Defective regulation of the epithelial Na⁺ channel by Nedd4 in Liddle's syndrome

Liddle's syndrome is an inherited form of hypertension linked to mutations in the epithelial Na⁺ channel (ENaC). ENaC is composed of three subunits (α, β, γ), each containing a COOH-terminal PY motif (xPPxY). Mutations causing Liddle's syndrome alter or delete the PY motifs of β- or γ-ENaC. We recently demonstrated that the ubiquitin–protein ligase Nedd4 binds these PY motifs and that ENaC is regulated by ubiquitination. Here, we investigate, using the Xenopus oocyte system, whether Nedd4 affects ENaC function. Overexpression of wild-type Nedd4, together with ENaC, inhibited channel activity, whereas a catalytically inactive Nedd4 stimulated it, likely by acting as a competitive antagonist to endogenous Nedd4. These effects were dependant on the PY motifs, because no Nedd4-mediated changes in channel activity were observed in ENaC lacking them. The effect of Nedd4 on ENaC missing only one PY motif (of β-ENaC), as originally described in patients with Liddle's syndrome, was intermediate. Changes were due entirely to alterations in ENaC numbers at the plasma membrane, as determined by surface binding and immunofluorescence. Our results demonstrate that Nedd4 is a negative regulator of ENaC and suggest that the loss of Nedd4 binding sites in ENaC observed in Liddle's syndrome may explain the increase in channel number at the cell surface, increased Na⁺ reabsorption by the distal nephron, and hence the hypertension.

J. Clin. Invest. 103:667–673 (1999)

The amiloride-sensitive epithelial Na⁺ channel (ENaC) plays an essential role in the reabsorption of fluid across epithelia of the kidney, colon, lung, and ducts of exocrine glands. It facilitates the entry of Na⁺ across apical membranes, a process driven by the basolateral Na⁺/K⁺-ATPase (1). Hence, it has a major impact on the control of whole body Na⁺ homeostasis, blood volume, and blood pressure. The activity of the Na⁺ channel is tightly controlled by various hormones such as aldosterone, vasopressin, and insulin, a number of intracellular mediators such as Na⁺, Ca²⁺, pH, cAMP, enzymes such as protein kinases A and C, and by extracellular proteases (1). Precise regulation of ENaC is essential, as illustrated by the number of human diseases that have been linked to malfunction or to mutations in ENaC, including Liddle's syndrome (2), pseudohypoaldosteronism type I (PHA-I) (3), cystic fibrosis (4), and pulmonary edema (5, 6). Liddle's syndrome is an autosomal dominant form of inherited human arterial hypertension, characterized by an early onset of severe hypertension, salt sensitivity, hypokalemia, metabolic alkalosis, and low aldosterone and renin plasma concentrations (7, 8). The disease has been genetically linked to mutations in either of two genes encoding the β or γ subunits of the ENaC complex (2, 9).

ENaC is composed of a combination of three similar subunits, α, β, and γ (10–13), assembled with a stoichiometry of 2α, 1β, 1γ (14). Each subunit consists of short intracellular NH₂ and COOH termini, two transmembrane domains, and a large extracellular loop (15–17). The COOH-terminal region of every subunit contains conserved proline-rich sequences, including the sequence xPPxY, called the PY motif (18). The original mutations identified to cause Liddle's syndrome, including frameshifts and premature stop codons, all lead to truncation of the COOH terminus, with deletion of the PY motifs of β- or γ-ENaC (2, 9). Moreover, in some kindred, specific point mutations within the PY motif were recently reported (19–21), pointing to this region as an important site for the regulation of ENaC and as a Liddle's syndrome “hot spot.” Indeed, an abnormal increase in channel activity was observed upon expression of ENaC harboring the Liddle's truncation or mutations within the PY motif of β- or γ-ENaC in Xenopus oocytes (22–24). This elevated ENaC activity has been shown to be caused by both an increase in the number of channels at the plasma membrane as well as an elevated mean open probability of each channel (25), and it was recently linked also to a defective Na⁺-dependent feedback inhibition (26).

