ENaC Degradation in A6 Cells by the Ubiquitin-Proteosome Proteolytic Pathway*

Bela MalikDagger , Lynn Schlanger§, Otor Al-KhaliliDagger , Hui-Fang BaoDagger , Guichun YueDagger , Stephen Russ Price§, William E. Mitch§, and Douglas Charles EatonDagger

From the Dagger  Department of Physiology and § Renal Division, Emory University, Atlanta, Georgia 30322

Received for publication, November 26, 2000, and in revised form, January 25, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The amiloride-sensitive epithelial Na+ channel (ENaC)1 is essential for control of fluid and electrolyte homeostasis by distal nephron kidney cells and fluid clearance from the lung alveolar spaces (1). The channel consists of three homologous subunits, alpha , beta , and gamma ; each subunit consists of two transmembrane domains and one large extracellular domain with several putative N-linked glycosylation sites (2, 3). ENaC activity is regulated either by altering the channel open probability or the number of functional ENaC molecules in the plasma membrane. Changes in the number of channels will result from an imbalance between the synthesis and degradation of ENaC proteins. Certain signals (e.g. vasopressin) cause the insertion of new channels into the plasma membrane of Xenopus distal nephron-derived A6 cells (4). Additionally, in several heterologous expression systems such as Xenopus oocytes and Madin-Darby canine kidney cells transfected with and overexpressing all three ENaC subunits, the number of channel molecules at the plasma membrane and ENaC activity is regulated by ENaC degradation (5-7). The physiological impact of these observations in terms of ENaC activity being regulated by degradation of channel proteins in native, sodium-transporting epithelial cells has not been tested. In our experiments, we sought to determine whether degradation could alter ENaC activity in native, sodium-transporting epithelial cells because as shown in prior studies linking ENaC activity (5-7) with its degradation might result from overexpression of ENaC in cells which do not normally express ENaC.

Membrane proteins are frequently degraded in lysosomes, but there are examples of transport proteins being degraded by the 26S ubiquitin-proteosome system (8-10). In fact, several different types of proteins with diverse functions, cellular localizations, and half-lives are degraded by this pathway (8). Protein degradation by the ubiquitin-proteosome pathway is initiated when a target protein is conjugated to ubiquitin, which leads to its recognition and degradation by the 26S proteosome (11). Conjugation of ubiquitin to the substrate protein is ATP-dependent through formation of an isopeptidic covalent linkage between the terminal glycine of ubiquitin and a lysine in the target protein. Several enzymes catalyze this process: a ubiquitin-activating enzyme, E1; a ubiquitin-conjugating enzyme, E2; and a ubiquitin ligase, E3 (11), but other proteins can substitute for E1, E2, and E3. For example, Neural precursor cell-expressed developmentally down-regulated protein (Nedd4) can act as a ubiquitin ligase.

ENaC beta  and gamma  subunits interact with Nedd4 in the yeast two-hybrid system and with ENaC in rat lung and kidney cells (12, 13). Nedd4 is homologous to ubiquitin ligase and contains three WW domains (12). Binding of these domains to the PPXY regions of ENaC beta  and gamma  subunits has been reported so far in Western binding assays and in mammalian cells transfected with ENaC subunits (5, 14). After these WW domains interact with the ENaC subunit PPXY domain, an E6-AP carboxyl terminus homologous domain in Nedd4 acts as a ubiquitin ligase and conjugates ubiquitin to the amino termini of alpha  and gamma  ENaC subunits (15). Ubiquitin coupling to ENaC alpha  and gamma  subunits occurs in Madin-Darby canine kidney cells transfected with all three ENaC subunits, and a reduction in ENaC protein levels occurs (5). Co-expressing Nedd4 and ENaC subunits in Xenopus oocytes reduces ENaC protein at the cell surface and decreases whole cell sodium current (6, 7). The simplest explanation of this observation would be that ENaC protein is degraded by ubiquitin-mediated proteosomal proteolysis but Abriel and colleagues (6) embed the change in channel activity to lysosomal degradation of ENaC. For some membrane proteins, ubiquitin does not act as a proteosomal degradation signal but rather as a signal for endocytosis of the proteins and subsequent degradation by lysosomal rather than proteosomal pathways (16). The reports of Staub and colleagues and Goulet and colleagues (5-7) provide evidence that artificially expressing ENaC in cells that normally do not express ENaC, leads to ubiquitin-mediated lysosomal degradation and, by implication, down-regulation of ENaC activity (5-7).

