Title: Nedd4-2 Induces Endocytosis and Degradation of Proteolytically Cleaved Epithelial Na+ Channels
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 α, β, and γENaC 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 αENaC. 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. 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 α, β, and γENaC 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 αENaC. 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. The epithelial Na+ channel ENaC forms a pathway for Na+ reabsorption across epithelia, including the kidney, lung, and colon. Therefore, it plays a critical role in Na+ homeostasis and blood pressure control (reviewed in Refs. 1Schild L. Rev. Physiol. Biochem. Pharmacol. 2004; 151: 93-107Crossref PubMed Scopus (101) Google Scholar and 2Snyder P.M. Endocrinology. 2005; 146: 5079-5085Crossref PubMed Scopus (196) Google Scholar). Defects in ENaC function or regulation cause inherited forms of hypertension and hypotension (3Lifton R.P. Science. 1996; 272: 676-680Crossref PubMed Scopus (563) Google Scholar) and may contribute to the pathogenesis of lung disease in cystic fibrosis (4Boucher R.C. Stutts M.J. Knowles M.R. Cantley L. Gatzy J.T. J. Clin. Investig. 1986; 78: 1245-1252Crossref PubMed Scopus (445) Google Scholar). ENaC is regulated by Nedd4-2, a HECT domain E3 ubiquitin ligase that decreases ENaC expression at the cell surface (5Kamynina E. Debonneville C. Bens M. Vandewalle A. Staub O. FASEB J. 2001; 15: 204-214Crossref PubMed Scopus (250) Google Scholar, 6Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster C. Chraibi A. Pratt J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (579) Google Scholar, 7Knight K.K. Olson D.R. Zhou R. Snyder P.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2805-2808Crossref PubMed Scopus (125) Google Scholar). This regulation requires the binding of Nedd4-2 WW domains to PY motifs (PPPXYXXL) located in the C terminus of each of the three subunits that form the channel (α, β, and γENaC). Mutations in the PY motifs of β or γENaC disrupt binding, causing Liddle syndrome (8Snyder P.M. Price M.P. McDonald F.J. Adams C.M. Volk K.A. Zeiher B.G. Stokes J.B. Welsh M.J. Cell. 1995; 83: 969-978Abstract Full Text PDF PubMed Scopus (398) Google Scholar, 9Schild L. Lu Y. Gautschi I. Schneeberger E. Lifton R.P. Rossier B.C. EMBO J. 1996; 15: 2381-2387Crossref PubMed Scopus (361) Google Scholar, 10Goulet 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). In this inherited form of hypertension, increased expression of ENaC at the cell surface results in excessive renal Na+ reabsorption (7Knight K.K. Olson D.R. Zhou R. Snyder P.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2805-2808Crossref PubMed Scopus (125) Google Scholar, 8Snyder P.M. Price M.P. McDonald F.J. Adams C.M. Volk K.A. Zeiher B.G. Stokes J.B. Welsh M.J. Cell. 1995; 83: 969-978Abstract Full Text PDF PubMed Scopus (398) Google Scholar, 11Firsov 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). Binding is also modulated by aldosterone and vasopressin via serum and glucocorticoid-regulated kinase and protein kinase A, respectively; both kinases phosphorylate Nedd4-2, which reduces its binding to ENaC (6Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster C. Chraibi A. Pratt J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (579) Google Scholar, 12Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 13Snyder P.M. Olson D.R. Kabra R. Zhou R. Steines J.C. J. Biol. Chem. 2004; 279: 45753-45758Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). However, the mechanism by which Nedd4-2 reduces ENaC surface expression is uncertain. It is possible that Nedd4-2 regulates ENaC trafficking in the biosynthetic pathway, targeting it for degradation in the proteasome. Consistent with this model, localization of Nedd4-2 at the cell surface is not required for Nedd4-2 to inhibit ENaC (14Itani O.A. Stokes J.B. Thomas C.P. Am. J. Physiol. 