for each of cells by surface biotinylation of an intracellular of intracellular of in and to surface and and and of from the the intracellular pool of in with H peptide N-glycosidase F to the by and to for and The the with with cycloheximide for at We one for each each the surface and in for each using the and total was of for at a to the to the intracellular three with and by in containing by to and with antibodies to the by was using and using where the cells and three with surface and in and in for for at and in a of to the three for each to the J. Biol. 1999; PubMed Scopus Google Scholar). for at and the the ENaC at the cell with of by for at of to the intracellular pool and in and from the and by and to for immunoblotting and of three with in and at in of and and Triton and for cells surface to in with of and with of each and in by S. T. V. C. H. H. U. J. Biol. 1999; PubMed Scopus Google Scholar). The at in an for at from the to the of the and of the in of was and the by and analyzed by to and from and for the receptor from in the two and are to be S. T. V. C. H. H. U. J. Biol. 1999; PubMed Scopus Google Scholar). in cells surface of was to each of the and for at and three with by in with cells and three with surface was by for with antibodies in in both the and three with and with in at for three with and in in for at three with and with for in the with for in the with and using in and with by was to and at for was and for at containing and using a electron The expression of the ENaC in native kidney epithelia is very low, of the channel in these analyze ENaC in kidney epithelial we form and are with to cellular trafficking C. C. E. J. Biol. PubMed Scopus Google Scholar). has of in the not to channel T. Y. Rotin D. J. Physiol. 1998; PubMed Scopus Google Scholar), but that cells a for We tagged each ENaC with a at the C terminus of the We also a at the ectodomain of in the shown previously not to channel containing a short (3Firsov D. Schild L. Gautschi I. Merillat A.M. Schneeberger E. Rossier B.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15370-15375Crossref PubMed Scopus (396) Google Scholar). cells from the was expressed the of a and the other two expressed The tagged ENaC stably the and expression of the tagged ENaC was verified with the presence of in antibodies the the of each ENaC that the tagged ENaC is to the we the cells and with antibodies to the surface of cells. that the tagged ENaC is to the membrane of cells. was not was in the surface of the cells the tagged ENaC are in these the of ENaC expressed at the cell surface very of total ENaC The cells not detectable ENaC T. Y. Rotin D. J. Physiol. 1998; PubMed Scopus Google Scholar). the cells a channel at the plasma we the amiloride-sensitive cell in these cells. We the cell the the and the with a containing the cell the of the from its we the for the of the amiloride-sensitive cell The amiloride-sensitive cell at and and of the amiloride-sensitive from that of the amiloride-sensitive channel was the of to not be results demonstrate the presence of ENaC channel at the plasma membrane of cells. has reported previously that cellular pool of ENaC the expressed is core glycosylated with detectable H-resistant pool that transmembrane (16Snyder P.M. McDonald F.J. Stokes J.B. Welsh M.J. J. Biol. Chem. 1994; 269: 24379-24383Abstract Full Text PDF PubMed Google Scholar, 18Canessa C.M. Merillat A.M. Rossier B.C. Am. J. Physiol. 1994; 267: C1682-C1690Crossref PubMed Google Scholar, Canessa C.M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). have two the pool of ENaC is that an H-resistant is the total cellular ENaC is analyzed ENaC to the cell surface the of these we surface biotinylation cells to the cell pool of ENaC is of we the the of to the channel is each of the other D. Gautschi I. Merillat A.M. Rossier B.C. Schild L. EMBO J. 1998; PubMed Scopus Google Scholar, S. J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), and is also tagged its Because and that of (for review, see Ref. D. V. Schild L. Am. J. Physiol. 