To identify the site of ENaC degradation and whether proteolysis can regulate wild type ENaC function, we studied a native distal nephron cell line (A6) derived from Xenopus kidney and examined how inhibiting proteosome activity affected the amount of cellular ENaC protein, the number of functional ENaC molecules in the plasma membrane, and the properties of single ENaC channels. Our results suggest that functional ENaC channel proteins are degraded by ubiquitin-proteosome proteolysis and that this pathway therefore, might be an important regulator of ENaC in native, sodium-transporting epithelial cells.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation and Characterization of Antibodies-- Polyclonal antibodies were raised in rabbits against synthetic peptides derived from xENaC (Xenopus epithelial Na+ channel) subunit sequences (Lofstrand Laboratories). Anti-alpha xENaC polyclonal antibodies were generated against a peptide corresponding to the amino acid sequence 137CIPNNQRVKRDRAGLPYLLELLPPGS161 present in the extra-cellular domain of alpha  xENaC. Anti-beta and anti-gamma xENaC polyclonal antibodies were directed against amino acid sequences 624CGTPPPNYDSLRVNTAEPVSSDEEN647 and 600CVDNPICLGEEDPPTFNSALQLPQSQDSHVPRTPPPKYNTLRIQSAF647, respectively, which are present in the carboxyl-terminal regions of the subunits. The antibodies were purified from serum using an antigenic peptide affinity column that had been synthesized by coupling immunizing peptides with SulfoLink agarose beads according to the manufacturer's instructions (Pierce).

Lysates of A6 cells were resolved on 7.5% SDS-PAGE and transferred to nitrocellulose, and bands of 86, 97, and 100 kDa were observed after adding alpha , beta , and gamma  subunit-specific antibodies, respectively; the bands were missing when the respective antigenic peptide for each of the three subunits was added before probing the membrane (Fig. 1). These results imply that the bands corresponding to the glycosylated forms of the three xENaC subunit proteins bind to the complimentary determining region of the antibodies. However, the anti-beta antibody also recognized a 78-kDa band that corresponds to the non-glycosylated form of the protein. We also constructed fusion proteins of all three subunits with glutathione S-transferase protein (GST) and expressed them in Escherichia coli BL-21 bacteria. The antibody recognized a fusion protein of the correct size only in the induced bacteria (Fig. 1); these bands were abolished by adding the antigenic peptide. Next, we repeated these experiments with Chinese hamster ovary cells that had been transfected with complete alpha , beta , and gamma  xENaC subunits. The same size bands were observed in the Chinese hamster ovary cellular lysates as in the A6 lysate. No such bands were observed in the untransfected Chinese hamster ovary cells, and adding the antigenic peptides suppressed recognition of all three xENaC subunits (results not included).


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Fig. 1.   Characterization of anti-xENaC subunit-specific polyclonal antibodies. The top panel shows reactivity of anti-ENaC subunit-specific antibodies with A6 cellular proteins in the absence and presence of antigenic peptides. Confluent A6 cells grown on permeable supports were harvested, lysed, and subjected to Western blot analysis with anti-alpha (137CIP ... PGS150), anti-beta (624CGT ... EEN647), and anti-gamma (599CVD ... SAF647) polyclonal antibodies. The bottom panel shows reactivity of the same antibody against ENaC alpha  (65QFGLLF ... WSLWFGS518), beta  (571TILKFLA ... QAATA647), and gamma  (562WVVLRQR ... VFTLTSMR660) fusion proteins with GST (29 kDa) expressed in E. coli BL21 cells. Bacterial lysates, induced to express the fusion proteins (lane a), showed bands of the correct size that were not observed in the presence of antigenic peptide (lane c) or in the uninduced bacteria (lanes b and d).

Cell Culture-- A highly transporting clone, 2F3, of Xenopus laevis distal nephron epithelial cells (A6) were maintained using standard tissue culture techniques as described previously (17). Cells within passage number 100-120 were grown to confluence on 0.02 µm of Anopore membranes (Nalge Nunc) in the presence of 1.5 µM aldosterone. Trans-epithelial voltage and resistance of A6 monolayers were measured by EVOM (World Precision Instruments) and the current/unit area was calculated as: I = [(V)/(R)]/filter area in cm2. After treatment of the A6 monolayer with proteosomal (MG-132, Biomol) or lysosomal (chloroquine and methylamine, Sigma) inhibitors, measurements for calculating the trans-epithelial current were made, cells were washed twice with PBS buffer, and then harvested at 4 °C by scraping them in the PBS buffer containing mixture A protease inhibitors, (100 µM leupeptin, 100 µM antipain, 1 mM phenylmethylsulfonyl fluoride, 100 µM 1-chloro-3-tosylamido-7-amino-2-heptanone, and 100 µM L-1-tosylamido-2-phenylethyl chloromethyl ketone).

