Control of ENaC ubiquitination

Abstract

Ubiquitination influences the expression of the epithelial Na⁺ channel (ENaC). We assessed the mechanisms of selective ubiquitination of the mature, cleaved form of γENaC in both native rodent kidneys and Fisher rat thyroid (FRT) cells expressing the channel heterologously. In both models, singly cleaved and fully cleaved γENaCs were strongly ubiquitinated, implying that the second cleavage releasing an inhibitory peptide was not essential for the process. To see whether location of the protein in or near the apical membrane rather than cleavage per se influences ubiquitination, we studied mutants of γENaC in which cleavage sites are abolished. These subunits were ubiquitinated only when coexpressed with α- and βENaC, facilitating trafficking through the Golgi apparatus. To test whether reaching the apical surface is necessary we performed in situ surface biotinylation and measured ENaC ubiquitination in the apical membrane of rat kidney. Ubiquitination of cleaved γENaC was similar in whole kidney and surface fractions, implying that both apical and subapical channels could be modified. In FRT cells, inhibiting clathrin-mediated endocytosis with Dyngo-4a increased both total and ubiquitinated γENaC at the cell surface. Finally, we tested the idea that increased intracellular Na⁺ could stimulate ubiquitination. Administration of amiloride to block Na⁺ entry through the channels did not affect ubiquitination of γENaC in either FRT cells or the rat kidney. However, presumed large increases in cellular Na⁺ produced by monensin in FRT cells or acute Na⁺ repletion in rats increased ubiquitination and decreased overall ENaC expression.

NEW & NOTEWORTHY We have explored the mechanisms underlying the ubiquitination of the γ subunit of epithelial Na⁺ channel (ENaC), a process believed to control channel internalization and degradation. We previously reported that the mature, cleaved form of the subunit is selectively ubiquitinated. Here we show that this specificity arises not from the cleavage state of the protein but from its location in the cell. We also show that under some conditions, increased intracellular Na⁺ can stimulate ENaC ubiquitination.

INTRODUCTION

Regulation of the epithelial Na⁺ channel (ENaC) in the kidneys helps to maintain both Na⁺ and K⁺ balance by altering rates of urinary excretion of these ions. One aspect of this regulation involves the ubiquitination of the ENaC subunits, presumably controlling trafficking and degradation rates of the protein (1–3). In particular, the ubiquitin E3 ligase Nedd4-2 can bind to the C-termini of βENaC and γENaC, facilitating the ubiquitination of the α and γ subunits (2).

As ENaC subunits move from the endoplasmic reticulum (ER) to the apical plasma membrane, the α and γ subunits undergo cleavage by both intracellular and extracellular proteases resident in the Golgi apparatus and in the extracellular (luminal) space (4, 5). This process also activates the channels by eliminating inhibitory domains in the extracellular region of the proteins (6). The mineralocorticoid aldosterone stimulates channel activity in part by increasing surface expression of ENaC protein with most of the channels at the surface containing cleaved α and γ subunits in rodent kidneys (7–10).

Recently we found that ubiquitination of γENaC in the kidney was specific for the cleaved form of the subunit (11). This suggested that ubiquitination could serve to limit the lifetime of the channels after they are moved to the apical membrane and activated. The mechanism of this specificity is unknown. It could involve increased accessibility of ubiquitination sites on the protein due to conformational changes induced by proteolysis. Another possibility is that ubiquitination depends on the location of the channels within the cell. In this scenario, cleaved subunits are preferentially modified because most of the channels at or near the apical membrane are cleaved, having moved through the Golgi apparatus. A recent report suggested a third possibility that Nedd4-2 can be activated by intracellular Na⁺ (12). Activation of ENaC by cleavage could increase Na⁺ in the cytoplasm, especially in the vicinity of the channels themselves, thereby stimulating ubiquitination.

Here we use both native kidney tissue and an epithelial cell line with heterologous expression of ENaC to assess the contributions of cleavage per se, of cellular location, and of intracellular Na⁺ to the specific ubiquitination of γENaC.

METHODS

Animals

All procedures using animals were approved by the Institutional Animal Care and Use Committee of Weill Cornell Medical College. We used adult C57BL/6N mice (20–30 g) of both sexes. We detected no significant differences in ENaC ubiquitination between males and females, so results from both sexes are combined for statistical analysis. We also used female Sprague–Dawley rats (140–200 g) (Charles River). To increase ubiquitination of ENaC, animals were maintained for 1 wk either on a Na⁺-deficient diet [low-Na⁺ (LN), Harlan-Teklad TD07904] or on a diet containing 5% KCl [high-K⁺ (HK), Harlan-Teklad TD 220593]. For Na⁺-repletion experiments, animals were maintained on the LN diet for 1 wk, then fasted overnight and refed with either the low-Na⁺ diet or an identical diet with 1% NaCl added for 4 h. The latter group also received 0.9% NaCl in their drinking water. In some experiments, animals were housed for 2–4 h in metabolic cages. Urine was collected and its Na⁺ content was measured with a Na⁺-selective electrode (Cole-Parmer).

Some mice on LN food were treated with the protease inhibitor camostat. About 5 mg of camostat mesylate (MedChemExpress, Monmouth Junction, NJ) were dissolved in 0.2 mL H₂O and injected intraperitoneally. The same dose was repeated 2 h later. Control animals received H₂O only. Urine was collected over the 4 h of treatment, after which the kidneys were harvested for analysis. Some rats on the HK diet received a single dose of amiloride (0.6 mg/kg body wt). Amiloride (Sigma) was dissolved in saline at 0.3 mg/mL and injected subcutaneously. Urine was collected for 2 h after which the kidneys were harvested for analysis.