2001; PubMed Google Scholar) has shown that the pool of ENaC at the cell surface is very to the total cellular we to that of the the cells intracellular surface for each we analyzed both surface biotinylation of ENaC and biotinylation of an intracellular a for the the of the surface biotinylation of but not of intracellular expression of both cell surface biotinylation cell surface expression of ENaC was both the cells Using this cell surface biotinylation we analyzed the of the cell ENaC to and H. in the surface pool of was and to the intracellular We not to cell surface pool of this subunit is expressed at the surface The intracellular pool of was to both and H surface expression of in the cells was to we to cell surface biotinylation of in the cells demonstrated of H-resistant cell surface intracellular pools of to analysis of the pool of ENaC revealed with the that ENaC the to the plasma membrane not these results demonstrate that ENaC not its from the the to the plasma ENaC has previously to in Triton its L.S. Welsh M.J. J. 1998; PubMed Scopus Google L.S. Welsh M.J. Am. J. Physiol. 1999; PubMed Google Scholar). analyze the cell surface pool of ENaC is we of surface and intracellular pools of of ENaC in of Triton X-100 and that with the of the cellular and of the surface pool of ENaC was soluble. and Triton of the intracellular and cell surface pools of the channel in the was unlike is associated with rafts and was a that is to a the of ENaC, we its with lipid rafts. was the Nedd4 is associated with lipid and this Nedd4 to the plasma membrane in cells S. P. Rotin D. J. Biol. PubMed Scopus Google Scholar). We floatation assays S. T. V. C. H. H. U. J. Biol. 1999; PubMed Scopus Google S. P. Rotin D. J. Biol. PubMed Scopus Google Scholar, S. P. J. Biol. 1998; PubMed Scopus Google Scholar) to for the presence of ENaC in the of an that was of Triton ENaC by we and two to Biol. 1996; 8: PubMed Scopus Google Scholar, T. V. 1994; PubMed Scopus Google Scholar), and is not associated with rafts A. C.M. J. 1996; PubMed Google Scholar). in both and in lipid rafts the and and the of with the is was by the of the also the was not associated with rafts. from the total cellular pool of ENaC not in the and its that of the and not of The results the cells Moreover, that the cell surface pool of ENaC is also not associated with lipid rafts. these results that ENaC not with rafts its intracellular trafficking at the plasma O. Gautschi I. Ishikawa T. A. Schild L. Rotin D. EMBO J. 1997; PubMed Scopus Google Scholar) and that of J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. Horisberger J.D. Rossier B.C. J. Am. 1997; 8: PubMed Google Scholar) has demonstrated a short half-life of the total pool of ENaC in cells. The stability of ENaC at the cell surface where is a the half-life of ENaC at the plasma we surface ENaC cycloheximide treatment (for h), and the and of to the plasma We not to of its in the in and other kidney cells and its I. Cell. Full Text PDF PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar). that the cell surface pool of the of is short with half-life of the intracellular pool is also turned over (t 1/2 also shown for the other two ENaC and previously O. Gautschi I. Ishikawa T. A. Schild L. Rotin D. EMBO J. 1997; PubMed Scopus Google Scholar). was in the half-life of ENaC cells to (t 1/2 for cell surface and short half-life of cell surface and intracellular in the was also The regulation of ENaC trafficking and cell surface stability is of to its the that these has are to a in native have Xenopus A6 and Xenopus mammalian expressing ENaC to ENaC trafficking and this we the of a cell stably expressing ENaC, to and trafficking of ENaC in these kidney epithelial cells. ENaC glycosylation C.M. Merillat A.M. Rossier B.C. Am. J. Physiol. 1994; 267: C1682-C1690Crossref PubMed Google Scholar), are in the role of glycosylation of this channel is Moreover, that the glycosylation pattern in transmembrane the of glycosylation is not in was demonstrated by the glycosylation pattern of the cell pool of ENaC we is and have the of a detectable H-resistant pool of total cellular ENaC but ENaC of is was not to this the of a pool its presence in of the cell surface pool of ENaC by this have transmembrane trafficking to the plasma membrane the S. Biol. Cell. 1999; PubMed Scopus Google Scholar), the receptor J. Biol. PubMed Scopus Google Scholar), and the channel L. Y. 1994; PubMed Scopus Google Scholar), in the a of glycosylation was expression in Xenopus in the presence of at the cell results of core glycosylation of the ENaC are not in with of Prince and Welsh L.S. Welsh M.J. J. 1998; PubMed Scopus Google Scholar, L.S. Welsh M.J. Am. J. Physiol. 