Fusion Protein Expression in Bacteria-- xENaC polymerase chain reaction products were cloned into PGEM-T Easy vector (Promega, Madison, WI), cut with EcoRI enzyme and subcloned into PGEX4T-3 vector (Amersham Pharmacia Biotech). PGEX4T-3 is a prokaryotic vector containing the GST gene. Fusion proteins were generated with GST protein and amino acids corresponding to the alpha  (65QFGLLF ... WSLWFGS518), beta  (571TILKFLA ... QAATA647), and gamma  (562WVVLRQR ... VFTLTSMR660) xENaC subunits. All constructs were expressed in E. coli BL21-Codon Plus RIL competent cells (Stratagene) in liquid cultures grown at 37 °C to an absorbance of 0.5-0.6 at 600 nm. Fusion protein expression was induced by adding isopropyl-1-thio-beta -D-galactopyranoside at a final concentration of 1 mM for 2 h, after which cells were collected by centrifugation. Bacteria were washed and lysed by sonication in PBS buffer containing mixture A protease inhibitors. Unbroken cells were separated by centrifugation and the bacterial proteins were resolved on 12% SDS-PAGE, transferred to nitrocellulose, and probed with anti-ENaC subunit-specific antibodies.

Cell Surface Biotinylation-- Confluent A6 cells grown on permeable supports were washed three times with cold PBS buffer, and then 0.5 mg/ml sulfo-SS-biotin (Pierce) in borate buffer (85 mM NaCl, 4 mM KCl, 15 mM Na2B4O7, pH 9.0) was added to the apical surface while the basolateral compartment was exposed to media containing 5% (v/v) fetal calf serum. The experiment was performed at 4 °C with gentle agitation for 15 min, and the procedure was repeated. The labeling was stopped by adding 5% fetal calf serum. Cells were extensively washed in PBS buffer, harvested, and lysed in buffer B (PBS with 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate) containing mixture A protease inhibitors. Cellular debris was removed by centrifugation (1200 × g, 5 min), and biotin-labeled proteins were precipitated by incubating with prewashed streptavidin coupled to agarose beads for 18 h at 4 °C with gentle agitation. These beads were washed five times with buffer B and the biotin-streptavidin complex lysed by boiling in buffer containing 100 mM dithiothreitol and 5% SDS. The precipitated proteins were separated on 7.5% SDS-PAGE, transferred to nitrocellulose, and probed with anti-ENaC subunit-specific antibodies.

Immunoblotting-- A6 cellular proteins were separated by 7.5% SDS-PAGE (bacterial proteins were separated by 12% SDS-PAGE) and transferred to nitrocellulose paper. The nitrocellulose was blocked in TBS buffer containing 5% milk and 0.1% Tween 20 and then probed with the anti-xENaC subunit-specific antibodies or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Biodesign International). In experiments involving peptide competition, the primary antibody was incubated with the antigenic peptide for at least 1 h at room temperature before using it to probe the nitrocellulose. The secondary antibody was goat anti-rabbit coupled to horseradish peroxidase (Kirkegaard and Perry Laboratories), and the antigen antibody complex was detected by a chemiluminescent detection system (Amersham Pharmacia Biotech). The films were scanned, and the band intensity was measured using Sigma Gel (Jandel Scientific).

Half-life Measurements-- Confluent A6 cells on permeable supports were washed three times with PBS buffer and incubated for 48 h in regular media but without serum and methionine. The cells were then incubated overnight with media containing [35S]methionine, washed three times with PBS, and incubated in regular media containing either MG-132 or the vehicle Me2SO. At various times, plates of cells were harvested, lysed, and immunoprecipitated as above. The immunoprecipitated proteins were resolved on a 7.5% SDS-PAGE, dried, and exposed to x-ray film. The films were scanned, and the recorded band intensities were measured using Sigma Gel (Jandel Scientific).