In Situ Biotinylation

For the analysis of ENaC protein at the cell surface, rat kidneys were biotinylated in situ as described previously (7, 8, 13). Animals were anesthetized with ketamine/xylazine. Kidneys were perfused via the aorta with PBS, pH 8.0, containing 0.5 mg/mL sulfo-NHS-SS-biotin (Campbell Science, Rockville, IL). Reactions were quenched with Tris buffer, pH 8.0. Microsomes were prepared from kidney homogenates by centrifugation at 100,000 g for 100 min and solubilized with 3% Triton X-100. Surface proteins from equal amounts of microsomal protein were isolated using UltraLink neutravidin beads (Invitrogen).

Western Blot Analysis of Kidney Tissue

Whole-kidney microsomes were prepared for Western blot analysis by mincing one kidney and disrupting the tissue with a Dounce homogenizer. The homogenate was filtered through a nylon cell strainer (70 µm mesh) to remove intact tissue and centrifuged at 100,000 g for 100 min to obtain a microsomal pellet. This was suspended in 3 mL of lysis buffer and frozen for later analysis. After measurement of protein concentration, samples were prepared for electrophoresis as described previously (14).

Samples were electrophoresed on 4–12% bis-TRIS gels (Invitrogen), and the proteins were transferred electrophoretically to PVDF membranes. After blocking, membranes were incubated overnight at 4°C with a rabbit polyclonal antibody, recognizing the C-terminus of γENaC (15). Anti-rabbit IgG conjugated with alkaline phosphatase was used as a secondary antibody. Bound antibody was visualized with a Syngene Pxi imager using a chemiluminescence substrate (Western Breeze, Invitrogen). Band densities were quantified using Photoshop.

Isolation of Ubiquitinated Subunits

We used a commercial kit for the detection of ubiquitinated proteins (Signal Seeker, Cytoskeleton, Denver, CO) following the manufacturer’s instructions. Normally, one mouse kidney or ∼½ rat kidney was quickly homogenized in 1.5 mL or 4 mL, respectively, of ice-cold BlastR lysis buffer with a glass Dounce homogenizer. The lysis buffer contained deubiquitination inhibitors [N-ethylmaleimide (10 mM) and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (50 µM)] and a protease inhibitor cocktail included in the kit. The remaining mouse kidney or half of the rat kidney was separately homogenized in sucrose lysis buffer, and a microsomal pellet was prepared for Western blotting. The Blast lysate was filtered to remove DNA and unbroken tissue fragments. About 1 mL of the filtered lysate was mixed with 4 mL of cold BlastR dilution buffer and centrifuged at 10,000 g for 10 min in the cold room. The supernatant was then collected, aliquoted, and frozen for further processing. Protein concentration of the diluted lysate was measured using the Lowry method. The lysate was further diluted to obtain a protein concentration of ∼1 mg/mL with a mixture of BlastR lysis (one part) and BlastR dilution buffers (four parts) for affinity isolation of ubiquitinated proteins. About 30 µL of either ubiquitin affinity or control bead suspension were incubated with 1.5 mL of lysate on an oscillating platform for 2 h in a cold room. Subsequently, the beads were washed three times with 1 mL of cold wash buffer for 5 min each. Finally, the captured proteins were eluted from the beads at room temperature for 5 min with 40 µL of 1X elution buffer (loading buffer without reducing agent). After spinning, the supernatant was saved, 5 µL of 500 mM DTT were added, and the mixture was heated at 70°C for 10 min.

Expression of ENaC in FRT Cells

Fisher rat thyroid (FRT) cells were routinely cultured in DMEM/F-12 medium supplemented with 9% FBS. Cells were seeded in a 6-well cell culture plate or 10-cm petridish and transfected with plasmid DNAs encoding mouse ENaC (αβγ) using Lipofectamine 3000 (Invitrogen, L3000008) according to the manufacturer’s instructions. In some experiments, the wild-type (WT) γ plasmid was replaced by a γ construct bearing mutations at the two proteolytic sites (R143A, RKRK186QQQQ) to express the full-length γ subunit (R/A Q4) in FRT cells. About 24–48 h after transfection, cells were treated with 10 µM MG132 (Sigma-Aldrich, No. 474791) for 2 h at 37°C to inhibit proteasomal degradation of ubiquitin-conjugated proteins and boost the signal of ubiquitinated γENaC. In some experiments, cells received 10 µM amiloride for 30 min to block ENaC activity or 40 µM monensin (Sigma-Aldrich, M5273), an Na⁺ ionophore that increases intracellular Na⁺. To cleave ENaC at the surface, cells were exposed to 10 µg/mL trypsin (Sigma-Aldrich, T1426) at 37°C for 5 min. In other experiments, cells were treated with 10 µM Dyngo-4a (Cayman Chemical Company, No. 29479) to inhibit clathrin-mediated endocytosis. At the end of the experiments, cells were labeled with biotin (see Surface Biotinylation, Streptavidin Pulldown, and Ubiquitination Pulldown) or extracted in Goldstein buffer supplemented with inhibitors for proteases, phosphatases, and deubiquitinases.

