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 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.

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 (6,12,13).
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 (14). Moreover, proteasome inhibitors decrease ENaC degradation (15)(16)(17) and increase ENaC surface expression. 2 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 (18,19).
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 ␣ and ␥ subunits (20 -23). Using electrophysiological and biochemical assays, we tested the effect of Nedd4-2 on endocytosis and degradation of proteolytically cleaved channels.
Electrophysiological Endocytosis Assay-Fischer rat thyroid (FRT) cells cultured on permeable filter supports were transfected (TFX50; Promega) (12) with the following human ENaC subunits; ␣ or ␣ Cl-1 (R177A, R178A), ␤, and ␥ or ␥ Cl (R135A, R137A, R138A, R178W, R180A, K181A). For overexpression experiments, the cells were cotransfected with 0.23 g of each ENaC subunit along with either green fluorescent protein * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed: 371 EMRB, University of Iowa College of Medicine, Iowa City, IA 52242. E-mail: peter-snyder@ uiowa.edu.
(GFP, control) or Nedd4-2 (0.3 g). For RNA interference experiments, cells were cotransfected with 0.27 g of each ENaC subunit and siRNA against GFP (control) or Nedd4-2 (0.2 g) (26). In some studies, ENaC subunits contained the following mutations: ␣ Y644A , ␤ R566X , ␤ Y620A , ␤ P616 -618A , ␥ Y627A , or ␥ T629A . Following transfection, the cells were cultured for 48 h as described previously (12). 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 CaCl 2 , 1.2 mM MgCl 2 , 2.4 mM K 2 HPO 4 , 0.6 mM KH 2 PO 4 , 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 M) in the apical bathing solution. To proteolytically cleave and activate ENaC at the cell surface, trypsin (10 g/ml) was added to the apical membrane for 10 min and then removed by vigorous washes with ϳ12 times the volume of the apical chamber. Amiloride-sensitive current was measured every 5 min by addition of amiloride (10 M) to the apical membrane followed by wash out of amiloride.
Degradation Assay-HEK 293T cells were transfected with ␣-FLAG, ␤, and ␥ENaC 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 ␣ENaC was isolated with Neutr-Avidin beads, separated by SDS-PAGE, and detected by immu-noblot (anti-FLAG M2 monoclonal antibody-peroxidase conjugate). Fig. 1A, we transfected FRT epithelia with ␣, ␤, and ␥ENaC 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 ␣ and ␥ subunits. The basal amiloride-sensitive current (Fig. 1A) reflects ENaC channels cleaved in the biosynthetic pathway (27,28). 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 (7,23,27). In Fig. 1B, we mutated a consensus site for cleavage by furin in ␣ENaC (␣ Cl ; R177A/178A), and in ␥ENaC we mutated sites (␥ Cl ) for furin (R135/137/138A) and CAP1 (R178W, K179A, R180A, K181A) (27,29). 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.

Proteolytic Cleavage of Cell Surface ENaC-In
Quantitation of Endocytosis of Proteolytically Cleaved ENaC-Expression of the ␣ Cl ␤␥ Cl 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 A and B, representative short circuit current traces in FRT epithelia transfected with ␣ENaC (wild-type or ␣ cl ), ␤ENaC, and ␥ENaC (wild-type or ␥ cl ) (0.33 g each). Amiloride (10 M) and trypsin (2 g/ml) were added to apical bathing solution as indicated. C, model showing strategy for endocytosis assay. Uncleaved ENaC at the cell surface is cleaved by trypsin, which activates the channel. The rate of disappearance of cleaved channels from the cell surface is quantitated as an assay of endocytosis.
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 amiloridesensitive current. This likely reflects previously uncleaved/inactive channels inserted into the plasma membrane during the 45-min interval following the initial trypsin exposure.
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 ␣ Cl and ␥ Cl along with a mutant ␤ subunit. In Fig. 2, B and C, we tested ␤ R566X , 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 ␤ subunit. The difference between the curves was mainly in the initial rapid phase, which was largely absent for ␤ R566X (Fig. 2C). In contrast, the curves for wild-type and ␤ R566X 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 ␤ENaC (Fig. 2B). Thus, Liddle syndrome mutations decrease ENaC exocytosis, consistent with previous work (30 -32).