2005; 289: F334-F346Crossref PubMed Scopus (51) Google Scholar). Moreover, proteasome inhibitors decrease ENaC degradation (15Staub O. Gautschi I. Ishikawa T. Breitschopf K. Ciechanover A. Schild L. Rotin D. EMBO J. 1997; 16: 6325-6336Crossref PubMed Scopus (597) Google Scholar, 16Malik B. Schlanger L. Al-Khalili O. Bao H.F. Yue G. Price S.R. Mitch W.E. Eaton D.C. J. Biol. Chem. 2001; 276: 12903-12910Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 17Malik B. Yue Q. Yue G. Chen X. Price S.R. Mitch W.E. Eaton D.C. Am. J. Physiol. 2005; 289: F107-F116Crossref PubMed Scopus (54) Google Scholar) and increase ENaC surface expression. 2R. Zhou and P. M. Snyder, unpublished observation.2R. Zhou and P. M. Snyder, unpublished observation. Alternatively, Nedd4-2 could regulate ENaC in the endocytic pathway, altering ENaC endocytosis and/or targeting to lysosomes for degradation. This model is suggested by recent data from our laboratory and others indicating that Nedd4-2 binds to ENaC at the cell surface, where it catalyzes ubiquitination of each ENaC subunit (18Zhou R. Patel S.V. Snyder P.M. J. Biol. Chem. 2007; 282: 20207-20212Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 19Wiemuth D. Ke Y. Rohlfs M. McDonald F.J. Biochem. J. 2007; 405: 147-155Crossref PubMed Scopus (54) Google Scholar). In this work, we investigated the mechanism(s) by which Nedd4-2 reduces ENaC surface expression. To overcome technical hurdles that have hindered progress in this area, we developed a novel strategy; we took advantage of the observation that ENaC is activated by proteolytic cleavage at specific sites in the extracellular domains of the α and γ subunits (20Chraibi A. Vallet V. Firsov D. Hess S.K. Horisberger J.D. J. Gen. Physiol. 1998; 111: 127-138Crossref PubMed Scopus (169) Google Scholar, 21Kleyman T.R. Myerburg M.M. Hughey R.P. Kidney Int. 2006; 70: 1391-1392Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 22Hughey R.P. Carattino M.D. Kleyman T.R. Curr. Opin. Nephrol. Hypertens. 2007; 16: 444-450Crossref PubMed Scopus (67) Google Scholar, 23Caldwell R.A. Boucher R.C. Stutts M.J. Am. J. Physiol. 2004; 286: C190-C194Crossref PubMed Scopus (169) Google Scholar). Using electrophysiological and biochemical assays, we tested the effect of Nedd4-2 on endocytosis and degradation of proteolytically cleaved channels. cDNA Constructs—Human α, β, γENaC (24McDonald F.J. Snyder P.M. McCray Jr., P.B. Welsh M.J. Am. J. Physiol. 1994; 266: L728-L734Crossref PubMed Google Scholar, 25McDonald F.J. Price M.P. Snyder P.M. Welsh M.J. Am. J. Physiol. 1995; 268: C1157-C1163Crossref PubMed Google Scholar) and Nedd4-2 (12Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar) in pMT3 were cloned as described previously. Mutations were generated by site-directed mutagenesis (QuikChange; Stratagene). α-FLAG was generated by insertion of a FLAG epitope (DYKDDDDK) at the C terminus (7Knight K.K. Olson D.R. Zhou R. Snyder P.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2805-2808Crossref PubMed Scopus (125) Google Scholar). Small interfering RNA (siRNA) 3The abbreviations used are:siRNAsmall interfering RNAFRTFischer rat thyroidGFPgreen fluorescent proteinPBSphosphate-buffered salineHEKhuman embryonic kidney. against Nedd4-2 was obtained from Qiagen and characterized previously (26Snyder P.M. Steines J.C. Olson D.R. J. Biol. Chem. 2004; 279: 5042-5046Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). small interfering RNA Fischer rat thyroid green fluorescent protein phosphate-buffered saline human embryonic kidney. Electrophysiological Endocytosis Assay—Fischer