From the 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 ( 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, 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 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.
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-
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 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 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),
Inhibition of Proteosome Activity Increases the Steady State Levels
of ENaC Subunits and Increases the Half-life of the 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
,
,
) 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
,
, and
; 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.
and
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
and
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
and
ENaC
subunits (15). Ubiquitin coupling to ENaC
and
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).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
xENaC polyclonal antibodies were generated against a peptide corresponding to
the amino acid sequence
137CIPNNQRVKRDRAGLPYLLELLPPGS161 present
in the extra-cellular domain of
xENaC. Anti-
and anti-
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).
,
, and
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-
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
,
, and
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).
View larger version (40K):
[in a new window]
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-
(137CIP ... PGS150), anti-
(624CGT ... EEN647), and anti-
(599CVD ... SAF647) polyclonal antibodies.
The bottom panel shows reactivity of the same antibody
against ENaC
(65QFGLLF ...
WSLWFGS518),
(571TILKFLA ...
QAATA647), and
(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).
(65QFGLLF ... WSLWFGS518),
(571TILKFLA ... QAATA647), and
(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-
-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.
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.
(Eq. 1)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Subunit
,
, and
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.
View larger version (30K):
[in a new window]
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 ,
, and
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 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
subunit. However, the
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
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.
|
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).
|
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.
|
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.
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 and
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 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
subunit affects the net functional pool of ENaC more than the
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 or
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
,
, and
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.
|
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 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | O'Brodovich, H. (1995) New Horizons 3, 240-247[Medline] [Order article via Infotrieve] |
2. | Canessa, C. M., Schild, L., Buell, G., Thorens, B., Gautschi, I., Horisberger, J. D., and Rossier, B. C. (1994) Nature 367, 463-467[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Snyder, P. M.,
McDonald, F. J.,
Stokes, J. B.,
and Welsh, M. J.
(1994)
J. Biol. Chem.
269,
24379-24383 |
4. |
Marunaka, Y.,
and Eaton, D. C.
(1991)
Am. J. Physiol.
260,
C1071-C1084 |
5. |
Staub, O.,
Gautschi, I.,
Ishikawa, T.,
Breitschopf, K.,
Ciechanover, A.,
Schild, L.,
and Rotin, D.
(1997)
EMBO J.
16,
6325-6336 |
6. |
Abriel, H.,
Loffing, J.,
Rebhun, J. F.,
Pratt, J. H.,
Schild, L.,
Horisberger, J. D.,
Rotin, D.,
and Staub, O.
(1999)
J. Clin. Invest.
103,
667-673 |
7. |
Goulet, C. C.,
Volk, K. A.,
Adams, C. M.,
Prince, L. S.,
Stokes, J. B.,
and Snyder, P. M.
(1998)
J. Biol. Chem.
273,
30012-30017 |
8. |
Mitch, W. E.,
and Goldberg, A. L.
(1996)
N. Engl. J. Med.
335,
1897-1905 |
9. | Coux, O., Tanaka, K., and Goldberg, A. L.A. L. A. L. (1996) Annu. Rev. Biochem. 65, 801-847[CrossRef][Medline] [Order article via Infotrieve] |
10. | Ward, C. L., Omura, S., and Kopito, R. R. (1995) Cell 83, 121-127[Medline] [Order article via Infotrieve] |
11. | Hershko, A., and Ciechanover, A. (1998) Annu. Rev. Biochem. 67, 425-479[CrossRef][Medline] [Order article via Infotrieve] |
12. | Staub, O., and Rotin, D. (1996) Structure 4, 495-499[Medline] [Order article via Infotrieve] |
13. |
Staub, O.,
Yeger, H.,
Plant, P. J.,
Kim, H.,
Ernst, S. A.,
and Rotin, D.
(1997)
Am. J. Physiol.
272,
C1871-C1880 |
14. |
Harvey, K. F.,
Dinudom, A.,
Komwatana, P.,
Jolliffe, C. N.,
Day, M. L.,
Parasivarum, G.,
Cook, D. I.,
and Kumar, S.
(1999)
J. Biol. Chem.
274,
12525-12530 |
15. | Staub, O., Dho, S., Henry, P., Correa, J., Ishikawa, T., McGlade, J., and Rotin, D. (1996) EMBO J. 15, 2371-2380[Abstract] |
16. |
Strous, G. J.,
and Gowen, C. W.
(1999)
J. Cell. Sci.
112,
1417-1423 |
17. | Eaton, D. C., Marunaka, Y., and Ling, B. N. (1991) in Membrane Transport in Biology (Schafer, J. , and Giebisch, G. H., eds) , pp. 73-165, Springer-Verlag New York Inc., New York |
18. |
Franch, H. A.,
Curtis, P. V.,
and Mitch, W. E.
(1997)
Am. J. Physiol.
273,
C843-C851 |
19. |
Aniento, F.,
Roche, E.,
Cuerve, A. M.,
and Knepel, W.
(1993)
J. Biol. Chem.
268,
10463-10470 |
20. | Hamilton, K. L., and Eaton, D. C. (1985) Am. J. Physiol. 249, C200-C207[Abstract] |
21. | Seglen, P. O. (1983) Methods Enzymol. 96, 737-764[Medline] [Order article via Infotrieve] |
22. | Hansson, J. H., Nelson-Williams, C., Suzuki, H., Schild, L., Shimkets, R., Lu, Y., Canessa, C. M., Iwasaki, T., Rossier, B., and Lifton, R. P. (1995) Nat. Genet. 11, 76-82[Medline] [Order article via Infotrieve] |
23. | Hansson, J. H., Schild, L., Lu, Y., Wilson, T. A., Gautschi, I., Shimkets, R., Nelson-Williams, C., Rossier, B. C., and Lifton, R. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11495-11499[Abstract] |
24. | Shimkets, R. A., Warnock, D. G., Bositis, C. M., Nelson-Williams, C., Hansson, J. H., Schambelan, M., Gill, J. R., Jr., Ulick, S., Milora, R. V., Findling, J. W., Canessa, C. M., Rossier, B. C., and Lifton, R. P. (1994) Cell 79, 407-414[Medline] [Order article via Infotrieve] |
25. |
Galan, J. M.,
Moreau, V.,
Andre, B.,
Volland, C.,
and Hague-nauer-Tsapis, R.
(1996)
J. Biol. Chem.
271,
10946-10952 |
26. | Egner, R., and Kuchler, K. (1996) FEBS Lett. 378, 177-181[CrossRef][Medline] [Order article via Infotrieve] |
27. | Kolling, R., and Hollenberg, C. P. (1994) EMBO J. 13, 3261-3271[Abstract] |
28. |
Berdiev, B. K.,
Shlyonsky, V. G.,
Karlson, K. H.,
Stanton, B. A.,
and Ismailov, I. I.
(2000)
Biophys. J.
78,
1881-1894 |