In Fig. 2D, we tested a missense mutation within the ␤ENaC PY motif (␤ Y620A ) that also disrupts binding to Nedd4-2. This mutation slowed the rate of decrease in trypsin-activated ENaC current, similar to ␤ R566X . Equivalent mutations of the PY motifs in ␣ENaC (Y644A) and ␥ENaC (Y627A) also slowed the decline in current (Fig. 2, E and F) although, interestingly, mutation of the ␣ENaC PY motif had a smaller effect than the mutations in ␤ or ␥ENaC. 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 ␣, ␤, and ␥ENaC in HEK 293T cells; the ␣ subunit contained a FLAG epitope at the C terminus. To detect ␣ENaC at the cell surface, we biotinylated cell surface proteins, isolated them with NeutrAvidin beads, and then immunoblotted with anti-FLAG antibody. For wildtype ␣ENaC, 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 (7,33). To prevent cleavage by furin, we mutated the two furin consensus sites (␣ Cl-2 ), which has a similar effect on ENaC current as mutation of a single furin site (␣ Cl-1 ) (34). This eliminated the 65-kDa band (Fig. 3A). However, extracellular trypsin (5 g/ml for 5 min) cleaved ␣ Cl-2 , generating a 65-kDa band very similar in mass to the cleaved form of wild-type ␣ENaC (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.   FIGURE 2. Electrophysiological endocytosis assay. FRT epithelia were cotransfected with ␣ Cl ENaC, ␤ENaC, and ␥ Cl ENaC (0.33 g each), and short circuit current was measured at 37°C. Liddle syndrome mutations were introduced in each ENaC subunit as indicated. A and B, representative current traces with wild-type (A) or R566X (B) ␤ENaC. Trypsin (10 g/ml, bars) and amiloride (10 M, arrowheads) were added to apical bathing solution as indicated. C-F, plots of trypsin-activated amiloride-sensitive current (relative to initial current after trypsin removal, I 0 , mean Ϯ S.E.) versus time after trypsin removal from the bathing solution. ENaC subunits contained the indicated mutations and were compared with wild-type ENaC studied on the same days. C, ␤ R566X (n ϭ 10); D, ␤ Y620A (n ϭ 8); E, ␣ Cl/Y644A (n ϭ 4); F, ␥ Cl/Y627A (n ϭ 4).

TABLE 1 ENaC endocytosis
Fraction of amiloride-sensitive current remaining (I/I 0 ) or fraction of proteolytically cleaved channels remaining at the cell surface (Cl/Cl 0 ) 5 or 15 min following removal of trypsin from the extracellular solution, as indicated (mean Ϯ S.E.). p values determined by t-test.  MARCH 7, 2008 • VOLUME 283 • NUMBER 10 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 ␣ Cl-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 ␣ENaC 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 ␣ENaC. The quantity of this band increased at 5 min (2.1 Ϯ 0.1-fold, n ϭ 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 ␣ Cl-2 -FLAG in the total cellular lysate during the course of the experiment (Fig. 3B, bottom panel). Minimal cleaved ␣ Cl-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 (7).

Nedd4-2-induced Endocytosis and Degradation of ENaC
In Fig. 3B, right panels, and 3C, we tested the effect of Liddle syndrome mutations on endocytosis. Deletion of the C terminus of ␤ENaC (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 Ͼ60 min. Mutation of the PY motif (␤ Y620A ) 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 (35). Second, it fits the PPXY PY motif consensus that binds to proteins containing WW domains, including Nedd4-2 (36). The Tyr (␤ Y620A ) 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 (37,38). 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 (␥ T629A ) 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 (39). Conversely, mutation of the three prolines (␤ P616 -618A ) prevents ENaC regulation by Nedd4-2 but does not disrupt clathrin-mediated endocytosis (39). We tested the effects of these mutations on ENaC endocytosis using both electrophysiological and biochemical assays. Compared with wild type, ␥ T629A did not alter the decline in trypsin-activated current (Fig. 4A) or the removal of the 65-kDa cleaved form of ␣ Cl-2 -FLAG from the cell surface (Fig. 4, B and C). In contrast, endocytosis was decreased by ␤ P616 -618A , similar to mutation of ␤ Y620 or deletion of the ␤ 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 (26). 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 ␣ Cl-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 (7). 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). . Biochemical endocytosis assay. HEK 293T cells were transfected with the following ENaC subunits; ␣-FLAG (wild-type or ␣ Cl-2 ), ␤ (wild-type, R566X, or Y620A), and ␥ (5.33 g each). 