Effect of subunit composition and Liddle's syndrome mutations
on biosynthesis of ENaC
Lawrence S.
Prince and
Michael J.
Welsh
Howard Hughes Medical Institute, Departments of Pediatrics, Internal
Medicine, and Physiology and Biophysics, University of Iowa College
of Medicine, Iowa City, Iowa 52242
 |
ABSTRACT |
The epithelial
Na+ channel (ENaC) is comprised of
three homologous subunits:
,
, and
, all of which are required
for formation of the fully functional channel. This channel is
responsible for salt reabsorption in the kidney, the airway, and the
large bowel. Mutations in ENaC can cause human disease by increasing
channel function in Liddle's syndrome, a form of hereditary
hypertension, or by decreasing channel function in
pseudohypoaldosteronism type I, a salt-wasting disease of infancy. We
previously showed that ENaC is expressed on the cell surface as a
minimally glycosylated, Triton-insoluble protein. In the present
study we found that ENaC existed initially as a Triton-soluble protein
that contained high-mannose glycosylation, presumably in the
endoplasmic reticulum. This form of the protein disappeared as the
Triton-insoluble, minimally glycosylated form became the more prevalent
species. In pulse-chase studies of individually expressed subunits, we
found that the Triton-soluble form of
-ENaC accumulated initially,
whereas the Triton-soluble form of
-ENaC decreased throughout the
time course. However, when all three subunits were coexpressed, the
- and
-subunits showed a similar pattern. The complex became
Triton insoluble at some point after the endoplasmic reticulum, as
incubation at 15°C blocked the conversion to the insoluble form.
Deletion of the carboxy-terminal tail of
-ENaC causes Liddle's
syndrome. This mutation increased the amount of newly synthesized
Triton-insoluble ENaC heteromultimers but did not affect the half-life
of insoluble protein. Therefore, subunit composition and mutations in
individual subunits can influence biosynthesis of the ENaC complex.
degenerin/epithelial sodium channel; sodium channel; subunit
assembly; Triton solubility
 |
INTRODUCTION |
THE RATE OF TRANSEPITHELIAL
Na+ transport is regulated by
apical expression of the epithelial
Na+ channel (ENaC) (2, 8, 10).
ENaC is composed of three homologous subunits,
,
, and
(3, 4,
13, 15, 16). Each subunit contains two hydrophobic transmembrane
domains, intracellular amino and carboxy termini, and a large,
cysteine-rich extracellular domain with numerous sites for N-linked
glycosylation. Generation of large amiloride-sensitive
Na+ currents in heterologous cells
requires coexpression of
-,
-, and
-ENaC. Expression of
-
and
-ENaC subunits alone produces no current, whereas expression of
-ENaC alone generates small Na+
currents. These findings suggest the formation of a heteromultimeric complex at the plasma membrane. Biochemical evidence of complex formation has come from the findings that ENaC subunits can be coimmunoprecipitated (1, 6, 18) and that they cosediment on sucrose
density gradients as large complexes (6, 7).
Our previous study showed that when
-,
-, and
-ENaC were
expressed alone, they could be detected in an intracellular compartment and on the plasma membrane (18). Cell surface ENaC resided in a
Triton-insoluble environment and contained only minimal glycosylation. Coexpression of all three ENaC subunits decreased the amount of
-ENaC found intracellularly without dramatically affecting the amount of ENaC at the cell surface. These observations led us to study
the kinetics of ENaC biosynthesis and how coexpression of the various
ENaC subunits might affect the overall biosynthetic process.
We also examined the trafficking of ENaC proteins containing a
disease-associated mutation. Specific mutations in the carboxy terminus
of
- and
-ENaC cause Liddle's syndrome, a genetic form of
hypertension resulting from increased
Na+ absorption in the distal
nephron (12, 24). Mutations associated with Liddle's syndrome increase
the number of ENaC channels at the apical membrane (25) and may also
increase the open probability of channels at the cell surface (9). As a
result, Na+ absorption increases
and hypertension ensues. We asked whether Liddle's mutations increase
levels of ENaC at the cell surface at least in part by altering biosynthesis.
 |
MATERIALS AND METHODS |
Reagents and cell culture.