Surface Biotinylation, Streptavidin Pulldown, and Ubiquitination Pulldown

To label surface ENaC subunits, FRT cells transfected with ENaC subunits were washed three times with cold Dulbecco’s PBS with 1.0 mM CaCl₂ and 0.5 mM MgCl₂ (ThermoFisher, No. 14040216) to remove culture media and stop endocytosis. To label surface ENaC, cells were incubated with EZ-Link Sulfo-NHS-SS-Biotin (1 mg/mL, ThermoFisher, No. 21331) freshly prepared in a buffer containing 137 mM NaCl and 15 mM sodium borate (pH 9.0) for 30 min on ice. Excess biotin was then quenched with 10% FBS in DMEM/F12 media and rinsed three times with cold PBS. Cells were lysed in Goldstein buffer supplemented with inhibitors for proteases, phosphatases, and deubiquitinases to extract the whole-cell total proteins. Whole-cell lysates were incubated with streptavidin agarose beads (ThermoFisher, No. 20353) to pull down biotinylated proteins or with ubiquitination affinity beads (Cytoskeleton, UBA01B) as described earlier. All pulldowns were carried out overnight at 4°C on a rotating mixer. In some experiments, surface pulldown was performed following ubiquitin pulldown to isolate surface-ubiquitinated proteins. Labeled proteins were eluted into Laemmli sample buffer (Bio-Rad, No. 1610737) by heating the beads at 60°C for 10 min. A small percentage of the whole-cell lysates was used as “input” in the immunoblot analysis of biotinylated and/or ubiquitinated ENaC. To separate the two γ cleavage products, whole-cell lysates or pulldowns were treated with PNGase (New England Biolabs, P0709L) following the manufacturer’s instructions.

Western Blot Analysis

Samples were mixed with equal volumes of 2X Laemmli sample buffer (Bio-Rad, No. 1610737) containing 10% β-mercaptoethanol (Bio-Rad, No. 1610710) and heated at 60°C for 5–10 min. Denatured samples were separated on 4–15% stain-free Tris-glycine gels (Bio-Rad, No. 5678081) and were transferred to nitrocellulose membranes. After being blocked with 5% skim milk in PBS, membranes were incubated overnight at 4°C with a rabbit polyclonal antibody recognizing the C-terminus of γENaC (Stressmarq, SPC405), followed by incubation with HRP-conjugated anti-rabbit IgG secondary antibodies for 2 h at room temperature. Chemiluminescence signals were detected using Clarity Western ECL Substrate (Bio-Rad, No. 1705061) and imaged with the Bio-Rad ChemiDoc system. Band densities were quantified using Image Lab, a Bio-Rad software.

Statistical Analyses

Data are expressed as means ± standard deviation in the main text and shown in scatterplots with bars. Data distribution was examined with a D'Agostino–Pearson normality test. Statistical comparisons between two groups were determined with Student’s t test. Statistical comparisons between three or more groups were determined with one-way or two-way ANOVA followed by Šidák’s multiple comparisons test, using GraphPad Prism software. A P value of <0.05 was considered statistically significant.

RESULTS

Previous results showed that in rat kidneys, ubiquitination of the γENaC subunit was specific for the cleaved form of the subunit (11). Because this form represents the fully cleaved subunit, presumably by furin and an extracellular protease (16), we asked whether both cleavage events were necessary to render the subunit accessible to the ubiquitination process.

To address this question, we made use of the mouse kidney in which both singly and fully cleaved forms of γENaC can be recognized (16). As shown in Fig. 1A, both these forms were identified in ubiquitin pulldown fractions, although the full-length form of the subunit was absent. We also increased the relative amount of the singly cleaved subunit using treatment with camostat, a protease inhibitor that is filtered in the glomerulus and can inhibit the final cleavage step (17). In whole-kidney microsomes, 4 h after camostat injection, the amount of the singly cleaved γENaC band increased, whereas that of the fully cleaved subunit decreased (Fig. 1B). A similar change was seen in the ubiquitin pulldown fraction (Fig. 1C). Quantitation of the results by dividing the signals from the ubiquitin pulldown by those of the whole kidney microsomal fraction indicated that these ratios were similar for the singly and fully cleaved forms, both in the presence and absence of camostat (Fig. 1D). These results indicate that the second cleavage of the γ subunit is not necessary for ubiquitination of the native ENaC in rodent kidneys.

Figure 1.

Ubiquitination of singly and fully cleaved γENaC in the mouse kidney. Mice were maintained on a low-Na⁺ diet for 6–8 days. They were then treated twice with 5 mg camostat or vehicle at 2-h intervals. After 4 h, animals were euthanized, kidneys were excised, and total microsomes and ubiquitin-binding eluates were prepared. A: Western blots were loaded with 60 µg of microsome protein or eluate from 1.5 mg total protein. Each lane corresponds to a different animal. In this blot, all animals were female. Blots were stained with anti-γENaC antibody. Band densities were normalized to values of the fully cleaved band under control conditions. B: camostat increased the amount of singly cleaved γENaC and decreased the amount of fully cleaved γENaC, with no change in the amount of the full-length form. C: camostat increased the ratio of singly to fully cleaved γ subunit in both total microsomes and in ubiquitin pulldown fractions. D: the ratio of ubiquitinated to total microsomes was similar for the singly and fully cleaved forms and was not significantly changed by camostat. Bars indicate means and standard deviation for 8 animals in each group. Pink and black symbols correspond to female and male mice, respectively. Comparisons between camostat-treated and control animals (B–D) or between singly and fully cleaved γENaC (D) were analyzed with two-way ANOVA followed by Sidak’s multiple comparisons test. P values are shown. γENaC, γ subunit of the epithelial Na⁺ channel.