Patch Clamp Experiments-- A6 cells were grown to confluence on glutaraldehyde-fixed, collagen-coated Millipore-CM filters (Millipore Corp.) attached to the bottom of small Lucite rings. The cells were visualized with Hoffman modulation optics (Modulation Optics, Inc.). At room temperature the pipette tip gently contacted the A6 cell surface, and negative pressure was applied to obtain a seal resistance of 10-20 gigaohms. Unitary channel events were measured using a List EPC-7 Patch Clamp (Medical Systems Corp.), digitized by DAS 601 Pulse Code Modulator (Dagan Corp.), and recorded on a SL-HF860D video recorder (Sony Corp. of America). Data were acquired using a 902LPF 8-pole Bessel filter (Frequency Devices, Inc.), TL-2 acquisition hardware, and Axotape software (Axon Instrument, Inc.). Analysis of data was performed on a 386SX computer (Mitsuba Southeast, Inc.). Pipette and extracellular bath solutions were physiological amphibian saline solutions containing 95 mM NaCl, 3.4 mM KCl, 0.8 mM CaCl2, 0.8 mM MgCl2, and HEPES, pH 7.4. MG-132 was added to both the apical and basolateral surfaces of the A6 cells (at a concentration of 6 µM) before patches were made.

Amiloride-sensitive Na+ channels were identified in apical cell-attached patches as low conductance (4 picosiemans) channels. A nonlinear current-voltage relationship and long mean open and closed times were obtained as described (17). The total number of channels (N) in the patch was estimated by observing the number of current levels or the number of peaks detected on current amplitude histograms. As a measure of channel activity, NPo (Number of channels × the open probability) was calculated using the following relationship (Equation 1),
NP<SUB><UP>o</UP></SUB>=<LIM><OP>∑</OP><LL>n = 0</LL><UL>N</UL></LIM><FR><NU>n−t<SUB>n</SUB></NU><DE>T</DE></FR> (Eq. 1)
where T is the total recording time, and t is the time during recording when there were n channels open. If channels open independently of one another and the exact number of channels in a patch are known, then the open probability (Po) of a single channel in a patch can be calculated by dividing NPo by the number of the channels in a patch. Values are expressed as S.E. Test values of p < 0.05 were considered significant.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Inhibition of Proteosome Activity Increases the Steady State Levels of ENaC Subunits and Increases the Half-life of the beta  Subunit

At 6 µM, MG-132 is a specific inhibitor of the proteosome activity in kidney cells (18). When A6 cells were treated with 6 µM MG-132 for 2-3 h, there was an increase in alpha , beta , and gamma  subunits by 376, 453, and 728% respectively (versus levels in untreated cells) (Fig. 2A). A more specific (and expensive) inhibitor of the proteosome activity, lactacystin, also caused an increase in all three ENaC subunits (data not shown). These results suggest that the total amount of each ENaC subunit (both the membrane-associated and intracellular pools) increases when proteosome activity is inhibited and that activity of this proteolytic pathway might be important in determining the steady state levels of ENaC in A6 cells.


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Fig. 2.   A, inhibition of proteosome activity increases total cellular xENaC. A6 cells were treated with a proteosome inhibitor or left untreated and harvested after 2.5-3 h, lysed, and analyzed by Western blot. Bands were obtained at 86, 96, and 100 kDa with alpha , beta , and gamma  subunit-specific antibodies, respectively. The band intensity was measured using Sigma Gel (Jandel Scientific), and the results are shown in a graph below each blot (n > 3); the dotted lines represent the untreated cells. Cells treated with proteosome inhibitor MG-132 (6 µM) showed an increase in the band intensity in all three subunits, and similar increases in band intensity of all three subunits were observed with 10 µM lactacystin (results not shown). B, proteosome inhibition increases the half-life of cellular xENaC. A6 cells labeled with [35S]methionine were treated with proteosome inhibitor or left untreated and harvested at 0, 1, 2, and 3 h, lysed, immunoprecipitated, and resolved on 7.5% SDS-PAGE, and the gel was exposed to x-ray film. The band intensity was measured using Sigma Gel (Jandel Scientific) and the results are shown in a graph below each blot.

MG-132 treatment substantially increased the half-life of beta  ENaC from 2.05 ± 0.366 h to no detectable decrease in subunit amount after 4 h (Fig. 2B). MG-132 did not affect our measurement of the half-life of the alpha  subunit. However, the alpha  subunit half-life in untreated cells was extremely long (there was no statistically significant decrease in this subunit even after 24 h), so we could not reliably detect any change induced by MG-132 in our experiments. Nonetheless, prolongation of the beta  subunit half-life supports our hypothesis that MG-132 changes ENaC degradation rather than ENaC synthesis.

