Article

• The EMBO Journal (1997) 16, 6325 - 6336
• doi:10.1093/emboj/16.21.6325

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

O. Staub¹, I. Gautschi², T. Ishikawa¹, K. Breitschopf³, A. Ciechanover³, L. Schild² and D. Rotin¹

1. The Hospital for Sick Children, Division of Respiratory Research, 555 University Avenue, Toronto, Ontario M5G 1X8 and Biochemistry Department, University of Toronto, Toronto, Canada
2. Institut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1015 Lausanne, Switzerland
3. Biochemistry Department, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel

Correspondence to:

D. Rotin, E-mail: drotin@sickkids.on.ca

Received 23 April 1997; Revised 11 August 1997

• Keywords:

  • ENaC,
  • half-Life,
  • lysosome,
  • proteasome,
  • ubiquitination

Introduction

Amiloride-sensitive epithelial Na⁺ channels, located at the apical membrane of Na⁺-transporting epithelia, play an essential role in the control of salt and fluid homeostasis in the kidney and the colon (Rossier and Palmer, 1992), as well as in fluid clearance from the alveolar space at birth and during pulmonary oedema (Saumon and Basset, 1993; O'Brodovich, 1995). These channels are tightly regulated by hormones such as aldosterone, vasopressin and insulin as well as PKA/cAMP, PKC, Ca²⁺ and G proteins (Garty and Palmer, 1997).

The amiloride-sensitive epithelial Na⁺channel (ENaC) was cloned recently from rat (Canessa et al., 1993, 1994a; Lingueglia et al., 1993, 1994), human (McDonald et al., 1994, 1995; Voilley et al., 1994) and other species, and shown to be composed of three homologous subunits, -, - and ENaC. Each subunit consists of two transmembrane regions, a large extracellular domain and cytosolic N- and C-termini (Canessa et al., 1994b; Renard et al., 1994; Snyder et al., 1994) including proline-rich regions in each C-terminus (Rotin et al., 1994; Staub et al., 1996). Proper function of ENaC is crucial, as indicated by the finding that a gene knockout of ENaC is lethal due to the inability of the -/- mice to clear fluid from their lungs shortly after birth (Hummler et al., 1996). Abnormally high ENaC activity has been also demonstrated in cystic fibrosis patients (Stutts et al., 1995). Moreover, several inherited human disorders were recently linked by genetic linkage analysis to mutations in the ENaC subunits. These include the salt-wasting disease pseudohypoaldosteronism type 1 (PHA1, Chang et al., 1996; Strautnieks et al., 1996) and Liddle syndrome, an inherited autosomal dominant form of human hypertension (Liddle et al., 1963; Botero-Velez et al., 1994). In all these disorders, alteration in channel gating and/or numbers has been associated with the disease (Chang et al., 1996; Firsov et al., 1996).

We have recently identified the ubiquitin–protein ligase Nedd4 as an interacting protein of ENaC (Staub et al., 1996), and therefore wanted to investigate whether ENaC is ubiquitinated in living cells and whether such putative ubiquitination is involved in regulating channel number and function. Ubiquitination of cellular proteins usually serves to tag them for rapid degradation (reviewed in Ciechanover, 1994; Jentsch and Schlenker, 1995). It involves the covalent attachment of ubiquitin or a polyubiquitin tree onto lysine residues in target proteins. Several enzymes are involved in this process, including a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a ubiquitin–protein ligase (E3). In most studies described to date, ubiquitinated proteins are degraded by the 26S proteasome, a cytosolic threonine protease complex (Jentsch and Schlenker, 1995; Hilt and Wolf, 1996). Extensive work has implicated the involvement of the ubiquitin-proteasome system in regulating/degrading numerous cytosolic proteins, including several cell cycle proteins (e.g. cyclin A, B, Clb5p, cln 1, 2, 3p, SIC1) (reviewed in Deshaies, 1995), NFB and IB (Palombella et al., 1994), and the nuclear proteins c-Jun (Treier et al., 1994) and p53 (Scheffner et al., 1993). In addition, recent evidence suggests that ER-associated protein degradation of either misfolded or improperly assembled protein complexes involves the proteasome in either a ubiquitin-dependent or -independent fashion (reviewed in Brodsky and McCracken, 1997). For example, the immature form of CFTR becomes ubiquitinated in the ER and degraded by the proteasome (Jensen et al., 1995, Ward et al., 1995).

In recent years, it has become apparent that some transmembrane proteins also become ubiquitinated, and that ubiquitination is involved in their subsequent degradation by the endosomal/lysosomal pathway (reviewed in Hochstrasser, 1996). Examples of mammalian transmembrane proteins which become ubiquitinated include several tyrosine kinase receptors (e.g. EGFR, PDGFR, c-kit), cytokine receptors (e.g. T cell and IgE receptors) (Cenciarelli et al., 1992; Mori et al., 1992; Paolini and Kinet, 1993; Miyazawa et al., 1994; Galcheva-Garbova et al., 1995) and the growth hormone receptor, in which ligand-induced, ubiquitination-dependent endosomal/lysosomal degradation of the receptor was recently demonstrated (Strous et al., 1996). In yeast, the membrane receptor Ste2 (Hicke and Riezman, 1996) and membrane transporters Ste6 and Pdr5 (Kölling and Hollenberg, 1994; Egner and Kuchler, 1996), as well as several yeast amino acid permeases including Gap1 and Fur4 (Hein et al., 1995; Galan et al., 1996), have been shown to become ubiquitinated, a necessary step for their subsequent degradation in the vacuoles. The exact degradation pathway for ubiquitinated transmembrane proteins, and the possible involvement of the proteasome complex in this process, is not known (Hochstrasser, 1996).

In this report we show that ENaC is a short-lived protein which becomes ubiquitinated in living cells on the and subunits. The rapid turnover of ENaC is sensitive to inhibitors of both the lysosomal and proteasomal degradation systems; whereas excess of unassembled (or misfolded) subunits are degraded by the proteasome, our data suggest that the assembled ENaC complex is targeted to the lysosome. Moreover, mutating a cluster of lysines (to arginines) at the N-terminus of ENaC, or - plus ENaC, causes reduced ubiquitination, slower turnover and increased Na⁺ channel activity due to increased number of channels at the plasma membrane. Using Brefeldin A treatment, we demonstrate that this increase is caused by an elevated number of mutant channels retained at the cell surface, as well as by increased incorporation of mutant channels into the plasma membrane. These results suggest that ubiquitination of ENaC controls the number of channels at the plasma membrane, and provides a powerful means to regulate channel function.

ENaC subunits are short-lived proteins with a rapid turnover rate

We have shown previously (Staub et al., 1996) that the ubiquitin protein ligase Nedd4 binds to ENaC. Since ubiquitination of proteins is usually associated with their rapid turnover, we initially investigated the turnover rate of ENaC using pulse–chase experiments. For these and all subsequent experiments, we used rat ENaC (rENaC), which share 85% overall amino acid sequence identity with the corresponding human ENaC (hENaC) subunits (Canessa et al., 1993, 1994a; McDonald et al., 1994, 1995). Pulse–chase experiments were performed with either kidney epithelial MDCK cells or with NIH-3T3 fibroblasts previously triple-transfected and stably expressing ENaC (Stutts et al., 1995). Cells were labelled for 2 h with [³⁵S]methionine/cysteine and then chased for various periods of time. The ENaC proteins were subsequently immunoprecipitated with antibodies directed against each of the ENaC subunits, and separated on SDS–PAGE (Figure 1). Following SDS–PAGE and autoradiography, bands were excised and radioactivity quantified by scintillation counting to determine half-life (t_1/2) of the proteins. As is evident from Figure 1A, - and ENaC expressed in MDCK cells have a rapid turnover rate with a half-life of 1 h; ENaC, on the other hand, has a longer half-life (t_1/2 >3 h). The half-life of the ENaC subunits measured following 1 h pulse was the same as that following 2 h pulse (not shown). In addition, the turnover rates of ENaC expressed in NIH-3T3 cells were very similar to those expressed in MDCK cells (data not shown).

Figure 1.