Both singly and fully cleaved γENaCs were clearly detected in the ubiquitination pulldowns of native mouse kidneys, resulting in two separate bands in the immune blot (Fig. 1A). In contrast, cleaved γENaC migrates as a single band around 75 kDa in both whole-cell lysates and ubiquitination pulldowns when expressed in FRT cells (Fig. 2B). To confirm whether both singly cleaved (furin-cleaved) and fully cleaved γENaCs are ubiquitinated in FRT cells, samples were treated with PNGase to remove glycans to separate the two cleaved γENaC bands (16). As shown in Fig. 2B, both singly and fully cleaved γENaC fragments were clearly detected in both whole-cell lysates and ubiquitin pulldowns, suggesting that both forms of the subunit can be ubiquitinated. Approximately 23 ± 16% of the singly cleaved γENaCs were ubiquitinated, almost three times higher than that of fully cleaved form (7 ± 6%; Fig. 2C). We also examined γENaC cleavage status at the cell surface with biotin labeling and streptavidin pulldown (Fig. 2A). There are more singly cleaved γENaC residues at the cell surface compared with the fully cleaved form (22 ± 12% vs. 9 ± 4%; Fig. 2C).

Figure 2.

Effect of trypsin on the ubiquitination of γENaC in FRT cells. FRT cells were transiently transfected with equal amounts of cDNA encoding the three mouse ENaC subunits (α, β, and γ). A: 24–48 h after transfection, cells were treated with MG132 (10 µM, 2 h) and incubated with biotin (1 mg/mL, 30 min) at 4°C to label surface proteins. About 3% of whole-cell lysates was saved as “input,” whereas the rest was split evenly for ubiquitin pulldown and streptavidin pulldown. B: a Western blot of whole-cell, surface, and ubiquitinated γENaC with or without PNGase treatment. C: the percentages of subunits that were expressed at the surface were similar to those that were ubiquitinated both for the singly and fully cleaved γENaC. Bars indicate means and standard deviations for 7 individual experiments. Comparisons between ubiquitinated and surface γENaC were analyzed with two-way ANOVA followed by Sidak’s multiple comparisons test for the exact P values. D: Western blot of whole-cell, surface, and ubiquitinated γENaC before and after trypsin treatment (10 µg/mL, 5 min, 37°C). All samples were treated with PNGase before loading. E: trypsin increased the amount of fully cleaved γENaC in both surface pulldown and ubiquitin pulldowns but not in whole-cell lysates. Bars indicate means and standard deviations for 7 control (Ctrl) and 3 trypsin treatment experiments. Comparisons between control and trypsin-treated samples were analyzed with two-way ANOVA followed by Sidak’s multiple comparisons test for the exact P values. F: the ratio between the singly and fully cleaved γENaC fragments in the ubiquitin pulldowns positively correlated with that in the surface pulldowns. Data were fit to a simple linear regression (Y = 0.648 × X + 0.0963, r² = 0.761). γENaC, γ subunit of the epithelial Na⁺ channel; FRT, Fisher rat thyroid.

To further test whether the second cleavage event promotes γENaC ubiquitination, trypsin was added to the media at a final concentration of 10 µg/mL for 5 min to cleave the channels at the cell surface. Trypsin treatment increased the relative abundance of fully cleaved γENaC in both ubiquitin pulldown and surface pulldown, but not in the whole-cell lysates (Fig. 2, D and E). The ratios of fully cleaved versus singly cleaved ubiquitinated γENaC positively correlate with the ratios of the two γENaC fragments at the surface (Fig. 2F), suggesting that γENaC ubiquitination happens at or near the cell surface.

The absence of ubiquitination in the full-length form of γENaC in mouse kidneys (Fig. 1A) could result from a ubiquitination-resistant conformation of the subunit or from a different location within the cell. Since the first cleavage event occurs in the Golgi apparatus (18), it is likely that the uncleaved full-length subunit resides in the ER or another pre-Golgi compartment. Consistent with this notion, stimulation of ENaC in vivo by aldosterone produces an increase in the cleaved form of γENaC, a decrease in uncleaved form, and a migration of ENaC from a diffuse cytoplasmic to an apical location (9, 15, 19). In surface fractions, most γENaC is in the fully cleaved form (7, 8). To test whether the full-length γENaC can be ubiquitinated if it is trafficked to the surface, we transfected FRT cells with a mutant γENaC (R/A_Q4) in which both the furin site and the putative extracellular protease cleavage site were mutated to prevent proteolytic cleavage (18, 20). Ubiquitination pulldown and subsequent surface pulldown were carried out as illustrated in Fig. 3A. When coexpressed with the α and β subunits, the R/A_Q4 mutant can be detected only as a full-length band in the surface biotinylated fraction (Fig. 3B), suggesting that furin cleavage is not required for ENaC surface trafficking.

Figure 3.