To determine whether ubiquitin-proteosome proteolysis influences ENaC proteins in the apical membrane, these proteins were labeled with biotin, precipitated with streptavidin, and subjected to Western blot analysis. When cells were treated with MG-132 before biotin labeling, the amount of ENaC subunit protein increased (Fig. 3). These results suggest that the inhibition of proteosome activity increases the number of functional ENaC molecules in the apical plasma membrane of A6 cells and, hence, should increase trans-epithelial Na+ transport.


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Fig. 3.   . A proteosome inhibitor increases membrane-associated cellular xENaC. Proteosome inhibitor-treated or untreated cells were labeled with biotin, harvested, and lysed. Biotin-labeled proteins were precipitated overnight using streptavidin coupled to agarose beads, and the precipitated proteins were eluted, resolved on SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were probed with anti-xENaC subunit-specific, polyclonal antibodies. The band intensity was measured using Sigma Gel (Jandel Scientific), and the results are shown in a graph below each blot (n > 3); the dotted lines represent the untreated cells. Increased band intensities were observed with all three xENaC subunit-specific antibodies.

Proteosome Inhibition Increases ENaC Activity

Inhibition of Proteosomal Complex Increases Trans-epithelial Current-- To determine whether the increase in ENaC protein is physiologically relevant, ENaC function was measured as trans-epithelial current after A6 cells were exposed to MG-132 for 4 h. There was an increase of trans-epithelial current by 2.5-3-fold in these cells compared with untreated cells (Fig. 4A). The increase in trans-epithelial current reached a maximum in about 2-2.5 h after the inhibitor was added, a time course consistent with that of a rapidly exchangeable pool of ENaC that regulates delivery of assembled ENaC to the membrane (a previously reported half-life of about 2-3 h) (5). To establish that this increase in the trans-epithelial current was due to an increase in amiloride-sensitive Na+ current, A6 cells treated with MG-132 were then exposed to 100 nM amiloride on the apical surface. Trans-epithelial current in both the experimental as well as the control cells was abolished by amiloride (Fig. 4B) indicating that the increase in trans-epithelial current following inhibition of the proteosome is predominantly due to increased ENaC activity. To confirm that the MG-132 results are due to inhibition of proteosome activity, we used a more specific inhibitor, lactacystin; results with 10 µM lactacystin showed an increase in trans-epithelial current in the same time course as observed with MG-132. This response was also abolished by 100 nM amiloride (data not shown).


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Fig. 4.   A proteosome inhibitor increases trans-epithelial current in A6 cells. Confluent A6 cells grown on permeable supports were treated with 6 µM MG-132 or left untreated. Trans-epithelial voltage and resistance were measured every 15 min for 4 h, and trans-epithelial current/unit area was calculated and plotted on the Y axis versus time on the X axis. A, MG-132 (filled circles) led to an increase in trans-epithelial current with a maximum value ~2.5-3-fold higher after 3 h. Untreated cells (empty circles) had no increase in their trans-epithelial current. Similar results were obtained with another proteosome inhibitor, 10 µM lactacystin, (results not shown). B, the increased trans-epithelial current was completely abolished by 100 nM amiloride indicating that the increase in trans-epithelial current was due to an increase in activity of xENaC. n = 6 and error bars represent S.E.

Degradation by Proteosomes Regulates the Number of ENaC Channels in A6 Membrane Patches-- The increase in trans-epithelial current implies an increase in the activity of ENaC in the apical membrane, but this response could be due to an increase in the number of channels in the membrane or to an increase in the open probability of existing channels. Although Western blots suggest that the number of channels increase, these channels may not be active; so we examined this possibility by patch clamp. Membrane patches were formed on the surface of paired sets of A6 cells that had been treated with MG-132 or left untreated. Recordings were made from 40 min to 4 h after application of MG-132 with alternate recordings from both treated and untreated cells (current levels in the patches were counted as a measure of the number of channels/patch, N) (Fig. 5A). There was a significant increase (p = 0.035) in the number of channels in MG-132 treated cells (mean for treated cells = 7.4 ± 1.3, n = 11; mean for untreated cells = 4.1 ± 0.71, n = 11) (Fig. 5B). There was also a significant increase (p = 0.049) in the activity of channels measured as the product of the number of channels × the open probability (NPo, mean for treated cells = 4.3 ± 1.0; mean for untreated cells = 1.0 ± 0.27). Based on these values of N and NPo for individual patches, it is possible to estimate the open probability (Po, mean for treated cells = 0.49 ± 0.089; mean for untreated cells = 0.21 ± 0.053). We recognize that caution is required in interpreting the Po values because they depend upon accurate estimates of N. However, if N in MG-132-treated cells were found to be larger than our calculated value, then Po for the treated cells would have been lower and not significantly different from that for untreated cells.