A monoclonal antibody against the FLAG epitope was obtained from Kodak
(Rochester, NY). COS-7 cells were obtained from the American Type
Culture Collection (Rockville, MD) and maintained in culture with DMEM
containing 10% FCS. Cells were incubated in a humidified atmosphere
containing 5% CO2. For
transfection of COS-7 cells, 1 × 107 cells were electroporated with
30 µg of plasmid DNA. The cells were evenly divided, plated onto five
100-mm dishes, and cultured for 24-48 h in medium containing 10 µM amiloride to prevent cell swelling before study. Cells were
60-80% confluent at the time of experiments.
DNA constructs.
Construction of the cDNAs encoding
-,
-, and
-subunits of
human kidney ENaC (all in the pMT3 expression vector) are described elsewhere (1, 15-17). A form of
-ENaC associated with Liddle's syndrome,
R566X-ENaC, was made
by inserting a stop codon after amino acid 565. The FLAG epitope
(DYKDDDDK) was introduced into full-length human kidney ENaC with use
of the Muta-Gene phagemid in vitro mutagenesis kit (Bio-Rad, Hercules,
CA). For experiments using
R566X-ENaC, the FLAG epitope
was inserted into the extracellular domain of
-ENaC at amino acid
397. Epitope-tagged subunits were then cloned into pMT3 for expression.
Immunoprecipitation.
For immunoprecipitation, cells were washed three times in ice-cold PBS
with 1 mM MgCl2 and 0.1 mM
CaCl2 (PBS c/m) and lysed in
Tris-buffered saline (TBS), pH 7.4, with 1% Triton X-100 (Pierce, Rockford, IL) containing the following protease inhibitors: 0.4 mM
phenylmethylsulfonyl fluoride, 20 µg/ml aprotonin, 20 µg/ml leupeptin, and 10 µg/ml pepstatin A. Lysates were centrifuged at
16,000 g at 4°C, and the
supernatant was incubated with 5 µg of anti-FLAG antibody. The pellet
was solubilized in 100 µl of 2% SDS, 1% 2-mercaptoethanol, 50 mM
Tris (pH 7.4), and 1 mM EDTA by heating to 90°C for 5 min. After
solubilization, 1.0 ml of TBS with 1% Triton X-100 was added and ENaC
was immunoprecipitated with anti-FLAG antibody. Antigen-antibody
complexes were precipitated with immobilized protein A (Pierce), and
precipitates were washed three times in TBS with 1% Triton X-100 and
eluted with Laemmli sample buffer [4% SDS, 65 mM Tris (pH 6.8),
100 mM dithiothreitol, 20% glycerol, and 0.005% bromphenol
blue]. Proteins were separated on 7% polyacrylamide gels by
SDS-PAGE.
Pulse-chase experiments and steady-state metabolic labeling.
For metabolic pulse-chase experiments, transfected COS-7 cells were
starved for 1 h in methionine-free medium and pulsed for 30 min with
100 µCi/ml
[35S]methionine (6,000 Ci/mmol; Amersham, Chicago, IL). After the pulse period, radioactive
medium was removed and replaced with complete medium for the chase
period. Cells were then lysed, and ENaC was immunoprecipitated as
described above. Steady-state labeling of ENaC in COS-7 cells was done
by starving the cells in methionine-free medium for 1 h and adding 50 µCi/ml of
[35S]methionine (6,000 Ci/mmol) to the medium for 4 h. The radiolabeled immunoprecipitates
were analyzed by SDS-PAGE on 7% polyacrylamide gels, then the gels
were fixed, dried, and developed overnight by autoradiography or
phosphorimaging with use of a Storm 600 PhosphorImager (Molecular
Dynamics, Sunnyvale, CA). Bands corresponding to ENaC proteins
typically contained an integrated pixel intensity of
105-106
units after subtraction of background values with use of Image Quant
software (version 1.2, Molecular Dynamics).
 |
RESULTS AND DISCUSSION |
Differences in pulse-chase kinetics of
- and
-subunits expressed alone.