Ubiquitination of full-length γENaC in FRT cells. FRT cells were transiently transfected with WT γ subunit alone or cotransfected with WT mouse α and β subunits with either the WT γ subunit or a mutant γ subunit containing mutations at both the furin site (R143A) and the extracellular cleavage site (RKRK186QQQQ) to express the full-length γ ENaC (R/A_Q4). Both WT γ and the R/A_Q4 mutant constructs contained an N-terminal HA tag and a C-terminal V5 tag. A: 24–48 h after transfection, cells were treated with MG132 (10 µM, 2 h) and incubated with biotin (1 mg/mL, 30 min) to label surface proteins. About 1% whole-cell lysates were saved as “input.” About 10% was used to pull down total surface protein, whereas the rest was used for ubiquitin pulldown. After being eluted from the ubiquitin beads, 10% was saved as the total ubiquitinated fraction, whereas the rest was subsequently incubated with streptavidin beads to isolate surface ubiquitinated γENaC. B: Western blot of whole-cell and total surface expression of the WT γENaC and R/A_Q4 mutant in the presence of WT α and β subunits. C: Western blot of whole-cell, total ubiquitinated, and surface ubiquitinated γENaC. D: total and surface ubiquitinated full-length γENaC was normalized to that of the WT γ subunit (R/A_Q4/WT). Bars indicate means and standard deviations for 5 individual experiments. The ratios were compared with a hypothetical value of 1 using a one-sample t test. P values are shown. γENaC, γ subunit of the epithelial Na⁺ channel; FRT, Fisher rat thyroid; WT, wild type.

When expressed in FRT cells alone (Fig. 3C), WT γ subunit with two epitope tags was detected as a full-length protein of 90 kDa in the whole-cell lysates and total ubiquitinated fraction but absent in the surface ubiquitinated fraction, presumably due to defects in channel assembling and trafficking in the absence of the α and β subunits (18). When coexpressed with the other two subunits, WT γENaC was cleaved and migrated faster than the γ subunit expressed alone or the R/A-Q4 mutant as expected (Fig. 3C). Ubiquitination of the R/A-Q4 mutant was readily detected both at the whole-cell level and in the surface fraction. In fact, the noncleaved R/A-Q4 mutant appeared to be more abundant than WT γENaC in both total and surface ubiquitination pulldowns (Fig. 3C). We observed ∼80% more R/A-Q4 mutant than the WT γ subunit in the total ubiquitinated pool, which rose to a 2.4-fold increase in the surface ubiquitinated fraction (Fig. 3D). Under these conditions, the ubiquitinated full-length mutant migrated at a higher molecular mass (∼130 kDa) than the predicted full-length γENaC (Fig. 2B), likely a result of the addition of multiple ubiquitins to accessible lysine residues. This increase is not observed for the cleaved subunits because the ubiquitination sites are on the N-terminus, which is separated from the immunodetected peptide (11). Overall, our data suggest that proteolytic cleavage of γENaC is not required for its modification by ubiquitin, but that trafficking of the protein to the plasma membrane is likely to be an important factor in controlling this process.

Given the apparent importance of cellular location in the process, we assessed the levels of ubiquitination in different compartments of the kidney. Here we used the rat kidney because of the relative ease of preparing cell surface fractions. Animals were fed a low-Na⁺ diet for 1 wk to increase both surface expression (7) and ubiquitination (11) of ENaC. Figure 4A shows ENaC in whole-kidney homogenate, in cell surface fractions, and in urinary exosomes, along with the ubiquitination pulldown from the respective starting materials. Quantitation of the blots (Fig. 4B) indicates that the ratio of ubiquitinated γENaC was similar in the total and surface fractions. The fraction ubiquitinated in exosomes was substantially lower (Fig. 4B). This was surprising since ubiquitination is presumably a signal for the retrieval of ENaC protein from the surface. Since urinary exosomes are thought to derive from multivesicular bodies containing internalized membrane protein (21), we expected the ratio of ubiquitinated to total subunits to be higher. Possibly, ENaC in this pathway undergoes deubiquitination either in the cell or in the urine.

Figure 4.

Ubiquitination of γENaC in surface fractions and exosomes from the rat kidney. Rats were fed a low-Na⁺ diet for 6–8 days to increase cleaved ENaC. Urine was collected for 4 h and used for preparation of exosomes. Kidneys were perfused with biotinylating reagent and excised. Exosomes and kidney tissue were homogenized in Blast buffer. Part of the homogenate was used to isolate ubiquitinated proteins. The rest was used to prepare a surface fraction from the biotinylated kidneys and subsequently ubiquitinated proteins were isolated from that fraction. A: Western blot of γENaC from three preparations. For whole-kidney homogenates, the total input lanes were loaded with 65 μg protein. The ubiquitin eluates were from 750 µg total protein. For the surface fraction, the total and ubiquitinated lanes were loaded with material from 2.1 and 3.5 mg total protein, respectively. For the urinary exosomes, total lanes were loaded with material from 32.5 µL and 400 µL, respectively. B: analysis of ubiquitinated/total input for γENaC in the three preparations. Bars indicate means and standard deviation for 4 animals in each group. P values are from one-way ANOVA followed by Sidak’s multiple comparisons test comparing whole-kidney homogenate, surface fractions, or urinary exosomes. γENaC, γ subunit of the epithelial Na⁺ channel.

To further explore the ubiquitination of surface channels, we treated ENaC-expressing FRT cells with Dyngo-4a, an inhibitor of clathrin-mediated endocytosis (Fig. 5). Dyngo-4a increased the amount of γENaC in the surface fractions modestly but significantly, with no significant change in whole-cell expression (Fig. 5B). We then isolated surface proteins from whole-cell and ubiquitin-pulldown fractions. The amount of surface γENaC in the ubiquitinated pool increased to a similar extent as in the whole-cell lysates (Fig. 5B). Together, our data suggest that inhibiting endocytosis led to more total and ubiquitinated γENaC subunits on the surface.