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Fig. 5.   A, typical single channel tracings from MG-132-treated and untreated A6 cells. A representation of single channel tracings from membrane patches formed on the surface of paired sets of A6 cells. An upward deflection represents an open channel, and these current levels were counted as a measure of the number of channels/patch (N). There was a significant increase (p = 0.035) in the number of channels in MG-132-treated cells versus untreated cells (mean for treated cells = 7.4 ± 1.3, n = 11, versus untreated cells = 4.1 ± 0.71, n = 11). B, a proteosome inhibitor increases the density of channels in membrane patches. Membrane patches were formed on the surface of paired sets of A6 cells and recording began at 40 min after application of MG-132 and continued until 4 h after treatment with alternate recordings from treated and untreated cells. Current levels in the patches were counted as a measure of the number of channels/patch (N). There was a significant increase (p = 0.035) in the number of channels in MG-132-treated cells versus untreated cells (mean for treated cells = 7.4 ± 1.3, n = 11, versus untreated cells = 4.1 ± 0.71, n = 11).

Inhibition of the Lysosomal Pathway Does Not Change the Level of the ENaC Subunit Protein or Its Activity

Because the lysosomal pathway is another degradation pathway for some membrane proteins and because the lysosomal pathway is reportedly important for ENaC degradation in cell systems transfected with ENaC subunits (5), we examined whether lysosomal proteolysis might regulate the number of ENaC in A6 cells. Chloroquine and methylamine are weak bases that prevent proteolysis in late lysosomes by increasing vacuolar pH to inhibit the activity of lysosomal enzymes and interfere with protein trafficking. A6 cells were incubated with 0.1 µM chloroquine or 10 µM methylamine for 2-3 h, and ENaC expression was examined by Western blotting. No significant increase was observed in any of the three ENaC subunits in cells treated with chloroquine (Fig. 6A) or methylamine (data not shown). We believe that the inhibitors did inhibit lysosomal function because there was a significant increase in GAPDH levels after incubation with methylamine, and GAPDH is known to be lysosomally degraded. Because the half-life of GAPDH is much longer than that of ENaC subunits, the cells were incubated for 72 h with methylamine before harvesting (19), but again, there was no significant increase in ENaC subunit levels (data not shown). Thus, lysosomal proteolysis does not appears to play an important role in the degradation of the endogenous ENaC proteins present in A6 cells.


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Fig. 6.   Lysosome inhibitors do not increase either cellular xENaC or trans-epithelial current in A6 cells. A6 cells were treated with lysosome inhibitors (chloroquine or methylamine) or left untreated, harvested after 2.5 h, and lysed. The lysate was analyzed by Western blot. Ai, bands were obtained at 86, 96, and 100 kDa with alpha , beta , and gamma  subunit-specific antibodies, respectively. Aii, GAPDH was used as a positive control. The band intensity was measured using Sigma Gel (Jandel Scientific), and the results are shown in a graph below each blot (n > 3), and the dotted lines represent the untreated cells. Ai, no significant change was observed between treated and untreated cells. Aii, the GAPDH protein increases in cells treated with methylamine after 72 h. Confluent A6 cells grown on permeable supports were treated with the lysosome inhibitor chloroquine (B), methylamine (C), or left untreated. Trans-epithelial voltage and resistance were measured at different time points, and then trans-epithelial current/unit area was calculated and plotted on the Y axis versus time on the X axis. No significant increase was observed with lysosome inhibitors, chloroquine and methylamine, n = 6 and error bars represent S.E.

