Article1 November 1997free access Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination O. Staub O. Staub The Hospital for Sick Children, Division of Respiratory Research, 555 University Avenue, Toronto, Ontario M5G 1X8 and Biochemistry Department, University of Toronto, Toronto, Canada Search for more papers by this author I. Gautschi I. Gautschi Institut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1015 Lausanne, Switzerland Search for more papers by this author T. Ishikawa T. Ishikawa The Hospital for Sick Children, Division of Respiratory Research, 555 University Avenue, Toronto, Ontario M5G 1X8 and Biochemistry Department, University of Toronto, Toronto, Canada Search for more papers by this author K. Breitschopf K. Breitschopf Biochemistry Department, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, 31096 Israel Search for more papers by this author A. Ciechanover A. Ciechanover Biochemistry Department, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, 31096 Israel Search for more papers by this author L. Schild L. Schild Institut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1015 Lausanne, Switzerland Search for more papers by this author D. Rotin Corresponding Author D. Rotin The Hospital for Sick Children, Division of Respiratory Research, 555 University Avenue, Toronto, Ontario M5G 1X8 and Biochemistry Department, University of Toronto, Toronto, Canada Search for more papers by this author O. Staub O. Staub The Hospital for Sick Children, Division of Respiratory Research, 555 University Avenue, Toronto, Ontario M5G 1X8 and Biochemistry Department, University of Toronto, Toronto, Canada Search for more papers by this author I. Gautschi I. Gautschi Institut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1015 Lausanne, Switzerland Search for more papers by this author T. Ishikawa T. Ishikawa The Hospital for Sick Children, Division of Respiratory Research, 555 University Avenue, Toronto, Ontario M5G 1X8 and Biochemistry Department, University of Toronto, Toronto, Canada Search for more papers by this author K. Breitschopf K. Breitschopf Biochemistry Department, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, 31096 Israel Search for more papers by this author A. Ciechanover A. Ciechanover Biochemistry Department, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, 31096 Israel Search for more papers by this author L. Schild L. Schild Institut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1015 Lausanne, Switzerland Search for more papers by this author D. Rotin Corresponding Author D. Rotin The Hospital for Sick Children, Division of Respiratory Research, 555 University Avenue, Toronto, Ontario M5G 1X8 and Biochemistry Department, University of Toronto, Toronto, Canada Search for more papers by this author Author Information O. Staub1, I. Gautschi2, T. Ishikawa1, K. Breitschopf3, A. Ciechanover3, L. Schild2 and D. Rotin 1 1The Hospital for Sick Children, Division of Respiratory Research, 555 University Avenue, Toronto, Ontario M5G 1X8 and Biochemistry Department, University of Toronto, Toronto, Canada 2Institut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1015 Lausanne, Switzerland 3Biochemistry Department, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, 31096 Israel *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:6325-6336https://doi.org/10.1093/emboj/16.21.6325 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The epithelial Na+ channel (ENaC), composed of three subunits (αβγ), plays a critical role in salt and fluid homeostasis. Abnormalities in channel opening and numbers have been linked to several genetic disorders, including cystic fibrosis, pseudohypoaldosteronism type I and Liddle syndrome. We have recently identified the ubiquitin-protein ligase Nedd4 as an interacting protein of ENaC. Here we show that ENaC is a short-lived protein (t1/2 ∼1 h) that is ubiquitinated in vivo on the α and γ (but not β) subunits. Mutation of a cluster of Lys residues (to Arg) at the N-terminus of γENaC leads to both inhibition of ubiquitination and increased channel activity, an effect augmented by N-terminal Lys to Arg mutations in αENaC, but not in βENaC. This elevated channel activity is caused by an increase in the number of channels present at the plasma membrane; it represents increases in both cell-surface retention or recycling of ENaC and incorporation of new channels at the plasma membrane, as determined by Brefeldin A treatment. In addition, we find that the rapid turnover of the total pool of cellular ENaC is attenuated by inhibitors of both the proteasome and the lysosomal/endosomal degradation systems, and propose that whereas the unassembled subunits are degraded by the proteasome, the assembled αβγENaC complex is targeted for lysosomal degradation. Our results suggest that ENaC function is regulated by ubiquitination, and propose a paradigm for ubiquitination-mediated regulation of ion channels. 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, α-, β- and γENaC. 