rat thyroid (FRT) cells cultured on permeable filter supports were transfected (TFX50; Promega) (12Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar) with the following human ENaC subunits; α or αCl-1 (R177A, R178A), β, and γ or γCl (R135A, R137A, R138A, R178W, R180A, K181A). For overexpression experiments, the cells were cotransfected with 0.23 μg of each ENaC subunit along with either green fluorescent protein (GFP, control) or Nedd4-2 (0.3 μg). For RNA interference experiments, cells were cotransfected with 0.27 μg of each ENaC subunit and siRNA against GFP (control) or Nedd4-2 (0.2 μg) (26Snyder P.M. Steines J.C. Olson D.R. J. Biol. Chem. 2004; 279: 5042-5046Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). In some studies, ENaC subunits contained the following mutations: αY644A, βR566X, βY620A, βP616–618A, γY627A, or γT629A. Following transfection, the cells were cultured for 48 h as described previously (12Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Short circuit current was measured using modified Ussing chambers (Warner Instrument Corp.). The apical and basolateral surfaces were bathed in 135 mm NaCl, 1.2 mm CaCl2, 1.2 mm MgCl2, 2.4 mm K2HPO4, 0.6 mm KH2PO4, 10 mm HEPES, pH 7.4, at 37 °C. Amiloride-sensitive short circuit current was determined as the current difference with and without amiloride (10 μm) in the apical bathing solution. To proteolytically cleave and activate ENaC at the cell surface, trypsin (10 μg/ml) was added to the apical membrane for 10 min and then removed by vigorous washes with ∼12 times the volume of the apical chamber. Amiloride-sensitive current was measured every 5 min by addition of amiloride (10 μm) to the apical membrane followed by wash out of amiloride. Biochemical Endocytosis Assay—HEK 293T cells were transfected (Lipofectamine 2000; Invitrogen) (18Zhou R. Patel S.V. Snyder P.M. J. Biol. Chem. 2007; 282: 20207-20212Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) with αCl-2-FLAG (R175A, R177A, R178A, R181A, R190A, R192A, R201A, R204A, C-terminal FLAG epitope), β, and γENaC. ENaC was cotransfected with cDNA encoding Nedd4-2, dominant negative Nedd4-2 (corresponding to amino acids 1 and 60–479 with Ser-221, Thr-246, and Ser-327 phosphorylation sites mutated to Ala), or GFP; or with siRNA against Nedd4-2 or GFP (quantities indicated in the figure legends). In some experiments, ENaC subunits contained the following mutations: βR566X, βY620A, βP616–618A, or γT629A. The cells were cultured in Dulbecco's modified Eagle's medium containing 10 μm amiloride. 48 h after transfection, the cells were washed with phosphate-buffered saline (PBS)-CM (PBS with 1 mm MgCl2 and CaCl2) and then incubated with trypsin (5 μg/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 then placed on ice. Cleaved channels remaining at the cell surface were labeled with biotin, as previously described (7Knight K.K. Olson D.R. Zhou R. Snyder P.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2805-2808Crossref PubMed Scopus (125) Google Scholar). Briefly, the cells were incubated with 0.5 mg/ml Sulfo-NHS-biotin (Pierce) in PBS-CM for 30 min on ice and then quenched with 100 mm glycine in PBS-CM for 10 min on ice. The cells were lysed in Nonidet P-40 lysis buffer (0.4% sodium deoxycholate, 1% Nonidet P-40, 63 mm EDTA, 50 mm Tris-HCl, pH 8, and protease inhibitor mixture). Biotinylated (cell surface) proteins were isolated by incubating the cell lysate with immobilized NeutrAvidin beads (Pierce) for 12 h at 4 °C. Following separation by SDS-PAGE, biotinylated αENaC was detected by immunoblot with anti-FLAG M2 monoclonal antibody-peroxidase conjugate and quantitated by densitometry. Degradation Assay—HEK 293T cells were transfected with α-FLAG, β, and γENaC with Nedd4-2 or GFP. 48 