48 h after transfection, cells were treated with or without trypsin (5 g/ml for 5 min), and then cell surface ␣-FLAG was biotinylated, isolated with NeutrAvidin beads, and detected by immunoblot (anti-FLAG). A and B, representative immunoblots. In B, following removal of trypsin from the medium, cells were incubated for 0 -60 min at 37°C prior to biotinylation. Data are quantitated (densitometry) in C; plot of cleaved ␣-FLAG at the cell surface versus time at 37°C (relative to 0 min) (mean Ϯ S.E., n ϭ 3-8 for each data point). (40), where it can undergo one of two fates, recycling back to the cell surface (41) or degradation in lysosomes (15,31). We hypothesized that Nedd4-2 controls the fate of cleaved ENaC by targeting it for degradation. To test this hypothesis, we transfected HEK 293T cells with ␣-FLAG, ␤, and ␥ENaC, labeled ENaC at the cell surface with biotin (at 4°C), and then incubated the cells at 37°C for 0 -80 min. The disappearance of biotinylated ␣-FLAG reflects the degradation rate of the cell surface pool of ␣ENaC. In the absence of Nedd4-2, cell surface ␣ENaC was slowly degraded; Ͻ50% of the cleaved form was degraded at 80 min (Fig. 7, A and B). In contrast, Nedd4-2 dramatically increased degradation; Ͼ75% was degraded at 80 min (Fig. 7, A  and B). This finding is similar to our previous work with ␤ENaC (18). Nedd4-2 also increased degradation of the full-length form of ␣ENaC (Fig. 7, A and B). Together the data suggest that Nedd4-2 reduces surface expression of cleaved ENaC by increasing both its endocytosis and its degradation.

Nedd4-2-induced Endocytosis and Degradation of ENaC
In contrast to this lack of effect of Nedd4-2 overexpression on ENaC endocytosis in FRT epithelia, our previous work found that Nedd4-2 overexpression decreased ENaC current in these cells (12). This indicates that increased endocytosis is not the sole mechanism by which Nedd4-2 regulates ENaC. Consistent with this conclusion, we found that Nedd4-2 overexpression increased degradation of the cell surface fraction of ENaC. The data support a model in which Nedd4-2 regulates ENaC at two distinct sites in the endocytic pathway. First, Nedd4-2 increases ENaC endocytosis from the cell surface into endosomes. This step is rapid, occurring over 5-15 min (depending on levels of Nedd4-2). Second, once in endosomes, Nedd4-2 targets ENaC for degradation in lysosomes, decreasing the ENaC pool available for recycling back to the cell surface. This is consistent with the recent report that a Liddle syndrome mutant decreased ENaC colocalization with a lysosomal marker (31). The net effect of both is to reduce the expression of ENaC at the cell surface and, hence, decrease epithelial Na ϩ transport.
Previous studies have reported a half-life for removal of ENaC from the cell surface ranging from 30 min to 3.6 h (31,32,(42)(43)(44). The half-life that we observed was shorter; a rapid phase of endocytosis occurred over 15 min in FRT cells and in HEK 293 cells the t1 ⁄ 2 for endocytosis was 15 min. This difference, as well as the wide variability in previous reports, could be explained by methodological differences. In each of the previous reports, the strategy was to inhibit protein synthesis (cycloheximide) or disrupt the Golgi (brefeldin A) and then measure decay of ENaC current or surface expression. These drugs have also been used to show that current decay is slowed by Liddle syndrome mutations (31,32,42). One limitation of this strategy is the significant (and unknown) time lag between addition of the drug and depletion of the biosynthetic pool of ENaC available for insertion into the plasma membrane. A recent study suggested that this pool was depleted very slowly following addition of cycloheximide in Madin-Darby canine kidney cells transfected with ENaC (31). This would lead to an underesti-mation of the endocytosis rate because the surface pool would be replenished by exocytosis of new channels. Moreover, cycloheximide and brefeldin A could inhibit synthesis/trafficking of a variety of proteins that mediate or regulate ENaC endocytosis. Rather than disrupt ENaC synthesis or trafficking, our strategy was to acutely modify a pool of channels at the cell surface (by proteolytic cleavage), which allowed us to follow the channels as they were removed from the surface using functional and biochemical assays. Somewhat analogous to a pulse-chase assay, this strategy has several advantages. Because of the discrete nature of the intervention, it provided improved time resolution compared with previous work. Moreover, the shift in molecular mass of ␣ENaC caused by cleavage allowed us to distinguish channels undergoing endocytosis from newly synthesized channels trafficking to the cell surface. In contrast to studies using brefeldin A or cycloheximide, our approach should not alter synthesis or exocytosis of proteins involved in ENaC trafficking, increasing specificity of the assay. Finally, this strategy allowed us to selectively quantitate endocytosis of the active (proteolytically cleaved) form of ENaC.