We had previously identified the ubiquitin–protein ligase Nedd4 (neuronal precursor cell expressed developmentally downregulated) as an interacting partner of ENaC, which binds to the PY motifs of αβγENaC via its WW domains (27). This protein, originally cloned by a subtractive screen (28), contains a C2/CalB domain (a calcium-dependent lipid-binding domain), three WW domains (29) (but four in the human and Xenopus laevis homologues) that are protein–protein interaction modules, and a HECT domain (homologous to E6-AP protein COOH-terminal, a ubiquitin–protein ligase domain) (30). Because ubiquitination of proteins has been associated with their rapid breakdown by the proteasome or by the endosomes/lysosomes in the case of some transmembrane proteins (31), we proposed that Nedd4 may act as a negative regulator of ENaC, which upon binding via its WW domains to the ENaC PY motifs, ubiquitinates the channel, leading to its subsequent endocytosis and lysosomal degradation (27). Indeed, we found that ENaC is a short-lived protein that is ubiquitinated in vivo and regulated by ubiquitination (32). Mutation of key lysine residues at the NH₂ termini of γ- and α-ENaC led to reduced ubiquitination and elevated channel activity when expressed in Xenopus oocytes, an elevation caused by an increase in the number of channels at the plasma membrane (32). Furthermore, we have demonstrated that the pattern of expression of Nedd4 in kidney and lung (33) is similar to that previously reported for ENaC (34).

Despite these lines of evidence, however, it has not yet been demonstrated directly that Nedd4 actually regulates ENaC function, which was therfore the goal of our current studies. Here we report that Nedd4 indeed negatively regulates ENaC activity and that this regulation is dependent on the presence of the PY motifs of ENaC and on Nedd4 with functional ubiquitin–protein ligase activity. Moreover, we find that Nedd4 affects ENaC activity predominantly by controlling the number of channels at the plasma membrane. This study provides an explanation at the molecular level for at least one aspect of the defective regulation of ENaC in Liddle's syndrome.

ENaC is regulated by Nedd4 in Xenopus oocytes. We have shown previously that the ubiquitin–protein ligase Nedd4 binds ENaC (27) and that ENaC is a short-lived protein regulated by ubiquitination (32). This prompted us to investigate whether Nedd4 is indeed a regulator of ENaC activity. For these studies, we used Xenopus laevis oocytes, which contain endogenous Nedd4 (ref. 27; see also Fig. 1a, left lane). We thus injected rat ENaC cRNA in Xenopus oocytes together with cRNA encoding wild-type Xenopus laevis Nedd4 (xNedd4) (35) or a mutant form bearing a Cys→Ser mutation (xN4C938S) at the conserved cysteine in the HECT domain, which inactivates its ubiquitin–protein ligase activity (30, 37). Both wild-type and mutant xNedd4 were overexpressed to similar levels, about 10- to 20-fold higher than that of endogenous xNedd4 (Fig. 1a). The overexpression of xNedd4 did not influence the biosynthesis of ENaC, as judged by metabolic labeling of Xenopus oocytes followed by immunoprecipitation with anti-ENaC antibodies (not shown). ENaC activity was determined by measuring amiloride-sensitive Na⁺ currents (I_Na) 20–24 hours after cRNA injection and oocyte incubation in a solution containing 80 mM sodium. Expression of ENaC alone (ENaC/H₂O) in Xenopus oocytes (control) resulted in I_Na between 1 and 10 μA. Upon coexpression of xNedd4-wt with ENaC (ENaC/Nedd4-wt), I_Na fell below 5% of the control values (Fig. 1b); this channel inhibition was dose-dependent, becoming more pronounced with increasing amounts of xNedd4 cRNA injected (Fig. 2). In contrast, coexpression of the inactive Nedd4 mutant (ENaC/Nedd4-CS) resulted in a two- to sixfold fold increase in I_Na relative to control oocytes. Expression of this mutant Nedd4 did not lead to obvious changes in other electrophysiological parameters, such as amiloride-sensitive current–voltage relationship, single-channel properties of ENaC, and non–amiloride-sensitive membrane conductance (not shown). Moreover, the activity of another channel of the ENaC/degenerin superfamily, FMRFamide pep-tide–gated Na⁺ channel (FaNaCh; ref. 38), which has no PY motifs, was not influenced by the expression of either wild-type or mutant xNedd4. In the same experimental conditions as for ENaC, the FaNaCh associated I_Na were 5.0 ± 1.8 μA/oocyte in control oocytes (FaNaCh cRNA injected alone); I_Na reached 4.3 ± 1.0 and 5.2 ± 1.2 μA/oocyte with expression of the Nedd4-wt and mutant Nedd4, respectively (n = 16 for each groups, in two batches of oocytes). Taken together, these results demonstrate that Nedd4 inhibits ENaC activity.