h later, cells were biotinylated as above and then incubated at 37 °C for times between 0 and 80 min. Following lysis of the cells in Nonidet P-40 lysis buffer, biotinylated αENaC was isolated with NeutrAvidin beads, separated by SDS-PAGE, and detected by immunoblot (anti-FLAG M2 monoclonal antibody-peroxidase conjugate). Proteolytic Cleavage of Cell Surface ENaC—In Fig. 1A, we transfected FRT epithelia with α, β, and γENaC and measured the short circuit current blocked by amiloride as an assay of ENaC activity. ENaC activity requires proteolytic cleavage of the extracellular domains of the α and γ subunits. The basal amiloride-sensitive current (Fig. 1A) reflects ENaC channels cleaved in the biosynthetic pathway (27Hughey R.P. Bruns J.B. Kinlough C.L. Harkleroad K.L. Tong Q. Carattino M.D. Johnson J.P. Stockand J.D. Kleyman T.R. J. Biol. Chem. 2004; 279: 18111-18114Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 28Hughey R.P. Bruns J.B. Kinlough C.L. Kleyman T.R. J. Biol. Chem. 2004; 279: 48491-48494Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Addition of trypsin to the extracellular solution increased amiloride-sensitive current 2-fold, reflecting acute proteolytic cleavage/activation of a pool of uncleaved (inactive) channels at the cell surface (Fig. 1A). This finding is consistent with previous work (7Knight K.K. Olson D.R. Zhou R. Snyder P.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2805-2808Crossref PubMed Scopus (125) Google Scholar, 23Caldwell R.A. Boucher R.C. Stutts M.J. Am. J. Physiol. 2004; 286: C190-C194Crossref PubMed Scopus (169) Google Scholar, 27Hughey R.P. Bruns J.B. Kinlough C.L. Harkleroad K.L. Tong Q. Carattino M.D. Johnson J.P. Stockand J.D. Kleyman T.R. J. Biol. Chem. 2004; 279: 18111-18114Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). In Fig. 1B, we mutated a consensus site for cleavage by furin in αENaC (αCl; R177A/178A), and in γENaC we mutated sites (γCl) for furin (R135/137/138A) and CAP1 (R178W, K179A, R180A, K181A) (27Hughey R.P. Bruns J.B. Kinlough C.L. Harkleroad K.L. Tong Q. Carattino M.D. Johnson J.P. Stockand J.D. Kleyman T.R. J. Biol. Chem. 2004; 279: 18111-18114Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 29Bruns J.B. Carattino M.D. Sheng S. Maarouf A.B. Weisz O.A. Pilewski J.M. Hughey R.P. Kleyman T.R. J. Biol. Chem. 2007; 282: 6153-6160Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). By preventing cleavage, these mutations nearly abolished basal amiloride-sensitive current. However, trypsin activated the mutant channel, generating a large amiloride-sensitive current. This presumably occurred via trypsin cleavage at remaining Arg or Lys residues adjacent to the mutated furin and CAP1 sites. Quantitation of Endocytosis of Proteolytically Cleaved ENaC—Expression of the αClβγCl mutant ENaC in FRT cells provided a strategy to measure the rate of ENaC endocytosis (Fig. 1C). A pool of channels at the cell surface is proteolytically cleaved and activated with a brief exposure to trypsin. Over time, cleaved/active channels are removed from the cell surface by endocytosis, resulting in decreased amiloride-sensitive current. The rate of decrease in current reflects the rate of ENaC endocytosis. During this time course, newly synthesized channels also undergo exocytosis, but they are uncleaved/inactive and therefore do not contribute significantly to the current. Fig. 2A shows a representative current trace. Trypsin activated a large amiloride-sensitive current (arrowheads indicate brief addition of amiloride to quantitate ENaC current). Following removal of trypsin, amiloride-sensitive current decreased over time. Average data are shown in Fig. 2C, which is a plot of amiloride-sensitive current (relative to current immediately after trypsin