ENaC activity (trans-epithelial current) was also measured in A6 cells treated with the lysosomal inhibitors chloroquine (0.1 µM) or methylamine (10 µM); there was no increase in trans-epithelial current when compared with untreated cells (Fig. 6B). Even when the drugs were tested at different doses and various exposure times, there was no increase in the trans-epithelial current. To confirm that lysosome degradation does not regulate activity of endogenous ENaC, we studied a lysosomal protease inhibitor, 10 µM leupeptin. There was no increase in trans-epithelial current of A6 cells (data not shown). Curiously, chloroquine caused some decrease in transport activity. This may be due to reduced ENaC trafficking to the membrane because chloroquine can inhibit endosomal trafficking. Alternatively, there may be a cytotoxic effect of chloroquine because it can also uncouple mitochondrial electron transport.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our objectives were to identify the proteolytic pathway primarily responsible for degradation of ENaC subunit proteins in native, sodium-transporting epithelial cells and to determine whether degradation could play a role in determining the number and activity of functional ENaC channels in renal A6 cells. We studied both the ubiquitin-proteosome pathway and lysosomal proteolysis because enzymes involved in ubiquitin conjugation can interact with the alpha  and beta  subunits of ENaC and because ubiquitin conjugation was linked to lysosomal degradation of ENaC subunits transfected into Madin-Darby canine kidney cells (6, 12). Moreover, lysosomes characteristically degrade membrane proteins and can degrade ENaC proteins under some circumstances (8). We used A6 cells because A6 epithelial cells in culture form polarized monolayers that express ENaC protein on the apical membrane and because ENaC activity is regulated by the same factors as in intact kidney (20).

Our results indicate that both the total pool of ENaC protein in A6 cells and the number of functional ENaC molecules in the apical membrane are predominantly degraded by the proteosome (Figs. 2A and 3). Based on the long half-life of the alpha  subunit and the increase in function of ENaC after blocking the proteosome for only 4 h, it is tempting to speculate that turnover of the beta  subunit affects the net functional pool of ENaC more than the alpha  subunit. The difference we observed between the increase in the total cellular and membrane-associated ENaC subunits (Figs. 2A and 3) is likely attributable to the techniques used (simple Western blotting versus double immunoprecipitation). The double immunoprecipitation experiments involved biotin labeling of cell surface ENaC subunits and their precipitation, first with streptavidin and then with anti-ENaC subunit-specific antibodies. Consequently, the effectiveness of ENaC subunit detection depended both on the efficiency of biotin labeling and subsequent immunoprecipitations compared with the detection efficiency of Western blots. Despite this caveat, we cannot rule out the possibility that proteosomes degrade ENaC localized at the cell surface at a slower rate than ENaC within the cell because intracellular mis-assembled or mis-folded subunits would be degraded more rapidly than properly assembled, functional channels would be within the apical membrane.

Our patch clamp results indicate that inhibition of proteosome activity increases channel density (Fig. 5) whereas inhibition of lysosomal or cathepsin activity produced no increase in ENaC amount or activity (Fig. 6, A and B). Interestingly, chloroquine, and to a much lesser extent methylamine, may actually reduce the functional ENaC activity (measured as trans-epithelial Na+ current). A potential explanation is that inhibitors of the lysosomal proteolytic pathway (like methylamine and chloroquine) operate by reducing the intralysosomal pH gradient but incidentally also reduce endosomal pH. Because a change in endosomal pH can interfere with protein trafficking (21), total cellular ENaC subunit protein levels might not change, but ENaC trafficked to the membrane would decrease. Chloroquine can also act as an uncoupler of mitochondrial electron transport and, therefore, could be toxic to cells over a prolonged period.