Half-life of ENaC. Pulse–chase experiments of ³⁵S-labelled (upper panel), (middle panel) and (lower panel) rat ENaC (called ENaC) stably expressed together in epithelial MDCK cells. Cells were labelled with [³⁵S]Met and Cys for 2 h and chased for the indicated times (hrs). The calculated t_1/2 values are: ENaC: 1.13 0.13 (mean SE, n = 9) h; ENaC: 1.23 0.09 (mean SE, n = 6) h; ENaC: t_1/2 >3 h. Similar results were obtained with NIH-3T3 cells (not shown).

View full figure (76 KB)

- and ENaC, but not ENaC, are ubiquitinated in vivo

A rapid turnover of proteins is often associated with ubiquitination-mediated degradation. We thus wanted to test whether the ENaC subunits are ubiquitinated in living cells. For these experiments, we used epitope-tagged - or ENaC because the antibodies available against these native subunits, unlike the antibodies against native ENaC, are not sensitive enough for immunoblotting. We subcloned HA-tagged ENaC, FLAG-tagged ENaC or untagged ENaC into CMV-based expression vectors. The HA or FLAG tags were added just upstream of the stop codons of - or ENaC respectively, since tagging at these sites had no effect on channel activity (not shown). We then transiently co-transfected HA-tagged ENaC, FLAG-tagged ENaC and ENaC together with a plasmid encoding His-tagged multiubiquitin (His-Ub) (Treier et al., 1994) (kindly provided by D.Bohmann) into Hek-293 cells. These cells were chosen because MDCK cells are not easily amenable to transient transfections. The ENaC subunits were expressed together (in Hek-293 cells) to allow for the formation of a functional channel complex that is able to reach the plasma membrane (Canessa et al., 1994a; Firsov et al., 1996). Twenty-four hours after transfection, cells were lysed, denatured with 2% SDS, diluted with lysis buffer and the diluted lysates incubated with Ni²⁺-NTA beads to allow for binding of the histidinated (and therefore ubiquitinated) proteins. After thorough washes, bound proteins were eluted and separated on SDS–PAGE, followed by immunoblotting with either anti-HA (i.e. anti-ENaC), anti-FLAG (i.e. anti-ENaC), or anti-ENaC antibodies directed against the C-terminus of ENaC (kindly provided by B.C. Rossier). As can be seen in Figure 2A, B and C (bottom panels, lysates), -, - and ENaC were all expressed in the transfected cells (in the same experiment). The precipitation of His-tagged (ubiquitinated) proteins from lysates of these transfected cells (with Ni²⁺-NTA beads) revealed high molecular weight ubiquitinated species of ENaC (Figure 2A, top panel, bracket) and ENaC (Figure 2C, top panel, bracket), as detected by anti-HA or anti-FLAG antibodies respectively; these ubiquitinated ENaC proteins were only detected in cells co-transfected with the His-Ub construct, and not in cells transfected with the ENaC subunits alone or with the His-Ub construct alone (Figure 2A, B and C, top panels). In contrast to - or ENaC, the ENaC subunit was not ubiquitinated (Figure 2B, upper panel), even though ENaC was strongly expressed in these cells (Figure 2B, lower panel). The ubiquitination of - and ENaC did not seem to be a peculiar property of one cell line, because it was also observed in transfected COS-7 cells (not shown). These results, therefore, demonstrate that in ENaC-transfected cells, and ENaC, but not ENaC, are ubiquitinated in vivo.

Figure 2.

Ubiquitination of and , but not , ENaC expressed in Hek-293 cells. Hek-293 cells transiently co-transfected (tfxn) with HA-tagged ENaC (), FLAG-tagged ENaC (), ENaC () and/or the His-Ub (Ub) construct were lysed and lysates incubated with Ni²⁺-NTA agarose beads (Ni²⁺-NTA) to precipitate histidinated (ubiquitinated) proteins. These proteins were separated on 7% SDS–PAGE, transferred to nitrocellulose and blotted with (A) anti-HA antibodies (to detect ubiquitinated ENaC), (B) anti-ENaC antibodies (to detect ubiquitinated ENaC) and (C) anti-FLAG (flag) antibodies (to detect ubiquitinated ENaC). Ubiquitinated species appear as a cluster of high molecular weight bands (upper panels, square brackets). Lower panels of each blot (Lysate) represent expression of the transfected protein (-, - or ENaC) in the lysates. Arrows indicate fully glycosylated forms of ENaC subunits.

View full figure (86 KB)

Mutation of conserved N-terminal lysines hyperactivates ENaC channel activity

Ubiquitination of proteins is mediated by the addition of ubiquitin or multiubiquitin groups onto Lys residues. Since we identified ubiquitination of - and ENaC in vivo, we wanted to investigate whether mutation of Lys residues, which may serve as the ubiquitin attachment sites, could lead to increased Na⁺ channel activity. Inspection of the sequences of rat (r) and human (h) ENaC (Canessa et al., 1994a; McDonald et al., 1994, 1995) revealed conserved Lys residues at the N-termini of these subunits (Figure 3). Most of these lysines are also conserved in xENaC, the Xenopus homologue of ENaC (Puoti et al., 1995). The cytosolic C-termini of these subunits are largely devoid of lysines, and the one or two lysines that are present in - or rENaC (respectively) are very close to the transmembrane domain and are not conserved. We therefore mutated the conserved lysines at the N-termini of -, - and rENaC (herein referred to as ENaC) to arginines either individually or in groups in those cases where the lysines were clustered close to each other. The mutants generated were as follows (see Figure 3): (i) In ENaC: K47 and K50 (K47,50R), K108 (K108R) or K47,50R plus K108R (K47,50,108R). An additional ENaC construct was also generated, in which lysines 23, 26 and 32 (which are not conserved between rat and human) of the K47,50R construct were mutated to Arg, yielding the mutant K23-50R. (ii) In ENaC: K4, K5 and K9 (K4,5,9R) or K16, K23, K39, K47, K48 and K49 (K16-49R). (iii) In ENaC: K6, K8, K10, K12, K13 (K6-13R), K26R (K26R) or K6-13 plus K26R (K6-13+26R).

Figure 3.

N-terminal Lys to Arg mutants of ENaC. Schematic representation of the conserved lysine residues at the N-termini of rENaC and hENaC. All conserved lysines (Bold letters, boxed), as well as three non-conserved lysine residues in ENaC, are numbered according to the published rat ENaC sequences (Canessa et al., 1994a). The numbered lysines were mutated to arginines to create the following mutant constructs: in ENaC: K47,50R, K108R, K47,50,108R and K23-50R; in ENaC: K4,5,9R and K16-49R; in ENaC: K6-13R, K26R and K6-13R + K26R. (TM1 = the first transmembrane domain of each ENaC subunit.)

View full figure (73 KB)

We then investigated the effect of the above Lys to Arg mutations in ENaC on ENaC channel activity. Two-electrode voltage clamp experiments were performed in which amiloride-sensitive Na⁺ currents (I_Na) were measured in Xenopus oocytes expressing the different Lys to Arg mutants. Our results show that none of the conserved Lys to Arg ENaC mutants, when expressed together with wild type (wt) ENaC, had any effect on increasing ENaC activity (Figure 4A). This lack of effect on ENaC function was also observed with the extended K23-50R mutant (not shown). Similarly, none of the Lys to Arg mutations within the N-terminus of ENaC, expressed together with wt-ENaC, caused any increase in amiloride-sensitive Na⁺ currents. In contrast, the expression of the ENaC mutants (K6-13R or K6-13+26R) together with wt-ENaC more than doubled Na⁺ channel activity (Figure 4A). Moreover, when this K6-13R mutant was co-expressed together with the K47,50R mutant (along with wt-ENaC), the hyperactivation of ENaC was potentiated, yielding up to 6-fold stimulation of Na⁺ channel activity when compared to the activity of the wt channel (Figure 4A). In contrast, such potentiation was not observed when the K4,5,9R or the K16-49R mutant was co-expressed with either K47,50R, K6-13R or both mutants (Figure 4A). These results therefore demonstrate that a cluster of conserved Lys residues particularly in ENaC, but also in ENaC, are responsible for the regulation of ENaC activity, and when mutated, lead to an increase in amiloride sensitive Na⁺ current (I_Na). Moreover our data show that the Lys to Arg substitutions increase channel activity only when performed on subunits previously shown to be ubiquitinated in vivo (- and ENaC).