Figure 5.

Effect of endocytosis inhibition on ubiquitination of γENaC in FRT cells. FRT cells were transiently transfected with WT mouse α, β, and γ subunits. About 24 h after transfection, cells were treated with 10 µM Dyngo-4a for 30 min. About 10 µL lysates were saved as whole-cell (WC) input; 200 µL lysates were used to pull down total surface protein, whereas another 1,000 µL were used for ubiquitin pulldown. After elution from the ubiquitin beads, 10% was saved as total ubiquitinated fraction (total Ubi), whereas the rest was incubated with streptavidin beads to isolate surface ubiquitinated (surface Ubi) γENaC. A: Western blots of whole-cell (WC), surface, total Ubi, and surface Ubi of γENaC in control or Dyngo-4a-treated cells. B: effect of Dyngo-4a treatment on γENaC abundance in whole-cell lysates, surface, total ubiquitinated, and surface-ubiquitinated pulldowns. γENaC band intensity of Dyngo-4a-treated cells was normalized to that of control cells of the same preparation (Dyngo-4a/Ctrl). The ratios were compared with a hypothetical value of 1 using a one-sample t test. γENaC, γ subunit of the epithelial Na⁺ channel; FRT, Fisher rat thyroid; WT, wild type.

One mechanism for coupling ubiquitination with location in the cell is through cytoplasmic Na⁺. A recent study indicated that the activity of Nedd4-2, the enzyme thought to catalyze ubiquitination of ENaC, is enhanced by increased Na⁺ concentrations (12). ENaC at the cell surface will conduct Na⁺ into the cell, increasing its concentrations throughout the cytoplasm but especially in the vicinity of the conducting channels themselves.

To test this idea, we used the Na⁺ ionophore monensin to increase intracellular Na⁺ concentration in FRT cells expressing ENaC. Monensin was added to the cells at a final concentration of 40 µM for 30 min at 37°C (Fig. 6A). We found that γENaC abundance at the whole cell level was significantly reduced by monensin treatment (Fig. 6B), whereas the percentage of ubiquitinated γENaC nearly doubled (7 ± 3% vs. 13 ± 6%; Fig. 6C). These data are consistent with the idea that raising intracellular Na⁺ concentration by monensin promotes γENaC ubiquitination in FRT cells.

Figure 6.

Effect of ionophore monensin on γENaC ubiquitination. FRT cells were transiently transfected with WT mouse α, β, and γ subunits. About 24–48 h after transfection, cells were treated with 10 µM MG132 for 1.5 h followed by 30-min cotreatment of 40 µM monensin. About 5% whole-cell lysates were saved as “input,” whereas the rest was used for ubiquitin pulldown. A: Western blot of whole-cell and ubiquitinated γENaC in control and monensin-treated cells. A stain-free gel image is shown to assess total protein loading. B: γENaC expression was reduced by monensin. γENaC abundance was normalized to total protein loading and shown as relative values to the control cells. C: γENaC ubiquitination was enhanced by monensin. Ubiquitinated γENaC was shown as a percentage of its whole-cell abundance. B and C: bars indicate means and standard deviations for 3 transfections of 8 individual samples. P values are shown for comparisons between control and monensin-treated cells analyzed with a Student’s t test. γENaC, γ subunit of the epithelial Na⁺ channel; FRT, Fisher rat thyroid; WT, wild type.

We also examined the effects of increasing cell Na⁺ in vivo. Here we followed a protocol in which rats were fed a Na⁺-deficient diet for 6–8 days, fasted overnight, and then salt repleted with food containing 1% NaCl and saline drinking water (22). As shown previously, this resulted in a large increase in Na⁺ excretion (Fig. 7B) and a marked decrease in the amount of cleaved ENaC in whole-kidney microsomes (Fig. 7, A and C). This response was presumed to be mediated, at least in part, by increased Na⁺ entering the ENaC-expressing cells in the distal nephron because it was observed even when aldosterone levels were maintained by infusion and was diminished by treatment with amiloride to block Na⁺ entry through the channels (22). We then measured the level of ubiquitination of ENaC under these conditions. The absolute amount of ubiquitinated γ subunit declined (Fig. 7D), but the ratio of ubiquitinated protein to total protein increased (Fig. 7E), similar to the effect of Na⁺ loading on FRT cells (Fig. 6). We also noted the expression of singly cleaved ENaC in the acutely Na⁺-repleted animals (Fig. 7A). This form of the subunit was also ubiquitinated. These results are consistent with the idea that elevated intracellular Na⁺ can promote ubiquitination by activating Nedd4-2.

Figure 7.

Effect of acute Na⁺ repletion on ubiquitination of γENaC. Rats were fed a low-Na⁺ (LN) diet for 6–8 days. They were fasted overnight and then offered either low-Na⁺ food plus tap water or control Na⁺ food plus saline (Resalt) for 4 h while urine was collected. Animals were euthanized, and kidneys were excised and used to prepare microsomes or ubiquitinated protein fractions. A: Western blots were loaded with 30 µg microsomes or eluate from 2 mg homogenate per lane and probed with anti-γENaC. Positions of quantified bands are indicated with ]. B: urinary Na⁺ excretion (U_NaV) in LN and resalted animals. C: quantitation of γENaC in whole-kidney microsomes. D: quantitation of ubiquitinated γENaC. E: ratios of ubiquitinated to total γENaC. Bars indicate means and standard errors for 5 animals in each group. P values are from a Student’s t test comparing resalted with control animals. γENaC, γ subunit of the epithelial Na⁺ channel.