Our earlier studies found that ENaC subunits at the plasma membrane
were Triton insoluble and contained only minimal N-linked glycosylation
(18). To investigate the biosynthetic process that generated this
protein, we conducted pulse-chase studies in COS-7 cells and followed
ENaC through the biosynthetic pathway. As previously shown (18), the
Triton-soluble fraction contained glycosylated and unglycosylated forms
of ENaC (Fig. 1). A minimally glycosylated form appeared in the Triton-insoluble fraction. Figure 1A shows that, when expressed alone,
the Triton-soluble, unglycosylated form of the
-subunit disappeared
very early during the chase. The soluble glycosylated
-subunit
disappeared more slowly than the unglycosylated form. During the course
of the chase, the Triton-insoluble, minimally glycosylated form
accumulated slowly and became the predominant species at later time
points.

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Fig. 1.
Pulse-chase studies of epithelial
Na+ current (ENaC). COS-7 cells
expressing FLAG
(A),
FLAG
(B),
FLAG
(C), or
 FLAG
(D) were labeled for 30 min with 100 µCi/ml of
[35S]methionine,
radiolabeled methionine was removed, and cells were incubated in
complete medium for indicated pulse period. At each time point, ENaC
subunits were immunoprecipitated from Triton-soluble (S) and
Triton-insoluble (I) fractions, separated by SDS-PAGE, and analyzed by
autoradiography. Glycosylated (Gly) and minimally glycosylated (U)
forms of each subunit are indicated by arrows.
|
|
We observed a similar overall pattern when the
-subunit of ENaC was
expressed alone (Fig. 1B). However,
interestingly, the glycosylated form of
-ENaC found in the
Triton-soluble fraction actually accumulated during the 1st h of the
chase and then began to disappear (Fig. 2).
This is different from soluble glycosylated
-ENaC, which disappeared
as soon as the chase period began. The observed patterns suggested that
the glycosylated, Triton-soluble form of ENaC represents a biosynthetic
intermediate as ENaC traffics to the cell surface. Because
glycosylated, Triton-soluble ENaC possesses high-mannose-type
oligosaccharides (1, 18), it probably represents protein resident in
the endoplasmic reticulum (ER) (19). Although
- and
-ENaC are
capable of trafficking to the cell surface when expressed alone, these
data suggest that
-ENaC has a longer residence time in the ER.

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Fig. 2.
Quantitation of ENaC pulse-chase studies. Experiments as shown in Fig.
1 were quantitated using a Storm 600 PhosphorImager. Values (means ± SE for 6-9 experiments) are expressed as amount of
glycosylated, Triton-soluble ENaC immunoprecipitated at each chase time
point divided by amount of glycosylated, Triton-soluble ENaC
immunoprecipitated at time 0.
Repeated-measures analysis with multiple means comparison (Supernova,
Abacus Concepts, Berkeley, CA) after time
0 indicates that
FLAG is different from
FLAG
(P = 0.0001),
FLAG is different from
FLAG
(P = 0.0001), and
FLAG is different from
 FLAG
(P < 0.03). Separate comparison of
individual time points by means of a paired
t-test showed that
FLAG was different from
FLAG
(P < 0.05) after
time 0. Comparison of
FLAG with
 FLAG shows that, at 60 min, P = 0.1, and at 180 min,
P = 0.08.
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Coexpression of subunits changes ENaC biosynthesis.
The differences in the time course of Triton-soluble glycosylated
-
and
-ENaC expressed alone raised the question of how coexpression of
all three ENaC subunits affects biosynthesis. As shown in Fig. 1,
C and
D, the findings from pulse chase of glycosylated
- and
-ENaC coexpressed with the other two subunits were very similar to each other. More Triton-soluble, glycosylated
-ENaC was present at each time point than when it was expressed alone (Fig. 2). Conversely, there was less Triton-soluble, glycosylated
-ENaC when it was coexpressed with
- and
-ENaC. These effects were dependent on which subunits were expressed and not which subunit
was immunoprecipitated. Therefore, the protein studied represented that
present in heteromultimeric complexes. These data suggest that
- and
-ENaC each contribute unique kinetic information to biosynthesis of
the heteromultimeric
-
-
complex. One possibility is that
-
and
-ENaC have different inherent rates of trafficking through the
biosynthetic pathway. Alternatively,
-ENaC could form multimeric
complexes more efficiently than
-ENaC, with the formation of the
complete complex limiting trafficking out of the ER. This second
hypothesis is supported by sucrose gradient sedimentation data (6).