In some ways, inhibition of the proteosome mimics changes in Na+ transport that are present in Liddle's syndrome because there is an increase in ENaC activity resulting predominately from an increase in the number of functional ENaC channels. In Liddle's syndrome, there is a mutation or deletion of the proline rich (PPXY) region of the beta  or gamma  subunit (22-24). Nedd4, a potential ubiquitin ligase is co-localized with ENaC in rat lung and kidney cells (13). Nedd4 functions as a negative regulator of whole cell current and of the ENaC residing in the plasma membrane in Xenopus oocytes expressing alpha , beta , and gamma  ENaC subunits (5). Because Nedd4 has regions that are homologous to ubiquitin-conjugating enzymes, we and others reasoned that ENaC could be degraded by the proteosome after Nedd4-dependent ubiquitin conjugation. Staub and et al. (13) transfected ENaC subunits into Madin-Darby canine kidney cells and examined how proteosome inhibition affected their turnover. They concluded that mis-folded and unassembled ENaC are degraded by the proteosome but that properly assembled and functional ENaC residing in the plasma membrane are degraded in lysosomes. This pathway would lead to an increase in non-functional cellular ENaC levels when proteosome activity is inhibited, but there would be no increase in membrane-associated ENaC proteins nor any gain in channel function. In contrast, in A6 cells that express functional ENaC channels there is an increase in channel function when proteosome activity is blocked (Fig. 5). Likewise, ENaC activity rises in oocytes injected with Liddle's mutant or inactivated Nedd4 (6, 7). These data implicate Nedd4-mediated ubiquitin conjugation in the degradation of ENaC (Fig. 7), but in cells transfected to express ENaC lysosomal inhibitors do appear to alter ENaC degradation (5) even though the endogenous ENaC protein levels and ENaC activities in native cells are not affected by blocking lysosomal activity or cathepsins (Fig. 6). Presumably, the results obtained in cells transfected to express ENaC result from the pathway that uses ubiquitin coupling as a signal for membrane protein endocytosis. Endocytosis leads to the degradation of these proteins by proteolysis in lysosomes as shown for uracil permease, multidrug transporter Pdr5, and Ste6 protein (25-27). However, we found that GAPDH degradation is inhibited by lysosomal inhibitors but that ENaC degradation is unaffected by the same lysosomal inhibitors. This leads to the conclusion that endogenous-functioning ENaC channel proteins in the cell membrane are not degraded by the lysosomes but are degraded by the proteosome. The difference in our results from those of Staub and colleagues (5) may lie in the fact that protein expression or cellular pathways in transfected cells change the pathways by which protein turnover is regulated.


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Fig. 7.   ENaC degradation through proteosomal pathway. Shown here is a schematic of ENaC degradation through the proteosomal pathway, which involves two distinct steps. In the first step, ENaC is coupled to ubiquitin in an ATP-dependent manner with involvement of E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase). In A6 cells, Nedd4 binds to ENaC and couples ubiquitin to the ENaC subunits. In the second step, ENaC conjugated to multiple ubiquitin molecules is unfolded and degraded into small peptides by the proteosomal complex. The proteosomal inhibitors we utilized bind to the active site of the complex and block its function.

We have also observed that blocking activity of the proteosome increased the open probability (Po) of ENaC. This observation needs to be interpreted cautiously because it is influenced by the accuracy of our estimate of N (the number of channels), and there are other possibilities for an increase in Po. One possibility is that the increase in Na+ transport alters the cellular environment in such a way as to increase ENaC Po. This possibility seems unlikely because "feedback" inhibition of ENaC by an increase in intracellular Na+ would be expected to decrease Po when Na+ transport increased. A second possibility is that there is a differential degradation of subunits leading to the formation of channels with a higher Po. This also seems an unlikely possibility because it would require a change in channel properties, but we observed no differences in other channel characteristics besides Po in MG-132-treated cells. A third possibility is that a ubiquitin-conjugated channel has a higher open probability. This is possible because mutations in the regions to which ubiquitin binds, at least in the alpha  subunit, can alter ENaC gating and hence, Po (28). The final possibility is that ENaC activity is maintained at low levels by a regulatory protein that is degraded by the proteosome. In this case, inhibition of the proteosome would increase the number of putative regulator molecules that increase Po. Distinguishing between the latter two possibilities might be difficult unless the regulatory protein is identified.

In summary, we find that inhibition of proteosome activity increases the cellular level of all three ENaC subunits, increases the trans-epithelial Na+ transport, the apical density of the channel, and possibly Po). These findings support the hypothesis that the pathway regulating membrane and cellular ENaC turnover in native, sodium-transporting epithelial involves ubiquitin conjugation and proteosome degradation rather than a lysosomal pathway.

    ACKNOWLEDGEMENTS

We thank Billie Jeanne Duke for tissue culture work and Nina C. Saxena for providing the transfected Chinese hamster ovary cells. We thank other laboratory colleagues for helpful discussions and especially thank Ollie Appleberry for her suggestions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK-37963-14 and DK-50268-4 (to D. C. E.), Grant DK-37175 (to W. E. M.), and DK-50740 (to S. R. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Physiology, Center for Cell and Molecular Signaling, Physiology Bldg., Rm. 074, 1648 Pierce Dr., Atlanta, GA 30322. Tel.:404-727-7247; Fax: 404- 727-0029; E-mail: bmalik@ccms-renal.physio.emory.edu.

Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M010626200

    ABBREVIATIONS

The abbreviations used are: ENaC, epithelial Na+ channel; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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