Figure 4.

Na⁺ channel activity of Lys to Arg mutants of ENaC expressed in Xenopus oocytes. (A) cRNA derived from the Lys to Arg mutants of -, - or ENaC (depicted in Figure 3) was injected individually or in combination into oocytes, together with wild type (wt) ENaC cRNA of the remaining ENaC subunits and amiloride-sensitive Na⁺ currents (I_Na) measured by the two electrode voltage clamp technique. Bars represent means SE of four independent experiments each with 8–10 oocytes. Asterisks denote statistically significant differences (P <0.05) from wt-ENaC. (B) Upper panel: Representative single channel recording of the K47,50R+K6-13R (plus wt-) ENaC double mutant in Xenopus oocytes using the patch–clamp technique in a cell-attached configuration, with Li⁺ ions as the major permeant ion in the patch pipet. Recording was performed at pipette potential (V_pip) of 100 V. At least six active channels are present in the patch as shown by the different current levels, and downward deflexions represent channel openings. Lower panel: Current–voltage (I/V) relationship of single channel Li⁺ currents measured between -50 and -150 mV and determined from five different channel recordings. Regression analysis of data points gives a single channel conductance for Li⁺ ions of 8.6 pS. Dotted line represents a fit to the constant field equation.

View full figure (48 KB)

Lys to Arg mutations in ENaC, or in - plus ENaC, lead to an increase of Na⁺ channel numbers at the cell surface

The elevated Na⁺ channel activity associated with the Lys to Arg mutations in ENaC or - plus ENaC was determined by an increase in amiloride-sensitive Na⁺ current. In theory, such an increase can result from either an increase in the number of channels (N) expressed at the cell surface, and/or higher Na⁺ influx per channel molecule when the open probability (Po) or single channel conductance are increased. Analysis by patch clamp technique of single channel currents of the K47,50R plus K6-13R double mutant (Figure 4B) revealed a high number of active channels per patch without significant changes in single channel conductance for Li⁺ ions, a highly permeant cation in ENaC. The conductance for Li⁺ ions of 8–9 pS was similar to that found for wt-ENaC (Schild et al., 1997), indicating that these Lys to Arg mutations did not affect the conductive properties of the channel. To distinguish between changes in the channel open probability and increased surface expression of ENaC, we used a quantitative binding assay to correlate in individual oocytes the amiloride-sensitive I_Na with the number of channel molecules present at the cell surface, as previously described (Firsov et al., 1996). This binding assay uses specific ¹²⁵I-labelled antibodies directed against a FLAG epitope introduced into the extracellular domain of each of the ENaC subunits (wt or the above Lys to Arg mutants), as described in Materials and methods. Such epitope tagging does not interfere with channel function (Firsov et al., 1996). Figure 6A and B show representative experiments in which amiloride-sensitive I_Na and surface expression of wt-ENaC or Lys to Arg mutants of - and ENaC (K47,50R; K6-13R; K47,50R + K6-13R) were measured in individual oocytes. The Lys to Arg mutants of ENaC were not tested since none of them was able to elevate channel activity (Figure 4A). Comparing the ENaC mutants with wt confirms that the K47,50R mutations did not increase the amiloride sensitive Na⁺ current (as shown in Figure 4A) or the number of channel molecules expressed at the cell surface (Figure 5A). For the K6-13R or the K47,50R plus K6-13R double mutant, the increase in I_Na relative to wt-ENaC was directly proportional to the increase in the number of binding sites recognized by the anti-FLAG antibodies. The significant linear correlation between the current expressed and the number of ENaC channel molecules indicates that the Na⁺ current per channel was not significantly altered in the Lys to Arg mutants relative to wt-ENaC. These results are summarized quantitatively in Figure 5C. Since the I_Na measured in the Lys to Arg mutants increased linearly with the specific binding of the anti-FLAG antibodies, we conclude that this increase was caused by an elevated number of channels at the cell surface, and not by changes in the channel open probability. This increase in number of channels at the plasma membrane induced by the Lys to Arg mutations in the and subunits would be consistent with an inhibition of ubiquitination of the channel protein.

Figure 6.

Inhibition of ubiquitination of the Lys to Arg mutants of - and ENaC. Hek-293 cells were transiently co-transfected (tfxn) with ENaC (), HA-tagged ENaC () or K23-50R (R), and FLAG-tagged ENaC () or K6-13R (R), with or without His-Ub (Ub). Cells were then lysed, and lysates incubated with Ni²⁺-NTA agarose beads to precipitate ubiquitinated proteins. Precipitated proteins were separated on 7% SDS–PAGE, transferred to nitrocellulose and blotted with either (A) anti-FLAG (ENaC), (B) anti-HA (ENaC) or (C) anti-ENaC antibodies (upper panels), to detect ubiquitination of each subunit or its Lys to Arg mutants in the same experiment. Lower panels represent levels of expression of the transfected proteins in the lysate. Square brackets mark the ubiquitinated species of and ENaC. The 105 kDa band across panel B (top) is probably a contaminant. (D) K6-13R mutant is more stable than wt-ENaC. MDCK cells stably expressing wt-ENaC (called WT) or K6-13R plus wt-ENaC (called K6-13R) (both subunits FLAG-tagged at the C terminus, as in Figure 2 above) were labelled with [³⁵S]Met/Cys and chased for the indicated times (hrs). Cells were then lysed, immunoprecipitated with anti ENaC antibodies, the immunoprecipitates separated on 7% SDS–PAGE and visualized by autoradiography, as represented by the autoradiogram. Histogram represents t_1/2 quantitation (mean SE) of three independent experiments similar to that depicted in the autoradiogram.

View full figure (63 KB)

Figure 5.

Correlation of surface labelling and function of Lys to Arg mutants of - and ENaC measured in individual oocytes. (A and B) Representative experiments in which amiloride-sensitive I_Na and surface expression of wt-ENaC, or the indicated Lys to Arg mutants, were measured in individual oocytes. Surface expression was quantified by binding of anti-FLAG ¹²⁵I-labelled antibody (Ab) to the FLAG-tagged ectodomains of (A) wt-ENaC (open circles), K47,50R (open triangles) or the K47,50R + K6-13R mutants (closed circles), and (B) wt-ENaC (open circles) or the K6-13R mutant (closed squares). Solid lines represent regression analysis of the data. Dotted lines represent the 95% confidence limits. (C) Summary histogram showing changes of the amiloride-sensitive current (I_Na) and specific cell surface binding of Ab, relative to the values obtained with wt-ENaC. Values are mean SE of four to five independent experiments in which average I_Na and antibody binding were determined from simultaneous measurements in eight to ten individual oocytes. Asterisks denote statistical significance at P <0.05 level.

View full figure (57 KB)

Inhibition of ubiquitination of the Lys to Arg mutants of ENaC, or of -plus ENaC

As we observed an increase in channel activity resulting from a greater number of ENaC at the plasma membrane in the K6-13R mutant, we wanted to test whether such increased cell surface number in ENaC channels is caused by impaired ubiquitination of this mutant. Since direct determination of ENaC ubiquitination in oocytes is not currently feasible due to limits of protein detection (since only a few oocytes can be analysed at a time, not sufficient for biochemical assays), we opted instead to use mammalian cells, which allow the use of a large number of cells per assay and are thus more suitable for biochemical studies. We therefore co-transfected Hek-293 cells with the His-Ub construct together with either wt-ENaC (control), or wt-ENaC plus the K6-13R mutants of ENaC. As before, the ENaC constructs were HA-tagged and the ENaC constructs were FLAG-tagged. Following transfection, cells were lysed, and lysates incubated with Ni²⁺-NTA beads to precipitate ubiquitinated proteins. These proteins were then separated on SDS–PAGE, transferred to nitrocellulose and immunoblotted with anti-FLAG antibodies, to detect the extent of ubiquitination of ENaC. Our results show that ubiquitination of ENaC was indeed inhibited in the K6-13R mutant (Figure 6A, upper panel, compare lanes 3 and 4), despite similar expression levels of the wt or mutant ENaC proteins in the lysate of the transfected cells (Figure 6A, lower panel, lanes 3 and 4). The same inhibition of ubiquitination of ENaC was also observed when the K6-13R mutant was expressed together with the K23-50R mutant of ENaC (Figure 6A, lanes 5 and 6). These results therefore demonstrate that those conserved Lys residues located at the N-terminus of ENaC which are involved in regulating channel stability at the plasma membrane (K6-13) indeed serve as attachment sites for ubiquitin.