We then asked whether reducing Na⁺ entry into the cell could inhibit ubiquitination. We tested this in FRT cells by treating the cultures with 10 µM amiloride, a concentration sufficient to almost completely block Na⁺ conductance through the channels. γENaC was blotted in 5% whole-cell inputs and in ubiquitin pulldowns (Fig. 8A). As shown in Fig. 8B, amiloride treatment for 30 min did not affect γENaC abundance at the whole cell level. Approximately 4% of γENaC was ubiquitinated in both the control and amiloride-treated cells (Fig. 8C), suggesting that amiloride treatment does not inhibit γ subunit ubiquitination.

Figure 8.

Effect of acute amiloride treatment on γENaC ubiquitination. FRT cells were transiently transfected with WT mouse α, β, and γ subunits. About 24–48 h after transfection, cells were firstly treated with 10 µM MG132 for 1.5 h and then treated with either 10 µM amiloride or 0.1% DMSO (Ctrl) for additional 30 min. About 10% whole-cell lysates were saved as “input,” whereas the rest was used for ubiquitin pulldown. A: Western blot of whole-cell and ubiquitinated γENaC in control and amiloride-treated cells. A stain-free gel image was shown for total protein loading. B: γENaC expression at the whole cell level was not altered by amiloride. γENaC abundance was normalized to total protein loading and shown as relative values to the control group. C: γENaC ubiquitination was not affected by amiloride. Ubiquitinated γENaC was shown as a percentage of its whole-cell abundance. B and C: bars indicate means and standard deviations of 5 individual experiments. Comparisons between the Ctrl and amiloride-treated cells were analyzed with a Student’s t test, and the exact P values are shown. γENaC, γ subunit of the epithelial Na⁺ channel; FRT, Fisher rat thyroid; WT, wild type.

We also examined the effects of amiloride in vivo. Rats were fed a diet with 5% KCl to increase the abundance of cleaved γENaC and to promote Na⁺ delivery to the distal nephron. This condition maximized amiloride-dependent urinary excretion of Na⁺ (23). Administration of amiloride to these animals for 2.5 h increased Na⁺ excretion (Fig. 9B) and also increased the amount of cleaved ENaC (Fig. 9, A and C). The latter effect could reflect the decreased feedback inhibition of the channels due to decreased Na⁺ entry into the cells. The amount of ubiquitinated ENaC also increased (Fig. 9D), whereas the ratio of ubiquitinated to total ENaC protein was not significantly different in control and drug-treated animals (Fig. 9E). This implies that under normal conditions, including those in which ENaC is active, Na⁺-dependent ubiquitination does not play a pivotal role in regulating channel processing.

Figure 9.

Effect of amiloride on ubiquitination of γENaC. Rats were fed a high-K⁺ (HK) diet for 6–8 days. They were injected with amiloride (0.6 mg/kg) or diluent and urine was collected for 2 h. Animals were euthanized, and kidneys were excised and used to prepare microsomes or ubiquitinated protein fractions. A: Western blots were loaded with 40 µg protein from microsomes or eluate from 2 mg homogenate per lane and probed with anti-γENaC. Positions of quantified bands are indicated with ]. B: urinary Na⁺ excretion (U_NaV) in HK and amiloride-treated animals. C: quantitation of γENaC in whole-kidney microsomes. D: quantitation of ubiquitinated γENaC. E: ratios of ubiquitinated to total γENaC. Bars indicate means and standard errors for 3 or 4 animals in each group. P values are from a Student’s t test comparing amiloride-treated with control animals. γENaC, γ subunit of the epithelial Na⁺ channel.

DISCUSSION

We previously assessed the ubiquitination of ENaC in the rodent kidney using a ubiquitin-affinity pulldown assay to detect ubiquitinated proteins (11). Consistent with the earlier studies with heterologous expression systems (24), we found evidence for ubiquitination of the α and γ subunits, but not β subunits of the channel protein. A surprising result was that, at least for γENaC, the ubiquitination was remarkably specific for the cleaved form of the subunit. We also found that the apparent molecular mass of the species isolated from the affinity beads was increased by the added ubiquitin moieties only when blots were run under nonreducing conditions. This suggested that the subunit was directly ubiquitinated on the N-terminal, which was dissociated from the C-terminal portion detected by the antibody under reducing conditions.

Here we consider several possible mechanisms that could account for the specificity of protein modification for the cleaved form of γENaC. One is that a conformation change that presumably occurs with the second cleavage, excising an inhibitory fragment and opening the channel (6), could expose the subunit to the ubiquitination process. Second is that ubiquitination could depend on the cellular location of the channels, since cleavage is associated with trafficking of the protein through the Golgi apparatus to the apical pole of the cell and eventually to the apical membrane itself (8). Third is an increase in the intracellular Na⁺ subsequent to the activation of the channels at the cell surface could activate Nedd-4-2 ubiquitin ligase (12), perhaps preferentially ubiquitinating channels that are conducting Na⁺ or that are nearby. These possibilities are not mutually exclusive.

