Interactions of beta and gamma ENaC with Nedd4 can be facilitated by an ERK-mediated phosphorylation.

Phosphorylation of the epithelial Na(+) channel (ENaC) has been suggested to play a role in its regulation. Here we demonstrate that phosphorylating the carboxyl termini of the beta and gamma subunits facilitates their interactions with the ubiquitin ligase Nedd4 and inhibits channel activity. Three protein kinases, which phosphorylate the carboxyl termini of beta and gammaENaC, have been identified by an in vitro assay. One of these phosphorylates betaThr-613 and gammaThr-623, well-conserved C-tail threonines in the immediate vicinity of the PY motifs. Phosphorylation of gammaThr-623 has also been demonstrated in vivo in channels expressed in Xenopus oocytes, and mutating betaThr-613 and gammaThr-623 into alanine increased the channel activity by 3.5-fold. Effects of the above phosphorylations on interactions between ENaC and Nedd4 have been studied using surface plasmon resonance. Peptides having phospho-threonine at positions beta613 or gamma623 bind the WW domains of Nedd4 two to three times better than the non-phosphorylated analogues, due to higher association rate constants. Using a number of different approaches it was demonstrated that the protein kinase acting on betaThr-613 and gammaThr-623 is the extracellular regulated kinase (ERK). It is suggested that an ERK-mediated phosphorylation of betaThr-613 and gammaThr-623 down-regulates the channel by facilitating its interaction with Nedd4.     

All three WW domains of murine Nedd4 are involved in the regulation of epithelial sodium channels by intracellular Na+

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPxY motif which is necessary for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle's syndrome where mutations delete or alter the PY motif of either the beta or gamma subunits, results in increased ENaC activity. We have recently shown using the whole-cell patch clamp technique that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. In this paper, we demonstrate that WW domains 2 and 3 bind alpha-, beta-, and gamma-ENaC with varying degrees of affinity, whereas WW domain 1 does not bind to any of the subunits. We further show using whole-cell patch clamp techniques that Nedd4-mediated down-regulation of ENaC in mouse mandibular duct cells involves binding of the WW domains of Nedd4 to three distinct sites. We propose that Nedd4-mediated down-regulation of Na+ channels involves the binding of WW domains 2 and 3 to the Na+ channel and of WW domain 1 to an unknown associated protein.     

Stimulation of the epithelial sodium channel (ENaC) by cAMP involves putative ERK phosphorylation sites in the C termini of the channel's beta- and gamma-subunit

The mechanisms involved in the regulation of the epithelial sodium channel (ENaC) via the cAMP pathway are not yet completely understood. The aim of the present study was to investigate cAMP-mediated ENaC regulation in Xenopus laevis oocytes heterologously expressing the three subunits (alphabetagamma) of rat ENaC and to determine the ENaC regions important for mediating the stimulatory effect of cAMP. In oocytes treated for about 24 h with 1 mm 3-isobutyl-1-methylxanthine (IBMX) and 1 microm forskolin (FSK) so as to increase intracellular cAMP, the amiloride-sensitive whole cell current (DeltaI(Ami)) was on average 10-fold larger than DeltaI(Ami) in matched control oocytes. This effect on DeltaI(Ami) was paralleled by an increase in ENaC surface expression caused by a reduced rate of ENaC retrieval. In addition, IBMX/FSK also enhanced ENaC open probability from about 0.2 to 0.5. The stimulatory effect of IBMX/FSK was dependent on the presence of intact PY motifs in the C termini of the channel. Mutagenesis of putative protein kinase A and CK-2 consensus motifs in the cytosolic domains of the channel did not reveal critical sites involved in mediating the stimulatory effect of IBMX/FSK. In contrast, site-directed mutagenesis of two putative ERK-consensus motifs (T613A in betaENaC and T623A in gammaENaC) largely reduced the stimulatory effect of IBMX/FSK. Phosphorylation of these ERK sites has previously been reported to enhance the interaction of ENaC and Nedd4 (Shi, H., Asher, C., Chigaev, A., Yung, Y., Reuveny, E., Seger, R., and Garty, H. (2002) J. Biol. Chem. 277, 13539-13547). Using co-expression experiments we demonstrated that mutating the two ERK sites attenuates the inhibitory effect of Nedd4-2 on ENaC currents. We conclude that an increase in intracellular cAMP favors the dephosphorylation of the two ERK sites, which reduces channel retrieval and increases P(O) by modulating ENaC/Nedd4 interaction. This defines a novel regulatory pathway likely to be relevant for cAMP-induced stimulation of ENaC in vivo.     