Effect of a 15°C block on solubility.
In earlier work we found that ENaC subunits became Triton insoluble at
some intracellular site before they reach the plasma membrane (18). To
further delineate the subcellular site at which ENaC becomes Triton
insoluble, we conducted pulse-chase experiments with incubation at
15°C during a prolonged (2-h) labeling period. Lowering the
temperature to 15°C blocks vesicular transport from the ER to the
cis-Golgi network (20). As shown in
Fig. 3, very little insoluble
-ENaC was
formed at 15°C. However, on warming of the cells to 37°C for
the chase, ENaC appeared in the Triton-insoluble fraction. Likewise,
maintaining cells at 15°C during the chase period continued to
inhibit formation of Triton-insoluble
-ENaC (Fig.
3B). ENaC therefore becomes Triton
insoluble at a site between the ER and the cell surface. These data
also support the hypothesis that ENaC in the ER is Triton soluble. At
this time, we do not know the basis for the insolubility; however, Triton insolubility has been observed with caveolar proteins, cytoskeletal proteins, and some high-molecular-weight oligomers (5, 14,
21, 22). The previous observation that some Triton-insoluble ENaC
(8-37%) may be intracellular (18) is consistent with insolubility occurring in a biosynthetic compartment such as the Golgi apparatus or
trans-Golgi network.

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Fig. 3.
Effect of reduced temperature on trafficking of -ENaC. COS-7 cells
were transfected with -ENaC 48 h before study. Cells in
methionine-free medium were labeled with 100 µCi/ml of
[35S]methionine for 2 h at 15°C. For chase, cells were warmed to 37°C
(n = 3;
A) or kept at 15°C for 2 h and
then warmed to 37°C (n = 4;
B). -ENaC was immunoprecipitated
from Triton-soluble and Triton-insoluble fractions at each time point.
A and
B: representative autoradiographs.
C: quantitation of Triton-insoluble
protein. Values are means ± SE.
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Effect of a Liddle's syndrome-associated mutation on ENaC
biosynthesis.
Mutations in
- and
-subunits associated with Liddle's syndrome
increase the number of ENaC channels in the plasma membrane (9, 12, 24,
25). Increased channel number at the plasma membrane could result from
increased biosynthesis, slowed degradation, and/or altered cellular
localization. Earlier work measuring amiloride-sensitive current in
brefeldin A-treated Xenopus oocytes
estimated the half-life of wild-type cell surface ENaC as ~3-4 h
(23, 27). Additional studies have shown that, in Liddle's syndrome,
reduced endocytosis contributes to the increase in cell surface protein
(23, 25). To study the biosynthesis of ENaC with Liddle's mutations,
we coexpressed wild-type
- and
-ENaC with
R566X-ENaC. The FLAG epitope
was placed in the extracellular domain of
-ENaC, rather than at an
intracellular site, to eliminate the possibility that the sequence
might interfere with interactions between ENaC and intracellular
proteins. By introducing the Liddle's mutation in
-ENaC and
assaying the biosynthesis of
-ENaC, we could be reassured that any
differences observed with
R566X
were due to biosynthesis of heteromultimeric complexes and not
-ENaC homomultimers.
Figure 4 shows that coexpression of
-
and
-ENaC with a
R566X-subunit increased the
amount of
-ENaC found in the Triton-insoluble fraction compared with
coexpression with a wild-type
-subunit. Earlier work showed that the
Triton-insoluble fraction of ENaC was present at the cell surface (18).
To determine whether expression of
R566X slowed degradation, we
did pulse-chase experiments comparing the metabolic half-lives of
-ENaC expressed with the
wt-subunit (Fig.
5A) and
the
R566X-subunit (Fig.
5B). Quantitation of four independent experiments is shown in Fig.
5C. The observed half-life for the
Triton-insoluble form of
-ENaC was ~10 h when it was expressed
with
wt- or
R566X-ENaC. This result
suggests that degradation of the Triton-insoluble
-
-
complex
is not affected by a Liddle's mutation.

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Fig. 4.