We next wanted to determine the extent of ubiquitination of the Lys to Arg mutants of ENaC when expressed either with wt-ENaC or with the K6-13R mutant of ENaC (plus wt-ENaC). This was done in order to test whether the potentiation of ENaC hyperactivation and retention at the cell surface observed in the double mutant (i.e. Lys to Arg mutations in both - and ENaC) is associated with reduced ubiquitination of not only ENaC, but also of ENaC. We therefore re-probed the blot shown in Figure 6A with HA antibodies, to determine the extent of ubiquitination of ENaC in the same experiment. Our results show that the K23-50R mutant [similar to the K47,50R mutant (data not shown)] was still ubiquitinated when co-expressed with wt-ENaC and His-Ub (Figure 6B, lane 3). In contrast, however, there was a clear and reproducible inhibition of this ubiquitination when the K23-50R mutant was co-transfected with the K6-13R mutant (and wt-ENaC plus His-Ub) (Figure 6B, lane 4). This reduction in ubiquitination was apparent despite similar expression levels of the wt or mutant ENaC proteins (Figure 6B, lower panel). Thus, the combined removal of lysines 6–13 from ENaC together with N-terminal lysines from ENaC caused a reduction of ubiquitination of both subunits, in agreement with the observed potentiation of cell surface retention and activity of ENaC under these conditions. None of the above Lys to Arg mutations in or ENaC, or both, caused any detectible ubiquitination of ENaC (Figure 7C).

Figure 7.

The effect of Brefeldin A on channel activity of wt-ENaC or the K47,50R+K6-13R mutant. cRNA from (A) wt-ENaC (WT) or (B) the K47,50R+K6-13R (plus wt-) mutant, was injected into Xenopus oocytes. Eighteen hours later, 10 g/ml Brefeldin A (BFA) were added (at time 0, arrow) to the bathing medium and amiloride-sensitive Na⁺ currents (I_Na) measured by the two-electrode voltage clamp technique at the indicated times. Data points represent mean SE of four separate experiments each performed with five oocytes.

View full figure (49 KB)

Our data show that ENaC turnover is regulated by ubiquitination, and that removal of potential ubiquitination sites on ENaC (K6-13R) leads to stabilization of ENaC at the plasma membrane. A prediction of these data is that the half-life of the K6-13R mutant should be prolonged. We therefore generated stable MDCK cell lines which express either FLAG-tagged wt- (control), or FLAG-tagged K6-13R mutant, together with wt-ENaC (the FLAG tag does interfere with channel function, data not shown). We then performed a pulse–chase experiment as described in Figure 1 above. Our results show that ENaC bearing the K6-13R mutations was indeed more stable (mean t_1/2 = 90 min) than the wt-ENaC (mean t_1/2 = 76 min), showing a small but statistically significant different (P = 0.027) increase in half-life of the protein (Figure 6D).

Collectively, these results show that ENaC stability and activity is regulated by ubiquitination. The primary target for this ubiquitination-mediated regulation is a cluster of lysine residues (K6–13) at the N-terminus of ENaC, since mutation of these lysine residues was sufficient to both increase channel number at the plasma membrane and to reduce its ubiquitination. ENaC, but not ENaC, is also playing a role in this regulation of channel stability by ubiquitination.

Accumulation of Brefeldin A-resistant pool of the Lys to Arg ENaC mutant at the cell surface

To test whether the impaired ubiquitination and increased channel number and activity of the Lys to Arg ENaC mutants is associated with increased arrival of newly synthesized channels at the plasma membrane, decreased degradation of cell surface-associated channels, or both, we tested the effect of Brefeldin A (BFA) on ENaC activity in Xenopus oocytes. Brefeldin A inhibits the ER to Golgi transport of newly synthesized membrane proteins, and has been previously shown to be effective in Xenopus oocytes (Geering et al., 1996). Thus, cRNA of wt-ENaC (wt) or K47,50R+K6-13R (with wt-) ENaC was injected into Xenopus oocytes and following overnight incubation, oocytes were treated with 10 g/ml BFA at time 0 (arrow) for up to 8 h, and amiloride-sensitive I_Na, representing active channels at the cell surface, measured by the two-electrode voltage clamp technique. Our results show that whereas the majority (80%) of wt ENaC activity disappeared by 8 h exposure to BFA, 43% (7%) of the K47,50R+K6-13R mutant channel activity was BFA-resistant (Figure 7). The accumulation of this BFA-resistant pool was apparent after 4 h incubation, and suggests that a significant fraction of the K47,50R+K6-13R mutant, unlike the wt ENaC, is retained at the cell surface or is recycled between endocytic vesicles and the plasma membrane. However, the amount of BFA-sensitive pool at 8 h incubation was also greater in the K47,50R+K6-13R mutant than in the wt channel (average of 9.9 A versus 5 A respectively), suggesting that the rate of arrival of newly synthesized channels to the plasma membrane is also elevated in the Lys to Arg mutant. Thus, loss of ubiquitination sites in ENaC likely leads to both an increase in incorporation and increased retention/recycling of the channel at the plasma membrane.

ENaC degradation is sensitive to endosomal/lysosomal and to proteasome inhibitors

Degradation of ubiquitinated proteins is carried out either by the proteasomes (usually of cytosolic or misfolded/misassembled proteins in the ER) or by the endosomal/lysosomal system in the case of several transmembrane proteins. In order to determine the mechanism(s) responsible for the rapid degradation (of total cellular pool) of ENaC seen in Figure 1, we tested the effect of inhibitors of either the proteasome or the endosomal/lysosomal system on the half-life of ENaC expressed in MDCK cells using pulse–chase experiments. Our results show that when the potent proteasome inhibitor lactacystin (10 M; Fenteany et al., 1995) was added during the 2 h pulse and the subsequent chase period, there was an attenuation of channel degradation (2.1- and 1.7-fold increase in half-life of and ENaC respectively; Figure 8A). This suggests an involvement of the proteasome in ENaC degradation. However, chloroquine (0.4 mM), a weak base that dissipates the late endosome/lysosome acidic pH thereby inhibiting proteolysis in these compartments, also led to a significant inhibition (2.7- and 1.3-fold increase in half-life of the and subunits respectively) of ENaC degradation when added during the chase period (Figure 8B). Our results, therefore, suggest that both the proteasomes and the endosomes/lysosomes are likely to play a role in the degradation of ENaC. One possible explanation for this dual mode of degradation may be that the individual ENaC subunits which are unassembled are targeted for proteasome degradation, whereas the properly assembled ENaC subunits are subject to degradation by late endosomes/lysosomes. To test whether the unassembled subunits are indeed degraded by the proteasomes and not by the lysosomes, we used a MDCK cell line which expresses high levels of HA-tagged ENaC alone (Staub et al., 1996). Despite high levels of expression, these cells display very little amiloride-sensitive Na⁺ currents when compared with MDCK cells expressing ENaC together, as determined by whole cell patch clamp analysis (T.Ishikawa, Y.Marunaka and D.Rotin, unpublished). Moreover, expression of ENaC alone in Xenopus oocytes leads to a severely reduced ENaC activity due to a reduced number of channels at the cell surface (Canessa et al., 1994a; Firsov et al., 1996). We therefore tested whether ENaC expressed alone is ubiquitinated and whether it is degraded by proteasomes. Figure 9B shows that transfection of ENaC into Hek-293 cells together with the His-Ub construct led to a massive ubiquitination of this subunit. Moreover, pulse–chase experiments performed with ENaC stably expressed in MDCK cells reveal a strong inhibition of ENaC degradation by 10 M lactacystin (Figure 9A, upper panel), whereas chloroquine (0.4 mM) was largely ineffective (Figure 9A, lower panel). These results, therefore, demonstrate that expression of the ENaC subunit alone, which can not properly assemble into a channel in the absence of ENaC, is targeted for proteasomal degradation.