The PY motif of ENaC, mutated in Liddle syndrome, regulates channel internalization, sorting and mobilization from subapical pool

The epithelial-Na(+)-channel (alphabetagammaENaC) regulates kidney salt-transport and blood pressure. Each ENaC subunit contains a PY motif (PPxY) and its mutation in beta/gammaENaC causes Liddle syndrome, a hereditary hypertension. These (extended) PY motifs (PP(616)xY(618)xxL(621)) serve as binding sites for the ubiquitin ligase Nedd4-2, which decreases cell-surface expression of ENaC by unknown route(s). Using polarized kidney epithelia [Madin-Darby canine kidney I (MDCK-I)] cells stably expressing extracellularly myc-tagged wild type (WT) or PY-motif mutants of betaENaC (P616A, Y618A or L621A, with WT-alphagammaENaC), and live-imaging plus enzyme-linked immunosorbent assay (ELISA)-type assays to analyze routes/rates of ENaC internalization/recycling, we show here that cell-surface half-life of all PY mutants was fourfold longer than WT-ENaC (approximately 120 versus 30 minutes), reflecting primarily reduced channel internalization but also attenuated replenishment of cell-surface ENaC from a large subapical pool. The Y618A mutant revealed more severe internalization and replenishment defects than the other PY mutants. Internalized WT-ENaC was detected in sorting/recycling and late endosomes/lysosomes, while the Y618A mutant accumulated in the former. Nedd4-2 ubiquitinated ENaC at the apical membrane causing channel internalization and degradation. Cyclic AMP (cAMP) accelerated mobilization of subapical ENaC to the cell surface and long-term ENaC recycling, but only mobilization, not recycling, was inhibited in the PY mutants. These results suggest that the ENaC PY motifs (and Nedd4-2) primarily regulate channel internalization but also affect cAMP-dependent replenishment, providing important insight into the Liddle syndrome defects.     

AMP-activated kinase inhibits the epithelial Na+ channel through functional regulation of the ubiquitin ligase Nedd4-2

We recently found that the metabolic sensor AMP-activated kinase (AMPK) inhibits the epithelial Na+ channel (ENaC) through decreased plasma membrane ENaC expression, an effect requiring the presence of a binding motif in the cytoplasmic tail of the beta-ENaC subunit for the ubiquitin ligase Nedd4-2. To further examine the role of Nedd4-2 in the regulation of ENaC by AMPK, we studied the effects of AMPK activation on ENaC currents in Xenopus oocytes co-expressing ENaC and wild-type (WT) or mutant forms of Nedd4-2. ENaC inhibition by AMPK was preserved in oocytes expressing WT Nedd4-2 but blocked in oocytes expressing either a dominant-negative (DN) or constitutively active (CA) Nedd4-2 mutant, suggesting that AMPK-dependent modulation of Nedd4-2 function is involved. Similar experiments utilizing WT or mutant forms of the serum- and glucocorticoid-regulated kinase (SGK1), modulators of protein kinase A (PKA), or extracellular-regulated kinase (ERK) did not affect ENaC inhibition by AMPK, suggesting that these pathways known to modulate the Nedd4-2-ENaC interaction are not responsible. AMPK-dependent phosphorylation of Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for modulation of Nedd4-2 and thus cellular ENaC activity. Moreover, cellular AMPK activation significantly enhanced the interaction of the beta-ENaC subunit with Nedd4-2, as measured by co-immunoprecipitation assays in HEK-293 cells. In summary, these results suggest a novel mechanism for ENaC regulation in which AMPK promotes ENaC-Nedd4-2 interaction, thereby inhibiting ENaC by increasing Nedd4-2-dependent ENaC retrieval from the plasma membrane. AMPK-dependent ENaC inhibition may limit cellular Na+ loading under conditions of metabolic stress when AMPK becomes activated.     