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 αENaC 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), NFκB and IκB (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 α and γ 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 αβγENaC complex is targeted to the lysosome. Moreover, mutating a cluster of lysines (to arginines) at the N-terminus of γENaC, or α- plus γENaC, 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. Results 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 αβγENaC using pulse–chase experiments. For these and all subsequent experiments, we used rat αβγENaC (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 αβγENaC (Stutts et al., 1995). Cells were labelled for 2 h with [35S]methionine/cysteine and then chased for various periods of time. The αβγENaC 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 (t1/2) of the proteins. As is evident from Figure 1A, α- and γENaC expressed in MDCK cells have a rapid turnover rate with a half-life of ∼1 h; βENaC, on the other hand, has a longer half-life (t1/2 >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 αβγENaC expressed in NIH-3T3 cells were very similar to those expressed in MDCK cells (data not shown). Figure 1.Half-life of αβγENaC. Pulse–chase experiments of 35S-labelled α (upper panel), β (middle panel) and γ (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 t1/2 values are: αENaC: 1.13 ± 0.13 (mean ± SE, n = 9) h; γENaC: 1.23 ± 0.09 (mean ± SE, n = 6) h; βENaC: t1/2 >3 h. Similar results were obtained with NIH-3T3 cells (not shown). Download figure Download PowerPoint α- and γENaC, but not βENaC, are ubiquitinated in vivo A rapid turnover of proteins is often associated with ubiquitination-mediated degradation. We thus wanted to test whether the αβγENaC subunits are ubiquitinated in living cells. For these experiments, we used epitope-tagged α- or γENaC because the antibodies available against these native subunits, unlike the antibodies against native βENaC, are not sensitive enough for immunoblotting. We subcloned HA-tagged αENaC, FLAG-tagged γENaC or untagged βENaC into CMV-based expression vectors. The HA or FLAG tags were added just upstream of the stop codons of α- or γENaC respectively, since tagging at these sites had no effect on channel activity (not shown). We then transiently co-transfected HA-tagged αENaC, FLAG-tagged γENaC and βENaC 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 αβγENaC 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-αENaC), anti-FLAG (i.e. anti-γENaC), or anti-βENaC antibodies directed against the C-terminus of βENaC (kindly provided by B.C. Rossier). As can be seen in Figure 2A, B and C (bottom panels, lysates), α-, β- and γENaC 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 αENaC (Figure 2A, top panel, bracket) and γENaC (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 α- or γENaC, the βENaC subunit was not ubiquitinated (Figure 2B, upper panel), even though βENaC was strongly expressed in these cells (Figure 2B, lower panel). The ubiquitination of α- and γENaC 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 αβγENaC-transfected cells, α and γENaC, but not βENaC, are ubiquitinated in vivo. Figure 2.Ubiquitination of α and γ, but not β, ENaC expressed in Hek-293 cells. Hek-293 cells transiently co-transfected (tfxn) with HA-tagged αENaC (α), FLAG-tagged γENaC (γ), βENaC (β) 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 αENaC), (B) anti-βENaC antibodies (to detect ubiquitinated βENaC) and (C) anti-FLAG (flag) antibodies (to detect ubiquitinated γENaC). 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 (α-, β- or γENaC) in the lysates. Arrows indicate fully glycosylated forms of ENaC subunits. Download figure Download PowerPoint 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 α- and γENaC 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) αβγENaC (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 β- or γrENaC (respectively) are very close to the transmembrane domain and are not conserved. We therefore mutated the conserved lysines at the N-termini of α-, β- and γrENaC (herein referred to as αβγENaC) 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 αENaC: K47 and K50 (αK47,50R), K108 (αK108R) or αK47,50R plus αK108R (αK47,50,108R). An additional αENaC construct was also generated, in which lysines 23, 26 and 32 (which are not conserved between rat and human) of the αK47,50R construct were mutated to Arg, yielding the mutant αK23-50R. (ii) In βENaC: K4, K5 and K9 (βK4,5,9R) or K16, K23, K39, K47, K48 and K49 (βK16-49R). (iii) In γENaC: K6, K8, K10, K12, K13 (γK6-13R), K26R (γK26R) or K6-13 plus K26R (γK6-13+26R). Figure 3.N-terminal Lys to Arg mutants of αβγENaC. Schematic representation of the conserved lysine residues at the N-termini of αβγrENaC and αβγhENaC. All conserved lysines (Bold letters, boxed), as well as three non-conserved lysine residues in αENaC, 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 αENaC: αK47,50R, αK108R, αK47,50,108R and αK23-50R; in βENaC: βK4,5,9R and βK16-49R; in γENaC: γK6-13R, γK26R and γK6-13R + K26R. (TM1 = the first transmembrane domain of each ENaC subunit.) Download figure Download PowerPoint We then investigated the effect of the above Lys