Figure 8.

The effect of inhibitors of the proteasome and late endosomes/lysosomes on the stability of total cellular pool of ENaC. ENaC expressed in MDCK cells were labelled with [³⁵S]Met and Cys for 2 h, and chased for the indicated times in the absence (-) or presence (+) of: (A) 10 M lactacystin added during the pulse and the chase periods, or (B) 0.4 mM chloroquine added during the chase period. The average increase in half-life for three to six independent experiments as represented in this figure are: ENaC: 2.7- and 2.1-fold increase for chloroquine and lactacystin inhibition respectively; ENaC: 1.3- and 1.7-fold increase for chloroquine and lactacystin. A similar pattern of inhibition of ENaC degradation was observed with either lactacystin or chloroquine treatment of ENaC expressed in NIH-3T3 cells (not shown).

View full figure (80 KB)

Figure 9.

Ubiquitination and mode of degradation of ENaC expressed alone in MDCK cells. (A) HA-tagged ENaC stably expressed in MDCK cells was labelled with ³⁵S Met and Cys for 2 h, and chased for the indicated times in the absence (-) or presence (+) of 10 M lactacystin added during the pulse and the chase periods (upper panel), or 0.4 mM chloroquine added during the chase period (lower panel), as described in Figure 8 above. (B) Ubiquitination of ENaC transfected with His-Ub (Ub) into Hek-293 cells. Histidinated (ubiquitinated) proteins were precipitated on Ni-NTA agarose beads, proteins separated on 7% SDS–PAGE and immunoblotted with anti-HA antibodies, to detect ubiquitinated species of ENaC. The square bracket depicts ubiquitinated ENaC. The control (, right lane) was transfected with ENaC without His-Ub, and reveals no detectable ubiquitination, similar to cells transfected with ENaC alone.

View full figure (87 KB)

The results presented here demonstrate collectively that ENaC stability and function are modulated by ubiquitination. Our current results show that ENaC is a short lived protein, with a half-life of 1 h for at least 2 of its subunits. This short t_1/2 was detected in ENaC heterologously expressed in Xenopus oocytes, in ENaC transfected into either epithelial (MDCK) cells or fibroblast (NIH-3T3), or in xENaC endogenously expressed in A6 cells (B.C.Rossier, personal communication). The accumulation of ENaC following treatment of cells with the weak base chloroquine (or with NH₄Cl; our unpublished data) which dissipate the acidic pH in late endosomes/lysosomes, suggests the involvement of the endosomal/lysosomal pathway in ENaC degradation. However, our findings that the half-life of the ENaC subunits is also prolonged when the cells are treated with lactacystin (or with MG132; data not shown), indicate that the proteasome is involved in ENaC breakdown as well. One possible explanation for these dichotomous data may be that a portion of the ENaC subunits which do not become properly assembled in the ER may be degraded in a proteasome-dependent manner, whereas the properly assembled ENaC is targeted to the cell surface and is subsequently degraded by late endosomes/lysosomes. Although we cannot currently preclude the possibility of a sequential course of events whereby lysosomal degradation is followed by proteasomal degradation, we believe different pools (assembled versus unassembled) of ENaC are targeted to the different pathways. This is based on our results demonstrating lactacystin-sensitive and chloroquine-insensitive degradation of ENaC when expressed alone, but chloroquine-sensitivity when expressed together with ENaC, as well as our preliminary results demonstrating a small additive stabilization of ENaC by lactacystin plus chloroquine relative to each inhibitor alone (O.Staub and D.Rotin, unpublished data). We believe our data represent the first documented case where the same ubiquitinated protein (e.g. ENaC) can be degraded by either the proteasome or the lysosome, depending on its assembly/disassembly status.

Although we propose a lysosomal-mediated degradation of ENaC when properly assembled, we currently do not know how much of that pool of protein is actually located at the plasma membrane at any one time. We believe, however, that the ubiquitination of - and ENaC we observed represents, at least in part, ubiquitinated mature channel at the plasma membrane and not just of excess unassembled ENaC chains. This conclusion is based on our finding of a tight correlation between ubiquitination of ENaC and its number (and function) at the cell surface: mutations of a cluster of Lys residues in ENaC (or - plus ENaC) lead to both a reduction of ubiquitination and increased number of channels at the plasma membrane, with a concomitant elevated Na⁺ channel activity. Moreover, our Brefeldin A experiments suggest that a significant fraction of this increase in channel numbers in the ubiquitination-defective ENaC mutant results from increased retention (or recycling) of ENaC at the cell surface. Thus, although we do not currently know at what point(s) during processing and plasma membrane incorporation of ENaC it becomes ubiquitinated, the consequences of such ubiquitination are clearly affecting channel stability at the cell surface.

The fact that the status of ENaC ubiquitination determines its stability at the plasma membrane is intriguing, because so far it is not known how ubiquitination signals for, or is involved in, degradation of transmembrane proteins. In this regard, the involvement of ubiquitination in regulating ENaC degradation at the cell surface is similar to that of other recently described transmembrane proteins. A notable example is the ubiquitination-dependent vacuolar degradation of the Saccharomyces cerevisae permeases Fur4 and Gap1, which require the presence of intact Rsp5 (the yeast homologue of Nedd4) for this degradation (Hein et al., 1995; Galan et al., 1996). We have previously demonstrated direct binding of ENaC to the ubiquitin-protein ligase Nedd4, by association of the WW domains of Nedd4 with the PY motifs of the ENaC subunits (Staub et al., 1996). We do not know yet whether Nedd4 participates in the ubiquitination of the ENaC subunits described in this report. We can speculate, however, that if it does, this may have implications for Liddle syndrome, an hereditary form of hypertension caused by deletions/mutations within the PY motifs of - or ENaC (Shimkets et al., 1994; Hansson et al., 1995a,b; Tamura et al., 1996). Such mutations lead to increased Na⁺ channel activity due to increased channel number and opening at the plasma membrane (Schild et al., 1995, 1996; Snyder et al., 1995; Firsov et al., 1996). The same mutations also lead to an inhibition of binding to Nedd4-WW domains (Staub et al., 1996). Thus, we can speculate that the increase in retention of ENaC at the plasma membrane associated with Liddle syndrome may involve reduced ubiquitination, possibly by reduced binding to Nedd4. Future experiments will address this possibility.

Regardless of the link to Liddle syndrome, the observation that ENaC is ubiquitinated in living cells, and that this ubiquitination is critical for the regulation of channel degradation or stability at the plasma membrane and hence to channel activity, is by itself important, as it provides the first demonstration of regulation of ion channel function by ubiquitination. Clearly, a tight regulation of ENaC is necessary for maintenance of salt and water homeostasis; impairment of such regulation has been linked to several diseases, including not only Liddle syndrome, but also PHA1 (Chang et al., 1996; Strautnieks et al., 1996), pulmonary oedema (Hummler et al., 1996) and cystic fibrosis (Stutts et al., 1995). It remains to be identified whether any subsets of genetically determined hypertensive patients (Liddle syndrome or otherwise) indeed have mutations in the conserved lysine residues at the N-termini of - or ENaC which serve as attachment sites for ubiquitin.