removal) versus time after trypsin removal (error bars are hidden by the symbols), and data at 15 min are shown in Table 1. Current decreased rapidly over the first 15 min, followed by a much slower decrease. This suggests that cleaved ENaC is rapidly removed from the cell surface. The slower phase could represent a second more stable population of channels or could reflect recycling of cleaved channels back to the cell surface, which would counter the decrease in current caused by endocytosis. Interestingly, re-addition of trypsin to the bathing solution generated a large increase in amiloride-sensitive current. This likely reflects previously uncleaved/inactive channels inserted into the plasma membrane during the 45-min interval following the initial trypsin exposure.TABLE 1ENaC endocytosisGroupI/I0 (15 min)pCl/Cl0 (15 min)p αβγ0.60 ± 0.010.48 ± 0.1 αβR566Xγ0.78 ± 0.024 × 10-60.99 ± 0.080.01 αβY620Aγ0.76 ± 0.013 × 10-110.83 ± 0.090.04 αY644Aβγ0.70 ± 0.012 × 10-3 αβγY627A0.76 ± 0.025 × 10-4 αβP616-618Aγ0.69 ± 0.014 × 10-50.83 ± 0.030.04 αβγT629A0.55 ± 0.0050.40.40 ± 0.140.4GroupI/I0 (15 min)pCl/Cl0 (15 min)p Control0.55 ± 0.0050.48 ± 0.1 Nedd4-2 siRNA0.68 ± 0.0071 × 10-110.67 ± 0.120.01 Nedd4-2 DN0.91 ± 0.045 × 10-4GroupI/I0 (5 min)pCl/Cl0 (5 min)p Control0.78 ± 0.030.83 ± 0.08 Nedd4-20.83 ± 0.020.20.30 ± 0.091 × 10-3 Open table in a new tab Liddle Syndrome Mutations Decrease ENaC Endocytosis—Liddle syndrome mutations disrupt Nedd4-2 binding to ENaC by altering or deleting a C-terminal PY motif. To test the effect of Liddle syndrome mutations on ENaC endocytosis, we cotransfected FRT cells with αCl and γCl along with a mutant β subunit. In Fig. 2, B and C, we tested βR566X, which deletes most of the C terminus, including the PY motif. Following activation by trypsin, there was a gradual decline in amiloride-sensitive current. However, the rate of decrease was dramatically slower than for the wild-type β subunit. The difference between the curves was mainly in the initial rapid phase, which was largely absent for βR566X (Fig. 2C). In contrast, the curves for wild-type and βR566X paralleled one another in the later phase. When trypsin was added to the bathing solution a second time, there was an increase in ENaC current, albeit much smaller than for wild-type βENaC (Fig. 2B). Thus, Liddle syndrome mutations decrease ENaC exocytosis, consistent with previous work (30Snyder P.M. J. Clin. Investig. 2000; 105: 45-53Crossref PubMed Scopus (137) Google Scholar, 31Lu C. Pribanic S. Debonneville A. Jiang C. Rotin D. Traffic. 2007; 8: 1246-1264Crossref PubMed Scopus (99) Google Scholar, 32Volk K.A. Husted R.F. Sigmund R.D. Stokes J.B. J. Biol. Chem. 2005; 280: 18348-18354Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). In Fig. 2D, we tested a missense mutation within the βENaC PY motif (βY620A) that also disrupts binding to Nedd4-2. This mutation slowed the rate of decrease in trypsin-activated ENaC current, similar to βR566X. Equivalent mutations of the PY motifs in αENaC (Y644A) and γENaC (Y627A) also slowed the decline in current (Fig. 2, E and F) although, interestingly, mutation of the αENaC PY motif had a smaller effect than the mutations in β or γENaC. Together, the data suggest that Liddle syndrome mutations decrease the rate of ENaC endocytosis. Biochemical Endocytosis Assay—As a second strategy to quantitate ENaC endocytosis, we used a biochemical assay to measure the rate of removal of proteolytically cleaved channels from the cell surface. We coexpressed α, β, and γENaC in HEK 293T cells; the α subunit contained a FLAG epitope at the C terminus. To detect αENaC at the cell surface, we biotinylated cell surface proteins, isolated them with NeutrAvidin beads, and then immunoblotted with anti-FLAG antibody. For wild-type αENaC, we detected a 90-kDa band corresponding to the full-length protein and a 65-kDa band corresponding to the C-terminal cleavage fragment (Fig. 3A), consistent with previous work (7Knight K.K. Olson D.R. Zhou R. Snyder P.