Regulation of the epithelial sodium channel by N4WBP5A,a novel Nedd4/Nedd4-2-interacting protein

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPXY motif that is believed to be important for interaction with the WW domains of the ubiquitin-protein ligases, Nedd4 and Nedd4-2. Disruption of this interaction, as in Liddle's syndrome where mutations delete or alter the PPXY motif of either the beta or gamma subunits, has been shown to result in increased ENaC activity and arterial hypertension. Here we present evidence that N4WBP5A, a novel Nedd4/Nedd4-2-binding protein, is a potential regulator of ENaC. In Xenopus laevis oocytes N4WBP5A increases surface expression of ENaC by reducing the rate of ENaC retrieval. We further demonstrate that N4WBP5A prevents sodium feedback inhibition of ENaC possibly by interfering with the xNedd4-2-mediated regulation of ENaC. As N4WBP5A binds Nedd4/Nedd4-2 via PPXY motif/WW domain interactions and appears to be associated with specific intracellular vesicles, we propose that N4WBP5A functions by regulating Nedd4/Nedd4-2 availability and trafficking. Because N4WBP5A is highly expressed in native renal collecting duct and other tissues that express ENaC, it is a likely candidate to modulate ENaC function in vivo.     

Casein kinase 2 specifically binds to and phosphorylates the carboxy termini of ENaC subunits.

A number of findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na+ channel (ENaC). A recent study has demonstrated that the C tails of the beta and gamma subunits of ENaC are subject to phosphorylation by at least three protein kinases [Shi, H., Asher, C., Chigaev, A., Yung, Y., Reuveny, E., Seger, R. & Garty, H. (2002) J. Biol. Chem. 277, 13539-13547]. One of them was identified as ERK which phosphorylates betaT613 and gammaT623 and affects the channel interaction with Nedd4. The current study identifies a second protein kinase as casein kinase 2 (CK2), or CK-2-like kinase. It phosphorylates betaS631, a well-conserved serine on the beta subunit. Such phosphorylation is observed both in vitro using glutathione-S-transferase-ENaC fusion proteins and in vivo in ENaC-expressing Xenopus oocytes. The gamma subunit is weakly phosphorylated by this protein kinase on another residue (gammaT599), and the C tail of alpha is not significantly phosphorylated by this kinase. Thus, CK2 may be involved in the regulation of the epithelial Na+ channel.     

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

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.     

The Nedd4-like protein KIAA0439 is a potential regulator of the epithelial sodium channel

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPxY, motif which is believed to be important for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle's syndrome, where mutations delete or alter the PPxY motif of either the beta or gamma subunits, has been proposed to result in increased ENaC activity. Here we present evidence that KIAA0439 protein, a close relative of Nedd4, is also a potential regulator of ENaC. We demonstrate that KIAA0439 WW domains bind all three ENaC subunits. We show that a recombinant KIAA0439 WW domain protein acts as a dominant negative mutant that can interfere with the Na(+)-dependent feedback inhibition of ENaC in whole-cell patch clamp experiments. We propose that KIAA0439 and Nedd4 proteins either play a redundant role in ENaC regulation or function in a tissue- and/or signal-specific manner to down-regulate ENaC.     

Identification of new partners of the epithelial sodium channel alpha subunit

A fine regulation of the amiloride-sensitive Epithelial Sodium Channel (ENaC), made of alpha, beta and gamma subunits, is crucial for maintenance of Na+ balance and blood pressure. Both beta- and gamma-ENaC participate in negative regulation by interacting with Nedd4-2, an E3 ubiquitin-ligase. Disruption of this interaction results in increased ENaC activity (Liddle syndrome). By two-hybrid screenings, we identified new potential partners of alpha-ENaC: WWP1 (E3 ubiquitin-ligase protein), UBC9 and TSG101 (E2 ubiquitin/SUMO-conjugating enzymes) and confirmed these interactions in GST pull-down assays. All these partners are implicated in protein trafficking and could be involved in the regulation of ENaC activity.     

14-3-3 isoforms are induced by aldosterone and participate in its regulation of epithelial sodium channels

Aldosterone increases sodium absorption across renal collecting duct cells primarily by increasing the apical membrane expression of ENaC, the sodium entry channel. Nedd4-2, a ubiquitin-protein isopeptide ligase, tags ENaC with ubiquitin for internalization and degradation, but when it is phosphorylated by the aldosterone-induced kinase, SGK1, Nedd4-2 is inhibited and apical ENaC density and sodium absorption increase. We evaluated the hypothesis that 14-3-3 proteins participate in the aldosterone-mediated regulation of ENaC by associating with phosphorylated Nedd4-2. Mouse cortical collecting duct (mCCD) epithelia cultured on filters expressed several 14-3-3 isoforms; this study focused on an isoform whose expression was induced 3-fold by aldosterone, 14-3-3beta. In polarized mCCD epithelia, aldosterone elicited significant, time-dependent increases in the expression of alpha-ENaC, SGK1, phospho-Nedd4-2, and 14-3-3beta without altering total Nedd4-2. Aldosterone decreased the interaction of alpha-ENaC with Nedd4-2, and with similar kinetics increased the association of 14-3-3beta with phospho-Nedd4-2. Short interfering RNA-induced knockdown of 14-3-3beta blunted the aldosterone-induced increase in alpha-ENaC expression, returned alpha-ENaC-Nedd4-2 binding toward prealdosterone levels, and blocked the aldosterone-stimulated increase in transepithelial sodium transport. Incubation of cell extracts with a selective phospho-Nedd4-2 antibody blocked the aldosterone-induced association of 14-3-3beta with Nedd4-2, implicating SGK1 phosphorylation at Ser-328 as the primary site of 14-3-3beta binding. Our studies show that aldosterone increases the expression of 14-3-3beta, which interacts with phospho-Nedd4-2 to block its interaction with ENaC, thus enhancing sodium absorption by increasing apical membrane ENaC density.     

