1 Department of Medicine, University of Pittsburgh, Pittsburgh 15261; 2 Department of Medicine, Maine Medical Center, Portland, Maine 04102; 3 Department of Physiology, Emory University, Atlanta, Georgia 30322; 4 Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and 5 Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The renal epithelial cell line A6,
derived from Xenopus laevis, expresses epithelial
Na+ channels (ENaCs) and serves as a model system to study
hormonal regulation and turnover of ENaCs. Our previous studies suggest that the -subunit of Xenopus ENaC (
-xENaC)
is detectable as 150- and 180-kDa polypeptides, putative immature and
mature
-subunit heterodimers. The 150- and 180-kDa
-xENaC were present in distinct fractions after
sedimentation of A6 cell lysate through a sucrose density gradient. Two
anti-
-xENaC antibodies directed against distinct domains
demonstrated that only 180-kDa
-xENaC was expressed at
the apical cell surface. The half-life of cell surface-expressed
-xENaC was 24-30 h, suggesting that once ENaC
matures and is expressed at the plasma membrane, its turnover is
similar to that reported for mature cystic fibrosis transmembrane
conductance regulator. No significant changes in apical surface
expression of
-xENaC were observed after treatment of A6
cells with aldosterone for 24 h, despite a 5.3-fold increase in
short-circuit current. This lack of change in surface expression is
consistent with previous observations in A6 cells and suggests that
aldosterone regulates ENaC gating and increases channel open probability.
epithelial sodium transport; epithelial sodium channels; ion channel; renal epithelium
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RENAL TUBULAR
EPITHELIAL CELLS transport enormous quantities of Na+
in a regulated manner, using a variety of Na+-selective
transport proteins. The final site of renal Na+
reabsorption is the collecting tubule, and the rate of Na+
absorption across the collecting tubule is modified by hormones, such
as aldosterone, that have key roles in the regulation of the
extracellular fluid volume and blood pressure (1, 13). Epithelial Na+ channels (ENaCs) mediate the transport of
Na+ across the apical plasma membrane of principal cells in
renal collecting tubules (1, 13). These channels,
originally cloned from rat distal colon, consist of at least three
structurally related subunits, ,
, and
.
The mineralocorticoid hormone aldosterone regulates transepithelial
Na+ transport in the collecting tubule by activating ENaC
(1, 13, 41). The mechanisms by which aldosterone regulates
ENaC likely involve both translational as well as posttranslational events (1, 13, 41). In addition, the mechanism of ENaC activation appears to be dependent on the length of time that renal (or
urinary) epithelia are exposed to aldosterone. Both early (~1.5 h)
and late (>6 h) effects have been described and reflect changes in the
biophysical properties of channels expressed at the cell surface or
increases in ENaC protein expression at the apical cell surface
(1, 12-13, 16). Patch-clamp analyses of single
Na+ channels in the A6 amphibian renal epithelial cell line
suggest that aldosterone activates channels by altering gating kinetics and increasing channel open probability (Po)
(16). Noise analysis is an important and complementary
method to examine ENaC Po and surface expression
in A6 cells in response to aldosterone. Previous studies using this
experimental approach suggested that aldosterone increases the density
of channels expressed at the plasma membrane, rather than
Po (15). Single-channel recordings
from rat cortical collecting ducts also indicate that aldosterone
increases the number of ENaC channels at the cell surface
(27). Aldosterone increases the message or protein
abundance of either the -subunit alone (4, 23) or both
the
- and
-subunits in renal epithelia (9, 24).
Subunit-specific ENaC antibodies have been used to localize
Na+ channels in the kidneys of animals maintained on low-
or high-NaCl diets (maneuvers that alter circulating aldosterone
levels). An increase in ENaC
-subunit protein expression, as well as
a redistribution of
- and
-ENaC from the cytoplasm to the apical
region of principal cells in the connecting tubule and cortical
collecting duct of rat and mouse kidney (21, 23), in
response to long-term dietary NaCl deprivation, has been reported by
several groups. However, an earlier study suggested that long-term
dietary NaCl deprivation did not alter the expression or localization
of
- or
-ENaC in rat kidney (31), and a recent study
demonstrated that activation of Na+ channels in rat
cortical collecting duct in response to short-term (15 h)
Na+ deprivation was not associated with a change in
-subunit protein expression (11). Taken together, these
data suggest that aldosterone increases apical Na+
transport in the collecting duct, at least in part, by increasing ENaC
cell surface expression in principal cells of the cortical collecting duct.
We previously raised an antibody directed against a 20-mer peptide
corresponding to residues within the extracellular domain of
Xenopus laevis ENaC (- xENaC) that
specifically recognizes in vitro translated
-xENaC. This
antibody specifically recognized three polypeptides in A6 cell lysate
with apparent molecular masses of ~70, ~150, and ~180 kDa
(46). In the present study, we used this
anti-
-xENaC antibody to examine the apical cell surface of
-xENaC in A6 epithelia. This antibody, as well as an
anti-
-xENaC antibody raised against a 35-mer peptide
corresponding to residues within the intracellular COOH-terminal domain
of
-xENaC, specifically recognized only 180-kDa
-xENaC at the apical cell surface. A pulse
(biotinylation)-chase protocol was used to examine the half-life of
Na+ channel
-subunits expressed at the cell surface. The
t1/2 was 24-30 h, suggesting that mature
(i.e., the ~180-kDa polypeptide)
-xENaCs have a
relatively slow turnover in A6 cells, in contrast to immature ENaC
subunits that turn over within hours (24, 45). No
significant change in surface expression of
-xENaC was
observed after treatment of cells with aldosterone for 24 h to
activate Na+ transport, despite a 5.3-fold increase in
short-circuit current (Isc). These data suggest
that increases in A6 cell Na+ transport in association with
long-term (24 h) aldosterone treatment reflect changes in ENaC
Po and are in agreement with the recent findings
of Weisz et al. (45).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture.