Our data suggest that ENaC is the important, if not the predominant subunit which is regulating ubiquitination-mediated degradation of the Na⁺ channel. Ubiquitination of ENaC, however, also plays a role in channel stability, which becomes significant only when the key lysine residues in ENaC (K6–13) are lost. We propose therefore that loss of the main ubiquitination target of ENaC (K6-13) allows the ubiquitination of ENaC to partially compensate for this loss; consequently, losing those N-terminal lysines in both - and ENaC greatly stabilizes the channel. Our measurements of turnover rate of ENaC indicate that the t_1/2 of ENaC is somewhat longer than that of - and ENaC. We currently do not know whether such a difference in t_1/2 is biologically meaningful. However, it is curious that ENaC (when expressed together with ENaC) does not become ubiquitinated despite the presence of multiple conserved N-terminal lysine residues, and that mutation to arginines of these conserved lysines did not lead to channel hyperactivation. This suggests that the ubiquitination-mediated regulation of channel stability is not targeting the subunit directly, but rather indirectly, via regulation of - and ENaC. Since ENaC is only efficiently exported to the plasma membrane in the presence of all three subunits (Canessa et al., 1994a; McDonald et al., 1995; Firsov et al., 1996), targeting any subunit for degradation is sufficient to dismantle the channel and abrogate its activity. The reason for the observed partial inhibition of ENaC activity in some of the Lys to Arg mutants of ENaC is not known, but we speculate such mutations may cause the formation of defective, less well functioning channels, perhaps similar to the recently described G37S mutation in the N-terminus of ENaC which causes PHA1 due to reduced ENaC activity (Chang et al., 1996).

In summary, the work presented here demonstrates a fundamental role for ubiquitination in the regulation of ENaC function, via regulation of channel stability at the plasma membrane. This could provide a previously undescribed mechanism to regulate function of ion channels at the cell surface.

Plasmids and constructs

Rat ENaC (rENaC, hereafter called ENaC) cDNA was used for all studies. Plasmids used for transient transfection into Hek-293 cells: ENaC was tagged with a triple haemagglutinin (HA) epitope (YPYDVPDY) at its carboxy terminal end just upstream of the stop codon and subcloned into a pCMV4 plasmid (Andersson et al., 1989). The addition of the tag at that position did not interfere with Na⁺ channel function as assessed by patch clamp analysis (T.Ishikawa, Y.Marunaka and D.Rotin, unpublished). Similarly, a FLAG epitope (DYKDDDDK) was introduced just before the stop codon at the carboxy terminus of ENaC and subcloned into the pCEP4 plasmid (Invitrogen). Lysine to arginine mutants of -, - and ENaC were generated by substituting specific lysine residues with arginines using the PCR based mutagenesis described by Nelson and Long (1989). These point mutants, mostly of conserved lysines and all located at the N-termini of the ENaC subunits, were designated as follows (see Figure 4): In ENaC: K47 and K50 (K47,50R), K108 (K108R), K47, K50 and K108 (K47,50,108R) and K23, 26, 32, 47 and 50 (K23-50R); In ENaC: K4,5,and 9 (K4,5,9R), and K16, 23, 39, 47, 48 and 49 (K16-49R); in ENaC: K6, 8, 10, 12, 13 (K6-13R), K26R (K26R), and K6–13 plus K26R (K6-13+26R). With the exception of K23, 27 and 32 in ENaC, all the above mutated lysines are conserved between rat, human and Xenopus (Canessa et al., 1994a; McDonald et al., 1994; Puoti et al., 1995). All the above lysine to arginine mutants were subcloned into pSD5 for expression into Xenopus oocytes (Canessa et al., 1994a). For expression in Hek-293 cells, K23-50R was subcloned into pCMV4 and K6-13R into pCEP4.

Transfections and cell lines

MDCK cells stably expressing rat -, - and ENaC together, or expressing only HA-tagged ENaC, were previously generated (Stutts et al., 1995; Staub et al., 1996) by transfecting cells with ENaC in pLKneo (Hirt et al., 1992) which contains a dexamethasone-inducible promoter, together with - and ENaC cloned into a CMV-promoter based vector and expressed constitutively. MDCK previously transfected with - and ENaC (kindly provided by Drs Canessa and Rossier) were stably transfected with pCEP4 encoding either FLAG-tagged wt-ENaC or K6-13R ENaC. Cells were maintained in DMEM + 10% fetal bovine serum (FBS) + G418 (0.3 mg/ml)+ 10 M amiloride, to prevent cellular Na⁺ overload (+ 0.1 mg/ml hygromycin for the FLAG-tagged ENaC-expressing cells). For transient transfections, Hek-293 cells were transfected using lipofectamine, according to manufacturer instructions (Life Sciences).

Pulse–chase experiments

For pulse–chase experiments, MDCK cells stably expressing -, - and ENaC, HA-tagged ENaC alone, or FLAG-tagged wt- or K6-13R ENaC, stably co-expressed with wt-ENaC, were grown to confluency in 6-well plates (Costar) and induced overnight in induction medium [DMEM/10% FBS/20 M amiloride containing 1 M dexamethasone and 2 mM Na⁺ butyrate (MDCK expressing ENaC), or 1 M dexamethasone (MDCK expressing HA-tagged ENaC)]. Cells were washed three times in depletion medium (Induction medium without FBS, cysteine and methionine) and labelled at 37°C (in 5% CO₂ in air) for 2 h in labelling medium [Depletion medium + 0.1 mCi/ml of ³⁵S-containing cell labelling mix (Promix, Amersham)]. Cells were then washed three times with ice-cold Induction medium containing 10 mM unlabelled methionine and cysteine, and then chased in the same medium for various periods of times at 37°C (in 5% CO₂ in air). Where indicated, lactacystin (10 M) was added to the pulse and chase solutions, and chloroquine (0.4 mM) was added to the wash and chase solution only. Cells were then washed three times with ice-cold phosphate buffer saline (PBS), and lysed in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 1 mM PMSF, 10 g/ml leupeptin, 10 g/ml pepstatin A, 10 g/ml aprotinin). Insoluble material was removed by centrifugation and the soluble fraction denatured with 2% SDS and boiling for 5 min at 95°C. The samples were then diluted with 11 volumes of lysis buffer and immunoprecipitations performed by adding polyclonal sera directed against either the N-terminus of ENaC (Rotin et al., 1994), the C-terminus of ENaC or the C-terminus of ENaC (Duc et al., 1994). Immunocomplexes were collected with protein A–sepharose, separated on 7% SDS–PAGE and detected by autoradiography.

Detection of ENaC ubiquitination in vivo

Hek-293 cells were transiently co-transfected with a mixture of plasmids encoding HA-tagged ENaC, untagged ENaC and FLAG-tagged ENaC together with a construct encoding His-tagged ubiquitin (His-Ub), which expresses eight His-tagged ubiquitin molecules from a CMV promoter (Treier et al., 1994). Twenty-four hours after transfection, cells were lysed in lysis buffer (containing also 50 M N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal, LLnL), insoluble material removed by centrifugation and the soluble fraction denatured in 2% SDS and boiling at 95°C for 5 min. Samples were then diluted with 11 volumes of lysis buffer, Ni²⁺-NTA agarose beads (Qiagen) added to the lysate, and the mixture incubated on a rotator for 4 h at 4°C. The beads containing the histidinated (and thus ubiquitinated) bound proteins were washed twice with HNTG (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.1% Triton X-100, 10% glycerol, 40 mM imidazole) and three times with lysis buffer. Bound proteins were separated on 7% SDS–PAGE, transferred to nitrocellulose and immunoblotted with either anti-HA antibodies (Babco), anti-FLAG antibodies (Kodak), or anti-ENaC antibodies directed against amino acids 559–636 at the C-terminus (Duc et al., 1994) followed by HRP-conjugated secondary antibody and chemiluminescence (ECL/Amersham) or Supersignal ULTRA/Pierce detection.

Expression and function of ENaC channels in Xenopus oocytes

Complementary RNA (cRNA) of each -, - and ENaC subunit, or their indicated mutants, were synthesized in vitro and equal amounts of subunit cRNA (5 ng total) at saturating concentration for maximal expression were injected into stage V oocytes. ENaC activity at the cell surface resulting from ENaC expression was determined with the two-electrode voltage-clamp technique by measurement of the amiloride-sensitive inward Na⁺ current (I_Na) at -100 mV holding potential, as previously described (Schild et al., 1995, 1996). Where indicated, Brefeldin A (BFA, 10 g/ml) was added to the oocyte bathing medium 18 h after cRNA injection, and I_Na measured as described above at 0, 2, 4, 6 and 8 h post BFA addition. A Patch clamp technique was used to record single channel currents in the cell-attached configuration with Li⁺ ions as permeant cation on defolliculated oocytes, as described previously (Canessa et al., 1994a; Schild et al., 1997).