Steady-state measurement of wild-type and Liddle's ENaC. COS-7 cells
expressing FLAG,
wt, and ( F ) or
FLAG,
R566X, and ( F R566X )
were labeled with 50 µCi/ml of
[35S]methionine for 4 h. ENaC subunits were immunoprecipitated and analyzed by SDS-PAGE and
phosphorimaging. A: autoradiograph of
1 experiment. B: quantitation of 4 independent experiments. Values (means ± SE) are expressed as
amount of Triton-insoluble -ENaC divided by total -ENaC.
* P < 0.05 (paired
t-test).
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Fig. 5.
Pulse-chase experiments of wild-type and Liddle's ENaC. COS-7 cells
expressing FLAG,
wt, and (A) or
FLAG,
R566X, and (B) were pulsed with 100 µCi/ml of
[35S]methionine and
chased for up to 12 h in complete medium. -ENaC was
immunoprecipitated from Triton-soluble and Triton-insoluble fractions
and analyzed by SDS-PAGE and phosphorimaging.
C: quantitation of 4 independent
experiments. Values (means ± SE) are expressed as amount of
Triton-insoluble -ENaC at each time point divided by amount of
Triton-insoluble -ENaC at time 0.
There were no statistically significant differences between the 2 subunits.
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Even though the half-life of Triton-insoluble
-ENaC was similar when
coexpressed with
wt- or
R566X-ENaC, there was a visible difference in the relative amount of insoluble
-ENaC at the end of
the 30-min pulse in Fig. 5, A and
B. The difference was similar to that
observed at steady state (Fig. 4). These observations suggested the
possibility of differences between
wt- and
R566X-ENaC early in the
biosynthetic pathway. To examine early biosynthetic events, we
conducted pulse-chase experiments using a short (5-min) pulse and chase
period up to 1 h. We measured the fraction of total
-ENaC that was
insoluble at each time point. Figure 6
shows that more Triton-insoluble
-ENaC was present at early time
points when coexpressed with the
R566X-subunit than with a
wild-type
-subunit. These findings suggest that Liddle's mutations
in
-ENaC may increase channel number in the plasma membrane at least
in part by increasing trafficking along the biosynthetic pathway.

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Fig. 6.
Rapid pulse-chase experiments of wild-type and Liddle's ENaC. COS-7
cells expressing FLAG,
wt, and (A) or
FLAG,
R566X, and (B) were pulsed with 100 µCi/ml of
[35S]methionine for 5 min and chased for up to 60 min in complete medium. -ENaC was
immunoprecipitated from Triton-soluble and Triton-insoluble fractions
and analyzed by SDS-PAGE and phosphorimaging.
C: quantitation of 4 independent
experiments. Values (means ± SE) are expressed as amount of
Triton-insoluble -ENaC divided by total amount of -ENaC at each
time point. * P < 0.05 (paired
t-test).
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How might a Liddle's mutation in ENaC affect biosynthesis? Earlier
work has shown that a PPXY motif in the carboxy terminus of ENaC
subunits binds to Nedd4 (26). Liddle's mutations delete or mutate this
motif and subsequent Nedd4 binding. The interaction of Nedd4 with ENaC
may decrease the amount of protein at the cell surface, may inhibit the
function of ENaC channels, and may increase the rate of ENaC
degradation (11, 26, 27). It is possible that Nedd4 first interacts
with ENaC during biosynthesis, while the subunits are still in the ER
or in the Golgi complex. We speculate that the loss of an interaction
with Nedd4 or some other protein may be responsible for the increased
relative amount of Liddle's ENaC that leaves the ER and becomes Triton insoluble.
 |
ACKNOWLEDGEMENTS |
We thank Andrea Mugge, Dan Vermeer, Suzy Dietz, and Theresa Mayhew
for assistance and our laboratory colleagues for helpful discussions.
 |
FOOTNOTES |
This work was supported by the Howard Hughes Medical Institute. L. S. Prince was supported by the Cystic Fibrosis Foundation and National
Heart, Lung, and Blood Institute Institutional Research Fellowship
HL-07121. M. J. Welsh is an Investigator of the Howard Hughes Medical Institute.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. J. Welsh,
Howard Hughes Medical Institute, 500 EMRB, University of Iowa College
of Medicine, Iowa City, IA 52242 (E-mail:
mjwelsh{at}blue.weeg.uiowa.edu).
Received 2 December 1998; accepted in final form 15 March 1999.
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