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2805-2808Crossref PubMed Scopus (125) Google Scholar, 33Hughey R.P. Mueller G.M. Bruns J.B. Kinlough C.L. Poland P.A. Harkleroad K.L. Carattino M.D. Kleyman T.R. J. Biol. Chem. 2003; 278: 37073-37082Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). To prevent cleavage by furin, we mutated the two furin consensus sites (αCl-2), which has a similar effect on ENaC current as mutation of a single furin site (αCl-1) (34Carattino M.D. Sheng S. Bruns J.B. Pilewski J.M. Hughey R.P. Kleyman T.R. J. Biol. Chem. 2006; 281: 18901-18907Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). This eliminated the 65-kDa band (Fig. 3A). However, extracellular trypsin (5 μg/ml for 5 min) cleaved αCl-2, generating a 65-kDa band very similar in mass to the cleaved form of wild-type αENaC (Fig. 3A). After removing trypsin from the extracellular medium, we measured the rate of disappearance of the 65-kDa band from the cell surface as an assay of ENaC endocytosis (Fig. 1C shows model). Because newly synthesized channels undergoing exocytosis are 90 kDa, we were able to distinguish them from cleaved channels undergoing endocytosis. In Fig. 3B, top panel, we generated a pool of trypsin-cleaved αCl-2-FLAG at the cell surface, incubated the cells at 37 °C for 0–60 min to allow endocytosis, and then biotinylated channels remaining at the cell surface. We observed a rapid decrease in the 65-kDa cleaved band at the cell surface (Fig. 3B, average data are quantitated in Fig. 3C); 50% was removed by 15 min and nearly all was removed at 60 min. Thus, the half-life of cleaved αENaC at the cell surface was short (15 min). In contrast to the cleaved band, the quantity of the full-length 90-kDa band at the cell surface reflects the net contributions of both endocytosis and exocytosis of full-length αENaC. The quantity of this band increased at 5 min (2.1 ± 0.1-fold, n = 3), likely resulting from exocytosis of newly synthesized channels. There was little change in the density of this band at later time points, indicating that a steady state between endocytosis and exocytosis was reestablished. As a control, there was no change in the quantity of αCl-2-FLAG in the total cellular lysate during the course of the experiment (Fig. 3B, bottom panel). Minimal cleaved αCl-2-FLAG was observed in the total lysate, indicating that only a small fraction of total ENaC in the cell was expressed at the cell surface, consistent with previous work (7Knight K.K. Olson D.R. Zhou R. Snyder P.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2805-2808Crossref PubMed Scopus (125) Google Scholar). In Fig. 3B, right panels, and 3C, we tested the effect of Liddle syndrome mutations on endocytosis. Deletion of the C terminus of βENaC (R566X) decreased the rate of disappearance of the cleaved 65-kDa band from the cell surface. At 15 min, there was no significant decrease in surface expression and the half-life lengthened to >60 min. Mutation of the PY motif (βY620A) had a similar effect (Fig. 3C). 