Surface labelling of ENaC in oocytes

For binding experiments, the lysine to arginine mutations at the N-termini of - or ENaC (K47-50R; K6-13R) were introduced into - or ENaC cDNA (respectively) which already contained a FLAG epitope at the ectodomains, located 30 residues downstream of the first transmembrane domain (Firsov et al., 1996). The addition of the FLAG tag at that position has no effect on channel function (Firsov et al., 1996). Surface expression of ENaC channels was determined by specific binding of [¹²⁵I]M₂IgG₁ (M₂Ab) to oocytes expressing FLAG-tagged ENaC, as described previously (Firsov et al., 1996). The equilibrium dissociation constant of specific [¹²⁵I]M₂IgG₁ (M₂Ab) binding was 3 nM. Accordingly, 12 nM M₂Ab were added to a Modified Barth's Saline (MBS) solution supplemented with 5% calf serum incubating the oocytes. After 1 h incubation at 4°C, each oocyte was washed eight times with 1 ml MBS solution and transferred individually to a gamma counter. The same oocyte was and then used to measure amiloride-sensitive Na⁺ current. M₂Ab non-specific binding was detected with oocytes injected with ENaC cRNAs encoding non-tagged subunits.

We would like to thank Drs C.M.Canessa and B.C.Rossier for MDCK cells expressing ENaC and anti-- and ENaC antibodies, Dr D.Firsov for preparation of iodinated antibodies, Drs M.J.Stutts and R.C.Boucher for the ENaC-expressing NIH-3T3 cells, and Dr D.Bohmann for the His-tagged ubiquitin construct. This work was supported by the Canadian CF Foundation (to D.R.), by the Medical Research Council (MRC) of Canada and a MRC Group Grant in Lung Development (to D.R.), by a grant from the International Human Frontier Science Program (HFSP) (to L.S. and D.R.), and by a grant from the Swiss National Foundation for Scientific Research to L.S. (No. 31-39435.93). A.C. is supported by the Israeli Academy of Sciences, the German–Israeli Foundation for Research and Scientific Development (G.I.F.), the Council for Tobacco Research, Inc. (CTR), and the UK–Israel Binational Biotechnology Foundation. K.B. is supported by MINERVA and by HFSP Fellowships, O.S. was supported by a Fellowship from the Canadian CF Foundation, and D.R. was a recipient of a Scholarship from the MRC of Canada.

Andersson S, Davis DL, Dahlbaeck H, Joernvall H and Russell DW (1989) Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem, 264, 8222–8229. | PubMed | ISI | ChemPort |

Botero-Velez M, Curtis JJ and Warnock DG (1994) Brief report: Liddles's syndrome revisited. New Engl J Med, 330, 178–181. | Article | PubMed | ChemPort |

Brodsky JL and McCracken AA (1997) ER-associated and proteasome-mediated protein degradation: how two topologically restricted events came together. Trends Cell Biol, 7, 151–156. | Article | ISI | ChemPort |

Canessa CM, Horisberger J-D and Rossier BC (1993) Epithelial sodium channel related to proteins involved in neurodegeneration. Nature, 361, 467–470. | Article | PubMed | ISI | ChemPort |

Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger J-D and Rossier BC (1994a) Amiloride-sensitive epithelial Na⁺ channel is made of three homologous subunits. Nature, 367, 463–467. | Article | PubMed | ISI | ChemPort |

Canessa CM, Mérillat A-M and RossierBC (1994b) Membrane topology of the epithelial sodium channel in intact cells. Am J Physiol, 267, C1682–C1690. | PubMed | ISI | ChemPort |

Cenciarelli C, Hou D, Hsu K, Rellahan BL, Wiest DL, Smith HT, Fried VA and Weissman AM (1992) Activation-induced ubiquitination of the T cell antigen receptor. Science, 257, 795–797. | Article | PubMed | ISI | ChemPort |

Chang SS et al. (1996) Mutations in the subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nature Genet, 12, 248–253. | Article

Ciechanover A (1994) The ubiquitin-proteasome proteolytic pathway. Cell, 79, 13–21. | Article | PubMed | ISI | ChemPort |

Deshaies RJ (1995) Make it or break it: The role of ubiquitin-dependent proteolysis in cellular regulation. Trends Cell Biol, 5, 428–434. | Article | PubMed | ISI | ChemPort |

Duc D, Farman N, Canessa CM, Bonvalet J-P and Rossier BC (1994) Cell-specific expression of epithelial sodium channel , and subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J Cell Biol, 127, 1907–1921. | PubMed | ChemPort |

Egner R and Kuchler K (1996) The yeast multidrug transporter Pdr5 of the plasma membrane is ubiquitinated prior to endocytosis and degradation in the vacuole. FEBS Lett, 378, 177–181. | Article | PubMed | ISI | ChemPort |

Fenteany G, Standaert RF, Lane WS, Chois S, Corey EJ and Schreiber SL (1995) Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science, 268, 726–731. | Article | PubMed | ISI | ChemPort |

Firsov D, Schild L, Gautschi I, Mérillat A-M, Schneeberger E and Rossier BC (1996) Cell surface expression of the epithelial Na⁺ channel and a mutant causing Liddle syndrome: a quantitative approach. Proc Natl Acad Sci USA, 93, 15370–15375. | Article | PubMed | ChemPort |

Galan JM, Moreau V, André B, Volland C and Haguenauer-Tsapis R (1996) Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease. J Biol Chem, 271, 10946–10952. | Article | PubMed | ISI | ChemPort |

Galcheva-Garbova Z, Theroux SJ and Davis RJ (1995) The epidermal growth factor receptor is covalently linked to ubiquitin. Oncogene, 11, 2649–2655. | PubMed | ChemPort |

Garty H and Palmer LG (1997) Epithelial sodium channels: function, structure, and regulation. Physiol Rev, 77, 359–396. | PubMed | ISI | ChemPort |

Geering K, Beggah A, Good P, Girardet S, Roy S, Schaer D and Jaunin P (1996) Oligomerization and maturation of Na,K-ATPase: Functional interaction of the cytoplasmic NH2 terminus of the beta subunit with the alpha subunit. J Cell Biol, 133, 1193–1204. | Article | PubMed | ISI | ChemPort |

Hansson JH et al. (1995a) Hypertension caused by a truncated epithelial sodium channel gamma subunit: Genetic heterogeneity of Liddle syndrome. Nature Genet, 11, 76–82.

Hansson JH, Schild L, Lu Y, Wilson TA, Gautschi I, Shimkets RA, Nelson-Williams C, Rossier BC and Lifton RP (1995b) A de novo missense mutation of the 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, 25, 11495–11499.

Hein C, Springael JY, Volland C, Haguenauer-Tsapis R and André B (1995) NPI1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin-protein ligase. Mol Microbiol, 18, 77–87. | Article | PubMed | ISI | ChemPort |

Hicke L and Riezman H (1996) Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell, 84, 277–287. | Article | PubMed | ISI | ChemPort |

Hilt W and Wolf DH (1996) Proteasomes: destruction as a programme. Trends Biochem Sci, 21, 96–102. | Article | PubMed | ISI | ChemPort |

Hirt RP, Poulain-Godefroy O, Billotte J, Kraehenbuhel J-P and Fasel N (1992) Highly inducible synthesis of heterologous proteins in epithelial cells carrying a glucocorticoid-responsive vector. Gene, 111, 199–206. | Article | PubMed | ISI | ChemPort |

Hochstrasser M (1996) Protein degradation or regulation: Ub the judge. Cell, 84, 813–815. | Article | PubMed | ISI | ChemPort |

Hummler E, Barker P, Gatzy J, Beermann F, Verdumo C, Schmidt A, Boucher RC and Rossier BC (1996) Early death due to defective neonatal lung liquid clearance in ENaC-deficient mice. Nature Genet, 12, 325–328. | Article

Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL and Riordan JR (1995) Multiple proteolytic systems including the proteasome, contribute to CFTR processing. Cell, 83, 129–135. | Article | PubMed | ISI | ChemPort |

Jentsch S and Schlenker S (1995) Selective protein degradation: a journey's end within the proteasome. Cell, 82, 881–884. | Article | PubMed | ISI | ChemPort |