Figure 1

Regulation of ENaC by Nedd4 overexpressed in Xenopus oocytes. (a) Immunoblot analysis showing expression of xNedd4 in oocytes injected with cRNA of ENaC alone (ENaC/H₂O), or ENaC coinjected with either 25 ng of xNedd4-wt cRNA (ENaC/Nedd4-wt) or the same amount of mutant xNedd4 lacking ubiquitin–protein ligase activity (ENaC/Nedd4-CS). Proteins from oocyte lysates were separated on SDS-PAGE and immunoblotted with anti-xNedd4 antibodies. The left lane shows the level of endogenous xNedd4. Faster-migrating protein bands in Nedd4-injected oocytes likely represent degradation products. (b) I_Na were measured in oocytes expressing either ENaC alone (ENaC/H₂O), ENaC plus xNedd4-wt (ENaC/Nedd4-wt), or ENaC plus xNedd4C938S (ENaC/Nedd4-CS). The measured currents were normalized to control oocytes (4.2 ± 0.6 μA/oocyte). n = 36 oocytes from six different batches. **P < 0.01 vs. ENaC/H₂O–injected oocytes. ENaC, epithelial sodium channel; I_Na, amiloride-sensitive Na⁺ current; wt, wild-type.

Figure 2

Dose–response relationship of the effect of Nedd4 on ENaC activity. (a) Immunoblot analysis showing expression of xNedd4 with increasing amounts of xNedd4 cRNA (ng/oocyte). (b) Corresponding normalized I_Na. The measured currents were normalized to control oocytes (6.1 ± 2.3 μA/oocyte). n = 16–18 oocytes from three different batches. **P < 0.01 vs. ENaC/H₂O–injected oocytes (i.e., 0 ng of xNedd4 cRNA). The difference in I_Na in oocytes injected with 0 and 0.2 ng cRNA/oocyte was not statistically significant.

The Nedd4-mediated downregulation of ENaC is dependent on the presence of the PY motifs. We have shown previously that the interaction between Nedd4 and ENaC occurs via Nedd4-WW domains binding to the PY motifs in ENaC (27). Therefore, we tested whether the functional regulation of ENaC by N edd4 is also dependent on the presence of intact PY motifs. To this end, we expressed a mutant ENaC channel in which the tyrosines of the PY motifs of all three subunits were mutated to alanines (in the case of α- and γ-ENaC), or to histidine in β-ENaC (Fig. 3); this ENaC mutant (ENaC-ΔPY₃) was thus missing all the binding sites for Nedd4 (27). When expressed in oocytes, such a channel showed an approximately fourfold increase in activity, as compared with ENaC-wt channels (Fig. 3, compare black columns). This elevated channel activity was unaltered by coexpression of either Nedd4-wt or of the inactive xN4C938S Nedd4 (Fig. 3), demonstrating that the PY motif–mutated ENaC has lost its responsiveness to Nedd4. Because deletion/mutation of the PY motifs of either β- or γ-ENaC is sufficient to cause Liddle's syndrome, we tested the responsiveness of such a Liddle's channel to Nedd4. Thus, we coexpressed Nedd4-wt or the xN4C938S mutant together with ENaC bearing a COOH-terminally truncated β-subunit (βR564stop), an originally described Liddle's mutation (2). Our results show that this Liddle's channel was only partially responsive to Nedd4-wt, with an effect intermediate between ENaC-wt possessing all PY motifs and ENaC lacking all of them (Fig. 3). Quantitatively, this βR564stop channel showed an approximately threefold increase in activity when compared with ENaC-wt (Fig. 3), and its coexpression with Nedd4-wt yielded only 71.5 ± 2.0% inhibition of I_Na compared with 95.1 ± 0.3% inhibition of ENaC-wt (this difference was significant, with P < 0.001; n = 24). Moreover, coexpression of the xN4C938S Nedd4 mutant with the βR564stop channel did not lead to the stimulation of channel activity seen with ENaC-wt. Thus, these data demonstrate that the absence of even one PY motif, as seen in Liddle's syndrome, is sufficient to interfere with Nedd4 regulation of ENaC, and that the greater the number of PY motifs (i.e., Nedd4-WW binding sites) present within the channel, the tighter the control of Nedd4 on ENaC activity.