Regulation of the epithelial Na+ channel by the protein kinase CK2

CK2 is a ubiquitous, pleiotropic, and constitutively active Ser/Thr protein kinase that controls protein expression, cell signaling, and ion channel activity. Phosphorylation sites for CK2 are located in the C terminus of both beta- and gamma-subunits of the epithelial Na(+) channel (ENaC). We examined the role of CK2 on the regulation of both endogenous ENaC in native murine epithelia and in Xenopus oocytes expressing rENaC. In Ussing chamber experiments with mouse airways, colon, and cultured M1-collecting duct cells, amiloride-sensitive Na(+) transport was inhibited dose-dependently by the selective CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). In oocytes, ENaC currents were also inhibited by TBB and by the structurally unrelated inhibitors heparin and poly(E:Y). Expression of a trimeric channel lacking both CK2 sites (alphabeta(S631A)gamma(T599A)) produced a largely attenuated amiloride-sensitive whole cell conductance and rendered the mutant channel insensitive to CK2. In Xenopus oocytes, CK2 was translocated to the cell membrane upon expression of wt-ENaC but not of alphabeta(S631A)gamma(T599A)-ENaC. Phosphorylation by CK2 is essential for ENaC activation, and to a lesser degree, it also controls membrane expression of alphabetagamma-ENaC. Channels lacking the Nedd4-2 binding motif in beta-ENaC (R561X, Y618A) no longer required the CK2 site for channel activity and siRNA-knockdown of Nedd4-2 eliminated the effects of TBB. This implies a role for CK2 in inhibiting the Nedd4-2 pathway. We propose that the C terminus of beta-ENaC is targeted by this essential, conserved pleiotropic kinase that directs its constitutive activity toward many cellular protein complexes.     

GRK2 interacts with and phosphorylates Nedd4 and Nedd4-2

Epithelial Na(+) channels (ENaC) mediate the transport of sodium (Na) across epithelia in the kidney, gut, and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4 and Nedd4-2, which bind to proline-rich motifs (PY motifs) present in the C-termini of ENaC subunits. Loss of inhibition leads to hypertension. ENaC channels are maintained in the active state by G-protein-coupled receptor kinase 2 (GRK2), an enzyme implicated in the development of essential hypertension. Here, we report that GRK2 interacts not only with ENaC, but also with both Nedd4 and Nedd4-2. Additionally, GRK2 is capable of phosphorylating both Nedd4 and Nedd4-2 at multiple sites. Of possible significance is the phosphorylation of the threonine at position 466 in Nedd4, which is located in the area of the ww3 domain that binds ENaC. These results support and extend the role of GRK2 in sodium transport regulation.     

Functional regulation of the epithelial Na+ channel by IkappaB kinase-beta occurs via phosphorylation of the ubiquitin ligase Nedd4-2