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. Nedd4-2 Binding Motif Mediates ENaC Endocytosis—The PPPXYXXL sequence mutated in Liddle syndrome fits the consensus for two motifs that have the potential to mediate ENaC endocytosis. First, it fits the YXXØ (Ø indicates hydrophobic amino acids) motif for endocytosis in clathrin-coated pits. This motif mediates endocytosis of the transferrin receptor (35Collawn J.F. Stangel M. Kuhn L.A. Esekogwu V. Jing S.Q. Trowbridge I.S. Tainer J.A. Cell. 1990; 63: 1061-1072Abstract Full Text PDF PubMed Scopus (392) Google Scholar). Second, it fits the PPXY PY motif consensus that binds to proteins containing WW domains, including Nedd4-2 (36Chen H.I. Sudol M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7819-7823Crossref PubMed Scopus (488) Google Scholar). The Tyr (βY620A) is common to both motifs; mutation of this residue decreased ENaC endocytosis (Figs. 2D and 3C). Previous work indicates that the Leu contributes to WW domain binding and is also part of both motifs (37Kanelis V. Rotin D. Forman-Kay J.D. Nat. Struct. Biol. 2001; 8: 407-412Crossref PubMed Scopus (186) Google Scholar, 38Henry P.C. Kanelis V. O'Brien M.C. Kim B. Gautschi I. Forman-Kay J. Schild L. Rotin D. J. Biol. Chem. 2003; 278: 20019-20028Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). To distinguish the relative contributions of these two motifs, we tested two additional mutations. Previous work indicates that mutation of a Thr within the sequence (γT629A) disrupts the effect of a dominant negative dynamin cDNA (which blocks endocytosis via clathrin-coated pits) but does not alter its regulation by Nedd4-2 (39Staruschenko A. Pochynyuk O. Stockand J.D. J. Biol. Chem. 2005; 280: 39161-39167Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Conversely, mutation of the three prolines (βP616–618A) prevents ENaC regulation by Nedd4-2 but does not disrupt clathrin-mediated endocytosis (39Staruschenko A. Pochynyuk O. Stockand J.D. J. Biol. Chem. 2005; 280: 39161-39167Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). We tested the effects of these mutations on ENaC endocytosis using both electrophysiological and biochemical assays. Compared with wild type, γT629A did not alter the decline in trypsin-activated current (Fig. 4A) or the removal of the 65-kDa cleaved form of αCl-2-FLAG from the cell surface (Fig. 4, B and C). In contrast, endocytosis was decreased by βP616–618A, similar to mutation of βY620 or deletion of the β C terminus. These findings support a critical role for Nedd4-2 in mediating ENaC endocytosis. We cannot exclude a role for the YXXØ motif in regulating ENaC endocytosis in other cells. Nedd4-2 Increases ENaC Endocytosis—As a more direct test of the role of Nedd4-2 in ENaC endocytosis, we transfected cells with Nedd4-2 siRNA. In previous work, we found that Nedd4-2 siRNA specifically silenced Nedd4-2 but not the related E3 ubiquitin ligase Nedd4 (26Snyder P.M. Steines J.C. Olson D.R. J. Biol. Chem. 2004; 279: 5042-5046Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Here we found that Nedd4-2 siRNA (compared with GFP siRNA) decreased the rate of removal of trypsin-activated channels from the cell surface in FRT epithelia, indicating a decrease in the rate of endocytosis (Fig. 5A). Likewise, Nedd4-2 siRNA reduced removal of cleaved αCl-2-FLAG from the cell surface in HEK 293T cells (Fig. 5, B and C). As a second approach, we overexpressed a dominant negative form of Nedd4-2 that contains the four WW domains that bind to ENaC but lacks the HECT domain that catalyzes ENaC ubiquitination (7Knight K.K. Olson D.R. Zhou R. Snyder P.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2805-2808Crossref PubMed Scopus (125) Google Scholar). The dominant negative Nedd4-2 also decreased removal of cleaved channels from the cell surface (Fig. 5, B and C). Together, the data indicate that endogenous Nedd4-2 mediates ENaC endocytosis. In Fig. 6, we tested the effect of Nedd4-2 overexpression on ENaC endocytosis. In HEK 293T cells, overexpression of Nedd4-2 increased endocytosis of cleaved channels (Fig. 6A). In contrast, overexpression did not alter the rate of ENaC endocytosis in FRT epithelia (Fig. 6B). Nedd4-2 Increases Degradation of Cell Surface ENaC—Following endocytosis, ENaC is local