The strategy we used also requires assumptions. First, the electrophysiological assay assumes that there are no time-dependent changes in ENaC gating (i.e. rundown); decreased current could result if P O decreased over time. We think this is a good assumption, as we have not observed evidence of significant rundown in FRT epithelia. Moreover, the electrophysiological data were corroborated by a biochemical approach (biotinylation) that counted channel number, rather than activity. Second, the strategy assumes that channels cleaved by trypsin are endocytosed at the same rate as channels cleaved by endogenous proteases. Two observations support this assumption; trypsin and endogenous proteases generate ␣ENaC cleavage fragments that are very similar in size, and functionally they appear to cleave an overlapping set of residues (7,27). We do not yet know whether full-length/inactive channels are endocytosed at the same rate as cleaved/active channels. In fact, our previous work raised the interesting possibility that Nedd4-2 may selectively regulate trafficking of proteolytically cleaved channels, because it altered that ratio of cleaved to full-length channels at the cell surface (7). Additional work will be necessary to determine whether this change in ratio resulted from differences in endocytosis. Third, the strategy assumes that the rate of endocytosis measured for ␣ENaC reflects endocytosis of functional ENaC channel complexes composed of all three subunits. In support of this assumption, we found that efficient expression of ␣ENaC at the cell surface in HEK 293T cells requires coexpression with ␤ and ␥ENaC, suggesting that the three subunits primarily traffic as a heteromultimeric complex in these cells. 4 Furthermore, the electrophysiological assay we used selectively quantitated endocytosis of functional channels; our previous work indicates that in FRT cells these channels are composed of all three ENaC subunits (30).
In FRT epithelia, ENaC endocytosis occurred over a biphasic time course. There are two potential explanations. First, there could be two or more distinct pools of ENaC with differing 4 R. Kabra, K. K. Knight, R. Zhou, and P. M. Snyder, unpublished observation. FIGURE 7. Nedd4-2 increases degradation of cell surface ENaC. HEK 293T cells were transfected with ␣-FLAG, ␤, and ␥ENaC subunits (1 g each) and either Nedd4-2 cDNA (ϩNedd4-2) or control GFP cDNA (ϪNedd4-2) (0.04 g). 48 h after transfection, the cells were biotinylated and then incubated for 0, 40, or 80 min at 37°C. Following lysis of the cells, biotinylated ␣-FLAG was isolated with NeutrAvidin beads and detected by immunoblot (anti-FLAG) (A), and the cleaved band was quantitated by densitometry (mean Ϯ S.E., n ϭ 4) (B). stability at the cell surface, one pool that is rapidly endocytosed and a second more stable pool. A variety of mechanisms could underlie this difference, including interactions with associated proteins that modulate endocytosis or localization in membrane microdomains with differing turnover rates. Second, the slower component could reflect recycling of endocytosed trypsin-activated channels late in the time course of the experiment, which would reduce the apparent rate of endocytosis. Our data certainly do not exclude a role for Nedd4-2 in the regulation of ENaC recycling. Although not the primary purpose of the current study, we observed a decrease in ENaC exocytosis when the channel contained a mutation in the Nedd4-2 binding sequence (PY motif, Fig. 2, A and B). However, based on trypsin sensitivity, this did not appear to arise from recycling of channels but reflected a pool of newly synthesized (trypsin-sensitive) channels. This finding is consistent with previous work from our laboratory and others (30 -32).
Elucidating the mechanisms by which Nedd4-2 regulates ENaC surface expression is important for our understanding of Na ϩ homeostasis and the pathogenesis of disease. Nedd4-2 is a critical convergence point for the regulation of epithelial Na ϩ transport. By decreasing the expression of ENaC at the cell surface, Nedd4-2 reduces Na ϩ reabsorption to maintain Na ϩ homeostasis. Defects in this regulation cause Liddle syndrome, an inherited form of hypertension. Moreover, single nucleotide polymorphisms in Nedd4-2 have been linked to hypertension (45), raising the intriguing possibility that Nedd4-2 may play a role in more common forms of hypertension. Our findings, together with previous work, focus attention on the endocytic pathway for identification of new candidate genes and potential therapeutic targets for hypertension and other diseases of Na ϩ homeostasis.