Figure 3

ENaC PY motifs are necessary for the effect of Nedd4 on ENaC activity. Oocytes were either injected with ENaC-wt (left three bars), ENaC bearing mutations in all three PY motifs (ENaC-ΔPY₃; middle three bars), or a Liddle's ENaC channel missing the COOH terminus of β-ENaC (βR564stop), including its PY motif (right three bars), together with either H₂O (closed bars), Nedd4-wt (open bars), or xNedd4C938S (hatched bars). Currents were normalized to control mean values (5.5 ± 1.2 μA/oocyte). Twenty-four oocytes from four different batches were measured per condition. *P < 0.05 and **P < 0.01 represent levels of significance relative to conditions indicated by the brackets.

Nedd4 affects the number of channels at the plasma membrane. Because we demonstrated regulation of ENaC activity by Nedd4, we next investigated the mechanism(s) by which this regulation is accomplished. There are three possible mechanisms by which Nedd4 could affect ENaC activity: (a) the number of channels at the plasma membrane, (b) the open probability of the channel, and (c) the single-channel conductance. To quantitate the number of channels at the cell surface, we expressed ENaC channels that were FLAG- tagged at the extracellular loops of the β and γ subunits (ENaC^F) (25). This allowed quantification of binding of ¹²⁵I-labeled anti-FLAG antibodies (i.e., determination of the number of channels at the cell surface) and simultaneous measurement of I_Na in the same oocyte, as described by Firsov et al. (25). As seen in Fig. 4, the anti-FLAG antibody binding was affected by Nedd4 proportionally to the macroscopic changes in I_Na, suggesting that it is primarily the number of channels at the cell surface that is controlled by Nedd4. This is different from the βR564stop (Liddle's) channel, where Firsov et al. had previously demonstrated that the increase in ENaC numbers could only account for about half of the total increase in channel activity, and changes in open probability were also contributing to this elevated activity (25). To further substantiate these findings, we performed immunostaining of the injected oocytes using anti-FLAG antibodies that stain ENaC^F. As can be seen in Fig. 5, in control oocytes expressing ENaC alone, ENaC-related immunofl uorescence was visible in the plasma membrane as well as in a mainly reticular, intracellular compartment. When ENaC was coexpressed with xNedd4-wt, ENaC-related immunofluorescence was no longer detectable at the plasma membrane, and the staining intensity in the intracellular compartments seemed to be reduced, suggesting that ENaC was not only internalized, but also more rapidly degraded. In contrast, when the inactive Nedd4 mutant (xNC938S) was coexpressed with ENaC, ENaC-related immunostaining was drastically increased at the plasma membrane, whereas staining intensity in the intracellular, reticular compartment appeared to be unaffected, suggesting retention of ENaC at the cell surface and impairment of channel internalization and degradation. In control experiments with uninjected oocytes, no staining was observed (not shown; see also ref. 36). Collectively, these studies show that Nedd4 regulates the number of ENaC channels at the plasma membrane.

Figure 4

Nedd4 regulates the number of ENaC channels at the cell surface. Oocytes were coinjected with ENaC^F and either H₂O (left two bars), 25 ng xNedd4-wt cRNA (middle two bars), or xNedd4C938S (Nedd4-CS; right two bars). To quantitate the number of channels a t the cell surface, I_Na (closed bars) and binding of iodinated anti-FLAG antibodies (open bars) were measured in the same oocytes, as described previously (25). Current and binding values were normalized to control values (5.5 ± 1.2 μA/oocyte and 0.10 ± 0.01 fmol/oocyte, respectively). **P < 0.01 vs. ENaC^F/H₂O–injected oocytes.

Figure 5

Immunostaining of ENaC expressed in Xenopus oocytes coexpressing Nedd4-wt or -CS. ENaC^F-related membrane immunostainings were carried out with anti-FLAG antibodies in oocytes expressing ENaC alone (a), ENaC plus Nedd4-wt (b), or ENaC plus xNedd4C938S (Nedd4-CS; c) as detailed in Methods. Scale bar: 20 μm.