Singly Versus Fully Cleaved γENaC

The idea that a second cleavage in γENaC is required for ubiquitination and presumably for channel degradation was attractive to us because this cleavage is thought to occur after the channels are delivered to the surface by membrane-associated or urinary proteases (6). This could serve as a natural brake on channel activity, akin to the inactivation of voltage-gated Na⁺ channels, albeit on a much slower timescale.

We used two preparations in which singly and fully cleaved γENaC can be detected and manipulated: heterologous expression in FRT cells with and without application of trypsin and native expression in the mouse kidney with and without treatment with camostat. In both cases, both singly and fully cleaved subunits were ubiquitinated. If anything, the singly cleaved species were more strongly modified.

In the case of the FRT cells, we increased the population of fully cleaved subunits using extracellular trypsin. This raised both the total amount of fully cleaved species and the amount that was ubiquitinated. Since the time of exposure to the protease was short (5 min), we cannot say whether the newly cleaved channels became ubiquitinated or whether ubiquitinated singly cleaved channels were cleaved by trypsin. This distinction does not affect the main conclusion that both forms of the subunit are substrates for the ubiquitinating enzymes.

Cellular Location

If the specificity of ubiquitination depends primarily on cellular location rather than cleavage per se, then even full-length γENaC should be modified if it undergoes apical trafficking. We tested this by mutating both furin and extracellular protease cleavage sites and expressing these constructs in FRT cells. The full-length mutant subunits moved to the cell surface and became ubiquitinated. This did not occur when WT γ subunit was expressed alone in the absence of the α and β subunits.

This suggests that apical location within the cell is a strong determinant of ubiquitination. The data do not distinguish an effect of location per se, as would occur if the ubiquitin ligase was confined to the apical pole of the cell, from one that depends on the association of ENaC with other proteins in the apical compartment that might affect the ubiquitination process.

These data also do not reveal whether the channels must be inserted into the apical membrane to be ubiquitinated. In the kidney, the level of ubiquitinated cleaved γENaC is similar in total cell extracts and in surface fractions. Since most of the cleaved species appear to not be at the surface (25), it is likely that the channels both at the surface and in the cytoplasmic vesicles are ubiquitinated. However, we cannot rule out the possibility that many of the channels in the cytoplasmic vesicles have been internalized from the surface and could have been ubiquitinated during their residence in the apical membrane.

Cellular Na⁺

A recent paper showed that Nedd4-2, the likely E3 ubiquitin ligase responsible for ENaC ubiquitination, is activated by increased cellular Na⁺ (12). This raised the attractive possibility that ubiquitination could play a role in the process of “feedback inhibition,” in which Na⁺ entry into the cells decreases the activity of the channels (22, 26–28). It might also contribute to the specificity of ubiquitination since Na⁺ concentrations could be higher where ions exit the channel at the cytoplasmic end, near the presumed sites of ubiquitination on the cytoplasmic N-terminus. This idea is speculative, however, in the absence of measurements of ions in this cytoplasmic subdomain.

We did indeed observe higher levels of ubiquitination in both FRT cells and in the kidney under conditions of large Na⁺ challenges. In the FRT cells, we used the Na⁺ ionophore monensin, and in the kidney, we invoked an acute increase in delivery of Na⁺ to the ENaC-expressing distal nephron cells by giving NaCl to Na⁺-depleted rats. In both cases, we observed decreases in overall expression of cleaved γENaC, consistent with a feedback inhibition response. In both cases, we also found a higher proportion of ubiquitinated γENaC. This could reflect increased rates of degradation of channels subsequent to ubiquitination, although we have no direct proof of this causal relationship.

Preventing Na⁺ entry into the cells with amiloride, and presumably lowering cell Na⁺, did not elicit the opposite response. In FRT cells, no changes in either expression or ubiquitination of ENaC could be detected. In the kidney, we did see an increase in cleaved γENaC with amiloride treatment consistent with the suppression of feedback inhibition. This differs from the FRT cells, possibly due to a longer exposure to amiloride or to a higher rate of Na⁺ entry, producing a larger feedback response under conditions of dietary K loading. However, we did not see a decrease in ubiquitination. Feedback inhibition is a complex process and likely has multiple mechanisms (28). Effects of changing Na⁺ influx from normal to low levels with amiloride may be independent of ubiquitination.

Overall, the most striking determinant of ubiquitination revealed by our experiments is that of cellular location. Channels are preferentially modified after traveling through the Golgi apparatus where they are cleaved by furin, but this cleavage per se is not a requirement. Channels removed from the cell through urinary exosomes have decreased levels of ubiquitination, suggesting partial deubiquitination during the retrieval process. Ubiquitination of channels after their arrival at the apical membrane, or at least at the apical pole of the cell, could serve to limit the amount of time they spend actively conducting Na⁺. This could allow more precise regulation of this activity by hormones and other factors.

DATA AVAILABILITY

Data will be made available upon reasonable request.

GRANTS

This work was supported by National Institutes of Health Grants RO1DK111380 (to L.G.P.), R01DK130901 (to S.S.), and U54DK137329 (Pittsburgh Center for Kidney Research).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.S., G.F., and L.G.P. conceived and designed research; S.S., G.F., S.C.M.W., and L.G.P. performed experiments; S.S. and L.G.P. analyzed data; S.S., G.F., and L.G.P. interpreted results of experiments; S.S. and L.G.P. prepared figures; S.S. and L.G.P. drafted manuscript; S.S., G.F., and L.G.P. edited and revised manuscript; S.S., G.F., S.C.M.W., and L.G.P. approved final version of manuscript.