We have previously shown that IkappaB kinase-beta (IKKbeta) interacts with the epithelial Na+ channel (ENaC) beta-subunit and enhances ENaC activity by increasing its surface expression in Xenopus oocytes. Here, we show that the IKKbeta-ENaC interaction is physiologically relevant in mouse polarized kidney cortical collecting duct (mpkCCDc14) cells, as RNA interference-mediated knockdown of endogenous IKKbeta in these cells by approximately 50% resulted in a similar reduction in transepithelial ENaC-dependent equivalent short circuit current. Although IKKbeta binds to ENaC, there was no detectable phosphorylation of ENaC subunits by IKKbeta in vitro. Because IKKbeta stimulation of ENaC activity occurs through enhanced channel surface expression and the ubiquitin-protein ligase Nedd4-2 has emerged as a central locus for ENaC regulation at the plasma membrane, we tested the role of Nedd4-2 in this regulation. IKKbeta-dependent phosphorylation of Xenopus Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for regulation of Nedd4-2 and thus ENaC activity. 32P labeling studies utilizing wild-type or mutant forms of Xenopus Nedd4-2 demonstrated that Ser-444, a key SGK1 and protein kinase A-phosphorylated residue, is also an important IKKbeta phosphorylation target. ENaC stimulation by IKKbeta was preserved in oocytes expressing wild-type Nedd4-2 but blocked in oocytes expressing either a dominant-negative (C938S) or phospho-deficient (S444A) Nedd4-2 mutant, suggesting that Nedd4-2 function and phosphorylation by IKKbeta are required for IKKbeta regulation of ENaC. In summary, these results suggest a novel mode of ENaC regulation that occurs through IKKbeta-dependent Nedd4-2 phosphorylation at a recognized SGK1 and protein kinase A target site.     

Cystic fibrosis transmembrane conductance regulator inhibits epithelial Na+ channels carrying Liddle's syndrome mutations.

Epithelial Na+ channels (ENaC) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) upon activation by protein kinase A. It is, however, still unclear how CFTR regulates the activity of ENaC. In the present study we examined whether CFTR interacts with ENaC by interfering with the Nedd4- and ubiquitin-mediated endocytosis of ENaC. Various C-terminal mutations were introduced into the three alpha-, beta-, and gamma-subunits of the rat epithelial Na+ channel, thereby eliminating PY motifs, which are important binding domains for the ubiquitin ligase Nedd4. When expressed in Xenopus oocytes, most of the ENaC stop (alpha-H647X, beta-P565X, gamma-S608X) or point (alpha-P671A, beta-Y618A, gamma-P(624-626)A) mutations induced enhanced Na+ currents when compared with wild type alpha,beta,gamma-rENaC. However, ENaC currents formed by either of the mutant alpha-, beta-, or gamma-subunits were inhibited during activation of CFTR by forskolin (10 micromol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l). Antibodies to dynamin or ubiquitin enhanced alpha,beta,gamma-rENaC whole cell Na+ conductance but did not interfere with inhibition of ENaC by CFTR. Another mutant, beta-T592M,T593A-ENaC, also showed enhanced Na+ currents, which were down-regulated by CFTR. Moreover, activation of ENaC by extracellular proteases and xCAP1 does not disturb CFTR-dependent inhibition of ENaC. We conclude that regulation of ENaC by CFTR is distal to other regulatory limbs and does not involve Nedd4-dependent ubiquitination.     

Hrs Controls Sorting of the Epithelial Na+ Channel between Endosomal Degradation and Recycling Pathways

Epithelial Na⁺ absorption is regulated by Nedd4-2, an E3 ubiquitin ligase that reduces expression of the epithelial Na⁺ channel (ENaC) at the cell surface. Defects in this regulation cause Liddle syndrome, an inherited form of hypertension. Previous work found that Nedd4-2 functions through two distinct effects on trafficking, enhancing both ENaC endocytosis and ENaC degradation in lysosomes. To investigate the mechanism by which Nedd4-2 targets ENaC to lysosomes, we tested the role of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a component of the endosomal sorting complexes required for transport (ESCRT)-0 complex. We found that α-, β-, and γENaC each interact with Hrs. These interactions were enhanced by Nedd4-2 and were dependent on the catalytic function of Nedd4-2 as well as its WW domains. Mutation of ENaC PY motifs, responsible for inherited hypertension (Liddle syndrome), decreased Hrs binding to ENaC. Moreover, binding of ENaC to Hrs was reduced by dexamethasone/serum- and glucocorticoid-inducible kinase and cAMP, which are signaling pathways that inhibit Nedd4-2. Nedd4-2 bound to Hrs and catalyzed Hrs ubiquitination but did not alter Hrs protein levels. Expression of a dominant negative Hrs lacking its ubiquitin-interacting motif (Hrs-ΔUIM) increased ENaC surface expression and current. This occurred through reduced degradation of the cell surface pool of proteolytically activated ENaC, which enhanced its recycling to the cell surface. In contrast, Hrs-ΔUIM had no effect on degradation of uncleaved inactive channels. The data support a model in which Nedd4-2 induces binding of ENaC to Hrs, which mediates the sorting decision between ENaC degradation and recycling.     