The goal of the present study was to provide a direct demonstration of the regulation of ENaC by Nedd4 and the implications of this regulation for Liddle's syndrome. The Xenopus laevis oocyte represents a convenient experimental system to study such questions, as we and others have previously shown that elevated Na⁺ channel (ENaC) activity can be observed when channels containing Liddle's syndrome mutations are expressed in these oocytes (22–24, 26). Moreover, because Xenopus oocytes express endogenous Nedd4 (27), they are likely to possess all the necessary machinery for proper Nedd4 function.

In our previous work, we demonstrated that rat Nedd4 is expressed in the same cells that express ENaC within the distal nephron and lung epithelia (27, 33), that Nedd4-WW domains bind to the PY motifs of ENaC (27), that point mutations within the PY motifs of ENaC identified in Liddle's syndrome patients (19–21) also abrogate binding to Nedd4-WW domains (27), and finally that ENaC stability and function are regulated by ubiquitination (32). In this study, we present several lines of evidence suggesting that it is indeed Nedd4 that is involved in the ubiquitination and the control of ENaC activity. First, overexpression of xNedd4-wt with ENaC in Xenopus oocytes caused a strong inhibition of I_Na when compared with oocytes expressing ENaC alone in the presence of endogenous xNedd4. Second, a catalytically inactive form of xNedd4 stimulates these currents, probably by competing with endogenous xNedd4. Third, the effect of xNedd4 is dependent on the presence of intact PY motifs within the ENaC subunits, suggesting an interaction between xNedd4 and the PY motifs, similar to our previous demonstration with rat Nedd4 (27). Moreover, FaNaCh, another channel of the ENaC/degenerin superfamily, which does not contain PY motifs (38), is not regulated by Nedd4.

The current findings provide some interesting insights into the mechanism by which Nedd4 mediates ENaC regulation. The observation that the PY motifs are necessary for Nedd4-dependent regulation strongly suggests that Nedd4 is indeed exerting its effect by binding via its WW domains to the ENaC PY motif. The completely opposite effects on channel function of Nedd4-wt versus the xN4C938S mutant support the hypothesis that Nedd4 is acting via ubiquitination, most likely by ubiquitinating ENaC directly, and thus leading to its retrieval from the plasma membrane and to degradation of the ENaC complex, as is clearly demonstrated by both quantitative surface labeling and immunostaining (Figs. 4 and 5). However, an effect of Nedd4 on ENaC during biosynthesis and recycling processes may also play a role. The inhibition of ENaC internalization in the catalytically impaired xNedd4 mutant is in agreement with our p revious findings demonstrating increased channel numbers and increased retention at the cell surface of ENaC bearing Lys→Arg mutations that render the channel ubiquitination defective (32). Recently, Shimkets et al. (39) have shown that ENaC is internalized via clathrin-coated pits and that this internalization is dependent on the presence of COOH-terminal endocytosis signals that encompass the PY motifs. It is interesting that a growing number of studies have now demonstrated a link between ubiquitination of transmembrane proteins at the cell surface and their subsequent endocytosis and degradation by the lysosomes or vacuoles in yeast (reviewed in ref. 31). Although it is not yet known how ubiquitination (which normally directs cytosolic or ER proteins to proteasomal degradation) provides a signal for endocytosis of transmembrane proteins, it is quite likely that ENaC also belongs to this class of cell-surface proteins in which ubiquitination and endocytosis and lysosomal degradation are tightly linked.