to Arg mutations in αβγENaC on ENaC channel activity. Two-electrode voltage clamp experiments were performed in which amiloride-sensitive Na+ currents (INa) were measured in Xenopus oocytes expressing the different Lys to Arg mutants. Our results show that none of the conserved Lys to Arg αENaC mutants, when expressed together with wild type (wt) βγENaC, had any effect on increasing ENaC activity (Figure 4A). This lack of effect on ENaC function was also observed with the extended αK23-50R mutant (not shown). Similarly, none of the Lys to Arg mutations within the N-terminus of βENaC, expressed together with wt-αγENaC, caused any increase in amiloride-sensitive Na+ currents. In contrast, the expression of the γENaC mutants (γK6-13R or γK6-13+26R) together with wt-αβENaC more than doubled Na+ channel activity (Figure 4A). Moreover, when this γK6-13R mutant was co-expressed together with the αK47,50R mutant (along with wt-βENaC), 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 βK4,5,9R or the βK16-49R mutant was co-expressed with either αK47,50R, γK6-13R or both mutants (Figure 4A). These results therefore demonstrate that a cluster of conserved Lys residues particularly in γENaC, but also in αENaC, are responsible for the regulation of ENaC activity, and when mutated, lead to an increase in amiloride sensitive Na+ current (INa). 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 (α- and γENaC). Figure 4.Na+ channel activity of Lys to Arg mutants of αβγENaC expressed in Xenopus oocytes. (A) cRNA derived from the Lys to Arg mutants of α-, β- or γENaC (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 (INa) 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 αK47,50R+γK6-13R (plus wt-β) 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 (Vpip) 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. Download figure Download PowerPoint Lys to Arg mutations in γENaC, or in α- plus γENaC, 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 γENaC or α- plus γENaC 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 αK47,50R plus γK6-13R 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 INa 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 αβγENaC 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 INa and surface expression of wt-ENaC or Lys to Arg mutants of α- and γENaC (αK47,50R; γK6-13R; αK47,50R + γK6-13R) were measured in individual oocytes. The Lys to Arg mutants of βENaC were not tested since none of them was able to elevate channel activity (Figure 4A). Comparing the ENaC mutants with wt confirms that the αK47,50R 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 γK6-13R or the αK47,50R plus γK6-13R double mutant, the increase in INa 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 INa 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 α and γ subunits would be consistent with an inhibition of ubiquitination of the channel protein. Figure 5.Correlation of surface labelling and function of Lys to Arg mutants of α- and γENaC measured in individual oocytes. (A and B) Representative experiments in which amiloride-sensitive INa 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), αK47,50R (open triangles) or the αK47,50R + γK6-13R mutants (closed circles), and (B) wt-ENaC (open circles) or the γK6-13R 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 (INa) 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 INa and antibody binding were determined from simultaneous measurements in eight to ten individual oocytes. Asterisks denote statistical significance at P <0.05 level. Download figure Download PowerPoint Figure 6.Inhibition of ubiquitination of the Lys to Arg mutants of γ- and αENaC. Hek-293 cells were transiently co-transfected (tfxn) with βENaC (β), HA-tagged αENaC (α) or αK23-50R (αR), and FLAG-tagged γENaC (γ) or γK6-13R (γR), 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 (γENaC), (B) anti-HA (αENaC) or (C) anti-βENaC 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 α and γ ENaC. The ∼105 kDa band across panel B (top) is probably a contaminant. (D) γK6-13R mutant is more stable than wt-γENaC. MDCK cells stably expressing wt-αβγENaC (called γWT) or γK6-13R plus wt-αβENaC (called γK6-13R) (both γ 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 γENaC antibodies, the immunoprecipitates separated on 7% SDS–PAGE and visualized by autoradiography, as represented by the autoradiogram. Histogram represents t1/2 quantitation (mean ± SE) of three independent experiments similar to that depicted in the autoradiogram. Download figure Download PowerPoint Inhibition of ubiquitination of the Lys to Arg mutants of γENaC, or of α-plus γENaC As we observed an increase in channel activity resulting from a greater number of αβγENaC at the plasma membrane in the γK6-13R 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-αβγENaC (control), or wt-αβENaC plus the γK6-13R mutants of γENaC. As before, the αENaC constructs were HA-tagged and the γENaC constructs were FLAG-tagged. Following transfection, cells were lysed, and lysates incubated with Ni2+-NTA beads to precipitate ubiquitinated proteins. These proteins were t