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 alpha, beta, and gammaENaC dramatically reduced endocytosis, whereas a mutation that disrupts a YXX? 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 alphaENaC. 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.     

Relative contribution of Nedd4 and Nedd4-2 to ENaC regulation in epithelia determined by RNA interference

Epithelial Na+ transport is regulated in large part by mechanisms that control expression of the epithelial Na+ channel (ENaC) at the cell surface. Nedd4 and Nedd4-2 are candidates to control ENaC surface expression, but it is not known which of these proteins contributes to ENaC regulation in epithelia. To address this question, we used RNA interference to selectively reduce expression of Nedd4 or Nedd4-2. We found that endogenous Nedd4-2, but not Nedd4, negatively regulates ENaC in two epithelial cell lines (Fischer rat thyroid and H441); small interfering RNA (siRNA) against Nedd4-2 increased amiloride-sensitive Na+ current (compared with control siRNA), but Nedd4 siRNA did not. A mutation associated with Liddle's syndrome (betaR566X) abolished the effect of Nedd4-2 siRNA, suggesting that a defect in ENaC regulation by Nedd4-2 contributes to the pathogenesis of this inherited form of hypertension. Previous work found that Nedd4-2 is phosphorylated by serum and glucocorticoid-regulated kinase, a Ser/Thr kinase induced by steroid hormones. Here we found that Nedd4-2 phosphorylation contributes to ENaC regulation by steroid hormones. Consistent with this model, ENaC stimulation by dexamethasone was reduced by Nedd4-2 siRNA and by overexpression of a mutant Nedd4-2 lacking serum and glucocorticoid-regulated kinase phosphorylation sites. Thus, endogenous Nedd4-2 negatively regulates ENaC in epithelia and is a component of a signaling pathway by which steroid hormones regulate ENaC. Defects in this regulation may contribute to the pathogenesis of hypertension.     

Serum and Glucocorticoid-induced Kinase (SGK) 1 and the Epithelial Sodium Channel Are Regulated by Multiple with No Lysine (WNK) Family Members

The four WNK (with no lysine (K)) protein kinases affect ion balance and contain an unusual protein kinase domain due to the unique placement of the active site lysine. Mutations in two WNKs cause a heritable form of ion imbalance culminating in hypertension. WNK1 activates the serum- and glucocorticoid-induced protein kinase SGK1; the mechanism is noncatalytic. SGK1 increases membrane expression of the epithelial sodium channel (ENaC) and sodium reabsorption via phosphorylation and sequestering of the E3 ubiquitin ligase neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), which otherwise promotes ENaC endocytosis. Questions remain about the intrinsic abilities of WNK family members to regulate this pathway. We find that expression of the N termini of all four WNKs results in modest to strong activation of SGK1. In reconstitution experiments in the same cell line all four WNKs also increase sodium current blocked by the ENaC inhibitor amiloride. The N termini of the WNKs also have the capacity to interact with SGK1. More detailed analysis of activation by WNK4 suggests mechanisms in common with WNK1. Further evidence for the importance of WNK1 in this process comes from the ability of Nedd4-2 to bind to WNK1 and the finding that endogenous SGK1 has reduced activity if WNK1 is knocked down by small interfering RNA.     

Enac degradation in A6 cells by the ubiquitin-proteosome proteolytic pathway

Amiloride-sensitive epithelial Na(+) channels (ENaC) are responsible for trans-epithelial Na(+) transport in the kidney, lung, and colon. The channel consists of three subunits (alpha, beta, gamma) each containing a proline rich region (PPXY) in their carboxyl-terminal end. Mutations in this PPXY domain cause Liddle's syndrome, an autosomal dominant, salt-sensitive hypertension, by preventing the channel's interactions with the ubiquitin ligase Neural precursor cell-expressed developmentally down-regulated protein (Nedd4). It is postulated that this results in defective endocytosis and lysosomal degradation of ENaC leading to an increase in ENaC activity. To show the pathway that degrades ENaC in epithelial cells that express functioning ENaC channels, we used inhibitors of the proteosome and measured sodium channel activity. We found that the inhibitor, MG-132, increases amiloride-sensitive trans-epithelial current in Xenopus distal nephron A6 cells. There also is an increase of total cellular as well as membrane-associated ENaC subunit molecules by Western blotting. MG-132-treated cells also have increased channel density in patch clamp experiments. Inhibitors of lysosomal function did not reproduce these findings. Our results suggest that in native renal cells the proteosomal pathway is an important regulator of ENaC function.