Our earlier demonstration of binding of Nedd4 to the regions (PY motifs) in ENaC that are deleted/mutated in patients with Liddle's syndrome (27) and our current demonstration of impaired regulation by Nedd4 of ENaC channels lacking one (as in Liddle's syndrome) of its PY motifs clearly implicate Nedd4 in the pathophysiology of this inherited form of hypertension. However, the increased retention of ENaC at the plasma membrane caused by loss of the PY motif, which leads to inhibition of internalization (39) and impaired binding to Nedd4 (thus likely to impaired ubiquitination), cannot provide a full explanation for the defective function of ENaC in Liddle's syndrome. As shown earlier by Firsov et al. (25), at least half of the increase in ENaC activity associated with the Liddle's mutations can be attributed to increased open probability of the channel. Whether or not Nedd4 can also affect channel gating is currently unknown. Interestingly, our recent work has demonstrated that ENaC is downregulated by elevated intracellular Na⁺ concentration (26, 40), and that this feedback inhibition is impaired in ENaC chains carrying the Liddle's mutations in their PY motifs (26). Because Nedd4 binds to these PY motifs, it is possible that it is involved in the negative regulation by intracellular Na⁺. In support of this notion, a recent report has suggested that Nedd4 mediates the control by intracellular Na⁺ of an epithelial Na⁺ channel in salivary ducts (41). The molecular identity of the latter channel, however, is unknown; hence deciphering its putative biochemical interactions with Nedd4 is currently not possible.

Despite the strong effect of wild-type or catalytically inactive xNedd4 on ENaC function, a much smaller effect of rat Nedd4 on ENaC activity was observed (data not shown) even though the protein was expressed at high levels and despite the fact that we used rat ENaC in our experiments. This suggests that either the presence of an additional WW domain in xNedd4 (which contains four WW domains versus three in rNedd4), which may allow better binding to the ENaC tetramer, or the presence of nonconserved NH₂-terminal sequences in xNedd4, which may play a role in localization or functioning of this ubiquitin–protein ligase, could contribute to these differences.

Although thus far there have not been any mutations in Nedd4 identified in patients with Liddle's syndrome, the search for such mutations in inherited forms of hypertension is nevertheless worthwhile in view of our clear demonstration of regulation of the channel by Nedd4. On the other hand, because Nedd4 is likely to have numerous cellular substrates, it is possible that it is an essential gene, as seen in yeast (42), and that mutations that severely interfere with its function may be lethal in higher organisms as well. A gene knockout of Nedd4 in mice, not yet available, may help shed some light on this issue.

In summary, our work provides strong evidence that Nedd4 is a suppressor of ENaC activity that regulates the number of ENaC channels at the plasma membrane. Moreover, it shows that the regulatory effect of Nedd4 requires the interaction between its WW domains and the PY motifs of ENaC, the same motifs that, when mutated, cause Liddle's syndrome. Accordingly, we have demonstrated that this suppressive effect is attenuated in Liddle's syndrome. Thus, this work has implications for our comprehension of the molecular and biochemical mechanisms underlying this disease, as well as for our understanding of ENaC function in general.

We would like to thank Dmitri Firsov for preparing the iodinated antibodies; Michel Lazdunski for providing FaNaCh cDNA; Lea Kläusli, Jerôme Dall’Aglio, and Ivan Gautschi for excellent technical assistance; and Bernard C. Rossier for critically reading the manuscript. This work was supported by grants of the Swiss National Science Foundation to O. Staub (31-52178.97), J.-D. Horisberger (31-45867.95), and L. Schild (31-049645.96); by a grant from the International Human Frontier Science Program to L. Schild, J.-D. Horisberger, and D. Rotin; by a Canadian Cystic Fibrosis Foundation grant to D. Rotin; by a Medical Research Council of Canada grant to D. Rotin; and by a National Institutes of Health grant (RO1-HL-35795) and a Veterans Affairs Merit Review grant to J.H. Pratt.