Kölling R and Hollenberg CP (1994) The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants. EMBO J, 13, 3261–3271. | PubMed | ISI | ChemPort |

Liddle GW, Bledsoe T and Coppage WS,Jr (1963) A familial renal disorder simulating primary aldosteronism but with negligible aldosterone secretion. Trans Assoc Am Physicians, 76, 199–213. | ISI | ChemPort |

Lingueglia E, Voilley N, Waldmann R, Lazdunski M and Barbry P (1993) Expression cloning of an epithelial amiloride-sensitive Na⁺ channel. A new channel type with homologies to Caenorhabditis elegans degenerins. FEBS Lett, 318, 95–99. | Article | PubMed | ISI | ChemPort |

Lingueglia E, Renard S, Waldmann R, Voilley N, Champigny G, Plass H, Lazdunski M and Barbry P (1994) Different homologous subunits of the amiloride-sensitive Na⁺ channel are differently regulated by aldosterone. J Biol Chem, 269, 13736–13739. | PubMed | ISI | ChemPort |

McDonald FJ, Snyder PM, McCray PB,Jr and Welsh MJ (1994) Cloning, expression, and distribution of a human amiloride-sensitive Na⁺ channel. Am J Physiol, 266, L728–L734. | PubMed | ISI | ChemPort |

McDonald FJ, Price MP, Snyder PM and Welsh MJ (1995) Cloning and expression of the - and -subunits of the human epithelial sodium channel. Am J Physiol, 268, C1157–C1163. | PubMed | ISI | ChemPort |

Miyazawa K, Toyama K, Gotoh A, Hendrie PC, Mantel C and Broxmeyer HE (1994) Ligand-dependent polyubiquitination of c-kit gene product: a possible mechanism of receptor down modulation in M07e cells. Blood, 83, 137–145. | PubMed | ISI | ChemPort |

Mori S, Heldin C-H and Claesson-Welsh L (1992) Ligand-induced polyubiquitination of the platelet-derived growth factor -receptor. J Biol Chem, 267, 6429–6434. | PubMed | ISI | ChemPort |

Nelson RM and Long GL (1989) A general method of site-specific mutagenesis using a modification of the Thermus aquatiqus polymerase chain reaction. Anal Biochem, 180, 147–151. | PubMed | ISI | ChemPort |

O'Brodovich H (1995) The role of active Na⁺ transport by lung epithelium in the clearance of airspace fluid. New Horizons, 3, 240–247. | PubMed | ChemPort |

Palombella V, Rando O, Goldberg AL and Maniatis T (1994) Ubiquitin and the proteasome are required for processing the NF-B1 precursor and the activation of NF-B. Cell, 78, 773–785. | Article | PubMed | ISI | ChemPort |

Paolini R and Kinet J-P (1993) Cell surface control of the multiubiquitination and deubiquitination of high-affinity immunoglobulin E receptors. EMBO J, 12, 779–786. | PubMed | ISI | ChemPort |

Puoti A, May A, Canessa CM, Horisberger J-D, Schild L and Rossier BC (1995) The highly selective low-conductance epithelial Na⁺ channel of Xenopus laevis A6 kidney cells. Am J Physiol, 38, C188–C197.

Renard S, Lingueglia E, Voilley N, Lazdunski M and Barbry P (1994) Biochemical analysis of the membrane topology of the amiloride-sensitive Na⁺ channel. J Biol Chem, 269, 12981–12986. | PubMed | ISI | ChemPort |

Rossier BC and Palmer LG (1992) Mechanisms of aldosterone action on sodium and potassium transport. In Seldin,D.W. and Giebisch,G. (eds), The Kidney: Physiology and Pathophysiology. Raven Press, New York, USA, pp. 1373–1409.

Rotin D, Bar-Sagi D, O'Brodovich H, Merilainen J, Lehto PV, Canessa CM, Rossier BC and Downey GP (1994) A SH3 binding region in the epithelial Na⁺ channel (ENaC) mediates its localization at the apical membrane. EMBO J, 13, 4440–4450. | PubMed | ChemPort |

Saumon G and Basset G (1993) Electrolyte and fluid transport across the mature alveolar epithelium. J Appl Physiol, 74, 1–15. | PubMed | ChemPort |

Scheffner M, Huibregtse JM, Vierstra R and Howley PM (1993) The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell, 75, 495–505. | Article | PubMed | ISI | ChemPort |

Schild L, Canessa CM, Shimkets RA, Warnock DG, Lifton RP and Rossier BC (1995) 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, 92, 5699–5703. | Article | PubMed | ChemPort |

Schild L, Lu Y, Gautschi I, Schneeberger E, Lifton RP and Rossier BC (1996) 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, 15, 2381–2387. | PubMed | ISI | ChemPort |

Schild L, Schneeberger E, Gautschi I and Firsov D (1997) Identification of amino acid residues in the alpha, beta and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation. J Gen Physiol, 109, 15–26. | Article | PubMed | ISI | ChemPort |

Shimkets RA et al. (1994) Liddles's syndrome: heritable human hypertension caused by mutations in the subunit of the epithelial sodium channel. Cell, 79, 407–414. | Article | PubMed | ISI | ChemPort |

Snyder PM, McDonald FJ, Stokes JB and Welsh MJ (1994) Membrane topology of the amiloride-sensitive epithelial sodium channel. J Biol Chem, 269, 24379–24383. | PubMed | ISI | ChemPort |

Snyder PM, Price MP, McDonald FJ, Adams CM, Volk KA, Zeiher BG, Stokes JB and Welsh MJ (1995) Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na⁺ channel. Cell, 83, 969–978. | Article | PubMed | ISI | ChemPort |

Staub O, Dho S, Henry PC, Correa J, Ishikawa T, McGlade J and Rotin D (1996) WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na⁺ channel deleted in Liddle's syndrome. EMBO J, 15, 2371–2380. | PubMed | ISI | ChemPort |

Strautnieks SS, Thompson RJ, Gardiner RM and Chung E (1996) A novel splice-site mutation in the gamma subunit of the epithelial sodium channel gene in three pseudohypoaldosteronism type 1 families. Nature Genet, 13, 248–250. | Article

Strous GJ, Van Kerkhof P, Govers R, Ciechanover A and Schwartz AL (1996) The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor. EMBO J, 15, 3806–3812. | PubMed | ISI | ChemPort |

Stutts MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier BC and Boucher RC (1995) CFTR as a cAMP-dependent regulator of sodium channels. Science, 269, 847–850. | Article | PubMed | ISI | ChemPort |

Tamura H, Schild L, Enomoto N, Matsui N, Marumo F, Rossier BC and Sasaki S (1996) Liddle disease caused by a missense mutation of beta subunit of the epithelial sodium channel gene. J Clin Invest, 97, 1780–1784. | Article | PubMed | ISI | ChemPort |

Treier M, Staszewski LM and Bohmann D (1994) Ubiquitin-dependent c-Jun degradation in vivo is mediated by the domain. Cell, 78, 787–798. | Article | PubMed | ISI | ChemPort |

Voilley N, Lingueglia E, Champigny G, Mattéi M-G, Waldmann R, Lazdunski M and Barbry P (1994) The lung amiloride-sensitive Na⁺ channel: Biophysical properties, pharmacology, ontogenesis, and molecular cloning. Proc Natl Acad Sci USA, 91, 247–251. | PubMed | ChemPort |

Ward CL, Omura S and Kopito RR (1995) Degradation of CFTR by the ubiquitin-proteasome pathway. Cell, 83, 121–127. | Article | PubMed | ISI | ChemPort |

Main navigation

• Search EMBO Journal

  Search

• Advanced search

• The EMBO Journal

  • Home
  • Aims and scope
  • Current issue
  • Advance online publication
  • Web focuses
  • Archive:
  • Browse by issue
  • Browse by subject
  • Browse by category
  • Free online sample issue
  • Press releases

• Authors and Referees

  • Editorial process
  • Guide for authors
  • Submit an article
  • Guide for referees
  • Editors and Editorial Board
  • Contact editorial office

• Customer Service

  • Subscribe
  • Order sample copy
  • Purchase articles
  • Reprints and permissions
  • Contact NPG
  • Advertising