1999; PubMed Google Scholar) that ENaC is of its at the plasma We not the for the expressed in with is from at in cells expressing ENaC Canessa C.M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google is that ENaC is very with a of to the cell that of total expressed in cells is at the cell surface results that both intracellular and cell surface pools of ENaC are largely in the Triton from L.S. Welsh M.J. J. 1998; PubMed Scopus Google Scholar, L.S. Welsh M.J. Am. J. Physiol. 1999; PubMed Google Scholar), that the ENaC is the cause of was not with of of ENaC we using floatation that ENaC is not associated with rafts in Triton X-100 in the We the that in other of the channel in rafts. rafts are and in that an role in of and in E. Nature. 1997; PubMed Scopus Google Scholar). We have demonstrated previously that a binding of ENaC, rafts to to the membrane of by its with an S. P. Rotin D. J. Biol. PubMed Scopus Google Scholar, H. Staub O. P. Rotin D. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). that the ENaC and Nedd4 at the plasma and that Nedd4 is not in trafficking of ENaC to the plasma membrane localization of ENaC is not that the of C terminus with ENaC at the membrane of epithelial cells D. D. H. J. Canessa C.M. Rossier B.C. EMBO J. 1994; PubMed Scopus Google Scholar). an is not for membrane of the C termini of ENaC not with plasma membrane localization of the have the in regulating ENaC transport to the cell surface J. C. R.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), but the regulation of this is not The stability of ENaC at the cell surface has critical for its and Liddle syndrome mutations in the PY motifs of are associated with increased channel at the plasma membrane C.C. Volk K.A. Adams C.M. Prince L.S. Stokes J.B. Snyder P.M. J. Biol. Chem. 1998; 273: 30012-30017Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 7Abriel H. Loffing J. Rebhun J.F. Pratt J.H. Schild L. Horisberger J.D. Rotin D. Staub O. J. Clin. Invest. 1999; 103: 667-673Crossref PubMed Scopus (327) Google Scholar, 8Kamynina E. Debonneville C. Bens M. Vandewalle A. Staub O. FASEB J. 2001; 15: 204-214Crossref PubMed Scopus (250) Google Scholar, R.A. Lifton R.P. Canessa C.M. J. Biol. Chem. 1997; 272: 25537-25541Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). The cell surface stability of ENaC is by O. Gautschi I. Ishikawa T. A. Schild L. Rotin D. EMBO J. 1997; PubMed Scopus Google Scholar) and the ubiquitin ligase Nedd4 (4Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (740) Google Scholar, 6Goulet C.C. Volk K.A. Adams C.M. Prince L.S. Stokes J.B. Snyder P.M. J. Biol. Chem. 1998; 273: 30012-30017Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 7Abriel H. Loffing J. Rebhun J.F. Pratt J.H. Schild L. Horisberger J.D. Rotin D. Staub O. J. Clin. Invest. 1999; 103: 667-673Crossref PubMed Scopus (327) Google Scholar, 8Kamynina E. Debonneville C. Bens M. Vandewalle A. Staub O. FASEB J. 2001; 15: 204-214Crossref PubMed Scopus (250) Google Scholar). the cell surface stability of the channel in epithelial cells is very results that the cell surface pool of ENaC is turned over with a half-life of ∼1 is in with in that rapidly from the plasma membrane transport of transmembrane from the (10Shimkets R.A. Lifton R.P. Canessa C.M. J. Biol. Chem. 1997; 272: 25537-25541Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, O. Gautschi I. Ishikawa T. A. Schild L. Rotin D. EMBO J. 1997; PubMed Scopus Google Scholar), channel numbers not analyzed to of a short half-life of the cell surface pool of ENaC, in A6 cells that is very at the plasma membrane (t 1/2 but for J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J.B. P. K.A. B. B. Am. J. Physiol. 2001; Scholar). A6 cells at a mammalian cells this half-life is long to we in cells in Xenopus O. Gautschi I. Ishikawa T. A. Schild L. Rotin D. EMBO J. 1997; PubMed Scopus Google Scholar), at was by using antibodies J.B. P. K.A. B. B. Am. J. Physiol. 2001; Scholar) to from A6 cells. The to three at and The was to be a of the glycosylated and its half-life at the cell surface was shown to be J.B. P. K.A. B. B. Am. J. Physiol. 2001; Scholar). long half-life to the ∼1 reported that the mode of regulation of the channel in A6 cells be from that in mammalian epithelial the of in regulation is the mode of trafficking and cell surface stability of ENaC expressed in mammalian kidney epithelial cells.

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