1. Garty, H, Palmer, LG. Epithelial sodium channels: function, structure, and regulation. Physiol Rev 1997. 77:359-396.
   View this article via: PubMed Google Scholar
2. Shimkets, RA, et al. Liddle's syndrome: heritable human hypertension caused by mutations in the β subunit of the epithelial sodium channel. Cell 1994. 79:407-414.
   View this article via: PubMed CrossRef Google Scholar
3. Chang, SS, et al. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet 1996. 12:248-253.
   View this article via: PubMed CrossRef Google Scholar
4. Stutts, MJ, et al. CFTR as a cAMP-dependent regulator of sodium channels. Science 1995. 269:847-850.
   View this article via: PubMed CrossRef Google Scholar
5. Hummler, E, et al. Early death due to defective neonatal lung liquid clearance in αENaC-deficient mice. Nat Genet 1996. 12:325-328.
   View this article via: PubMed CrossRef Google Scholar
6. O’Brodovich, HM. The role of active Na⁺ transport by lung epithelium in the clearance of airspace fluid. New Horizons 1995. 3:240-247.
   View this article via: PubMed Google Scholar
7. Liddle, GW, Bledsoe, T, Coppage (Jr), WS. A familial renal disorder simulating aldosteronism but with negligible aldosterone secretion. Trans Assoc Am Physicians 1963. 76:199-213.
8. Botero-Velez, M, Curtis, JJ, Warnock, DG. Brief report: Liddle's syndrome revisited. N Engl J Med 1994. 330:178-181.
   View this article via: PubMed CrossRef Google Scholar
9. Hansson, JH, et al. Hypertension caused by a truncated epithelial sodium channel gamma subunit: genetic heterogeneity of Liddle syndrome. Nat Genet 1995. 11:76-82.
   View this article via: PubMed CrossRef Google Scholar
10. Canessa, CM, Horisberger, J-D, Rossier, BC. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 1993. 361:467-470.
    View this article via: PubMed CrossRef Google Scholar
11. Canessa, CM, et al. Amiloride-sensitive epithelial Na⁺ channel is made of three homologous subunits. Nature 1994. 367:463-467.
    View this article via: PubMed CrossRef Google Scholar
12. Lingueglia, E, Voilley, N, Waldmann, R, Lazdunski, M, Barbry, P. Expression cloning of an epithelial amiloride-sensitive Na+ channel. A new channel type with homologies to Caenorhabditis elegans degenerins. FEBS Lett 1993. 318:95-99.
    View this article via: PubMed CrossRef Google Scholar
13. Lingueglia, E, et al. Different homologous subunits of the amiloride-sensitive Na+ channel are differently regulated by aldosterone. J Biol Chem 1994. 269:13736-13739.
    View this article via: PubMed Google Scholar
14. Firsov, D, Gautschi, I, Mérillat, A-M, Rossier, BC, Schild, L. The heterotetrameric architecture of the epithelial sodium channel (ENaC). EMBO J 1998. 17:344-352.
    View this article via: PubMed CrossRef Google Scholar
15. Canessa, CM, Mérillat, A-M, Rossier, BC. Membrane topology of the epithelial sodium channel in intact cells. Am J Physiol 1994. 267:C1682-C1690.
    View this article via: PubMed Google Scholar
16. Renard, S, Lingueglia, E, Voilley, N, Lazdunski, M, Barbry, P. Biochemical analysis of the membrane topology of the amiloride-sensitive Na+ channel. J Biol Chem 1994. 269:12981-12986.
    View this article via: PubMed Google Scholar
17. Snyder, PM, McDonald, FJ, Stokes, JB, Welsh, MJ. Membrane topology of the amiloride-sensitive epithelial sodium channel. J Biol Chem 1994. 269:24379-24383.
    View this article via: PubMed Google Scholar
18. Chen, HI, Sudol, M. The WW domain of Yes-associated protein binds a novel proline-rich ligand that differs from the consensus established for SH3-binding modules. Proc Natl Acad Sci USA 1995. 92:7819-7823.
    View this article via: PubMed CrossRef Google Scholar
19. Hansson, JH, et al. A de novo missense mutation of the β subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity. Proc Natl Acad Sci USA 1995. 25:11495-11499.
20. Tamura, H, et al. Liddle disease caused by a missense mutation of beta subunit of the epithelial sodium channel gene. J Clin Invest 1996. 97:1780-1784.
    View this article via: JCI PubMed Google Scholar
21. Inoue, J, et al. A family with Liddle's syndrome caused by a new missense mutation in the β subunit of the epithelial sodium channel. J Clin Endocrinol Metab 1998. 83:2210-2213.
    View this article via: PubMed CrossRef Google Scholar
22. Schild, L, et al. A mutation in the epithelial sodium channel causing Liddle's disease increases channel activity in the Xenopus laevis oocyte expression system. Proc Natl Acad Sci USA 1995. 92:5699-5703.
    View this article via: PubMed CrossRef Google Scholar
23. Snyder, PM, et al. Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na⁺ channel. Cell 1995. 83:969-978.
    View this article via: PubMed CrossRef Google Scholar
24. Schild, L, et al. Identification of a PY-motif in the epithelial Na+ channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome. EMBO J 1996. 15:2381-2387.
    View this article via: PubMed Google Scholar
25. Firsov, D, et al. Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach. Proc Natl Acad Sci USA 1996. 93:15370-15375.
    View this article via: PubMed CrossRef Google Scholar
26. Kellenberger, S, Gautschi, I, Rossier, BC, Schild, L. Mutations causing Liddle-syndrome reduce sodium-dependent downregulation of the epithelial sodium channel in the Xenopus oocyte expression system. J Clin Invest 1998. 101:2741-2750.
    View this article via: JCI PubMed Google Scholar
27. Staub, O, et al. WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na⁺ channel deleted in Liddle's syndrome. EMBO J 1996. 15:2371-2380.
    View this article via: PubMed Google Scholar
28. Kumar, S, Tomooka, Y, Noda, M. Identification of a set of genes with developmentally down-regulated expression in the mouse brain. Biochem Biophys Res Commun 1992. 185:1155-1161.
    View this article via: PubMed CrossRef Google Scholar
29. Staub, O, Rotin, D. WW domains. Structure 1996. 4:495-499.
    View this article via: PubMed CrossRef Google Scholar
30. Huibregtse, JM, Scheffner, M, Beaudenon, S, Howley, PM. A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc Natl Acad Sci USA 1995. 92:2563-2567.
    View this article via: PubMed CrossRef Google Scholar
31. Hershko, A, Ciechanover, A. The ubiquitin system. Annu Rev Biochem 1998. 67:425-479.
    View this article via: PubMed CrossRef Google Scholar
32. Staub, O, et al. Regulation of stability and function of the epithelial Na⁺ channel (ENaC) by ubiquitination. EMBO J 1997. 16:6325-6336.
    View this article via: PubMed CrossRef Google Scholar
33. Staub, O, et al. Immunolocalization of the ubiquitin-protein ligase Nedd4 in tissues expressing the epithelial Na⁺ channel (ENaC). Am J Physiol 1997. 272:C1871-C1880.
    View this article via: PubMed Google Scholar
34. Duc, C, Farman, N, Canessa, CM, Bonvalet, J-P, Rossier, BC. Cell-specific expression of epithelial sodium channel αβ and γ subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J Cell Biol 1994. 127:1907-1920.
    View this article via: PubMed CrossRef Google Scholar
35. Rebhun, J.F., and Pratt, J.H. 1998. Molecular cloning of Nedd4 from Xenopus laevis.DNA Seq. In press.
    View this article via: PubMed Google Scholar
36. Mastroberardino, L, et al. Ras pathway activates epithelial Na⁺ channel and decreases its surface expression in Xenopus oocytes. Mol Biol Cell 1998. 9:3417-3427.
    View this article via: PubMed Google Scholar
37. Scheffner, M, Nuber, U, Huibregtse, JM. Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature 1995. 375:81-83.
    View this article via: PubMed CrossRef Google Scholar
38. Lingueglia, E, Champigny, G, Lazdunski, M, Barbry, P. Cloning of the amiloride-sensitive FMRFamide peptide-gated sodium channel. Nature 1995. 378:730-733.
    View this article via: PubMed CrossRef Google Scholar
39. Shimkets, RA, Lifton, RP, Canessa, CM. The activity of the epithelial sodium channel is regulated by clathrin-mediated endocytosis. J Biol Chem 1997. 272:25537-25541.
    View this article via: PubMed CrossRef Google Scholar
40. Ishikawa, T, Marunaka, Y, Rotin, D. Electrophysiological characterization of the rat epithelial Na⁺ channel (rENaC) expressed in MDCK cells. Effects of Na⁺ and Ca²⁺. J Gen Physiol 1998. 111:825-846.
    View this article via: PubMed CrossRef Google Scholar
42. Dinudom, A, et al. Nedd4 mediates control of an epithelial Na⁺ channel in salivary duct cells by cytosolic sodium. Proc Natl Acad Sci USA 1998. 95:7169-7173.
    View this article via: PubMed CrossRef Google Scholar
43. Hein, C, Springael, J-Y, Volland, C, Haguenauer-Tsapis, R, Andre, B. NPI1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin-protein ligase. Mol Microbiol 1995. 18:77-87.
    View this article via: PubMed CrossRef Google Scholar