A6 renal epithelial cells, derived from the distal nephron of X. laevis, were cultured as previously described (46).
A6 cells were subcultured onto 4.7-cm2 Vitrogen-coated
Costar polycarbonate filters (Corning, Acton, MA) for analysis of the
effects of aldosterone on -xENaC expression and for
analysis of
-xENaC turnover. A6 cells were subcultured onto Millipore HAWP filter rings (Bedford, MA) for analysis by sucrose
density gradient centrifugation. Monolayers were used 10-14 days
after plating. To examine the effect of aldosterone on ENaC expression,
monolayers were transferred to serum-free media for 3 days. The
monolayers were then subsequently placed in either serum-free media
supplemented with 1 µM aldosterone (experimental monolayers) or
serum-free media alone (control monolayers) for 24 h before
biochemical or electrophysiological studies. This concentration of
aldosterone is sufficient to lead to saturating the binding of both the
mineralocorticoid and glucocorticoid receptors. However, previous
studies suggest that the response of A6 cells to aldosterone is
mediated by glucocorticoid receptors, as these cells may not express
mineralocorticoid receptors (33).
Sucrose density gradient centrifugation.
A6 cell monolayers grown on HAWP filters were extracted in 0.5% Triton
X-100 extraction buffer (26). The 0.5% Triton
X-100-soluble fraction was concentrated to a volume of 1 ml using
Centricon 10 concentrators (Amicon, Beverly, MA). Linear sucrose
gradients (5-20%) were overlain with 200 µl of the concentrated
0.5% Triton X-100-soluble fraction and centrifuged in a SW 50.1 rotor
(Beckman Instruments, Fullerton, CA) at 40,000 rpm for 22 h at
4°C, as previously described (46). Gradients were
fractionated from bottom to top into 20 fractions (250 µl/fraction).
Gradient fractions (1-3, 4-8, 12-13) were
pooled before analysis. The distribution of -xENaC within
the pooled fractions was determined by immunoblot analysis.
Cell surface biotinylation.
Cell surface biotinylation was used to examine both the turnover of
-xENaC and aldosterone-mediated changes in
-xENaC cell surface expression. Briefly, A6 cell
monolayers grown on Costar filters were washed extensively with
amphibian Ringer solution. Sulfonated
N-hydroxysuccinimide-biotin (sulfo-NHS-biotin; Pierce, Rockford, IL), 0.5 mg/ml, in a borate-buffered solution (85 mM NaCl, 4 mM KCl, 15 mM Na2B4O7, pH 9.0) was
added to the apical compartment and incubated with gentle shaking at
4°C (7, 8). The basolateral compartment received an
equivalent volume of tissue culture medium with 5% FCS to bind the
sulfo-NHS-biotin that leaked across the epithelium. Monolayers were
then extensively washed with amphibian Ringer solution containing
protease inhibitors (1 µM antipain, 1 µM leupeptin, 1 µM
pepstatin A, and 100 µM phenylmethylsulfonyl fluoride). Filters were
excised from their supports, and cells were solubilized in a lysis
buffer containing 0.4% sodium deoxycholate, 1% Nonidet P-40, 50 mM
EGTA, and 10 mM Tris · HCl (pH 7.4) supplemented with protease
inhibitors. Lysates (500 µl/well) were precleared for 30 min by
inverting on a rotator at 4°C with 50 µl of a 50% suspension of
protein A-agarose beads. Lysates were subsequently centrifuged for 2 min, transferred to new tubes, and incubated with a 1:100 dilution of
the anti-
-xENaC antibody for 12-16 h at 4°C.
Protein A-agarose beads (50 µl) were then added for an additional 30 min. Immunoprecipitates were washed extensively with high-stringency
wash buffer and were followed by low-stringency wash buffer
(26). The beads were resuspended in 50 µl of 2× sample
buffer containing 100 mM dithiothreitol, heated at 95°C for 2 min,
and centrifuged for 2 min to pellet the beads. Supernatants were
analyzed by 7.5% SDS-PAGE and transferred to polyvinylidene difluoride
(PVDF) membranes. Alternatively, after cell surface biotinylation,
streptavidin-agarose beads were used to precipitate biotinylated
proteins, as previously described. The precipitated proteins were
subjected to 7.5% SDS-PAGE and transferred to PVDF membranes.
Antibodies.
The rabbit polyclonal antibody directed against the -subunit of
xENaC was generated by Lofstrand Laboratories (Bethesda, MD)
by using a 20-mer synthetic peptide corresponding to amino acids
107-125 (CQNDLQELDKETQRTLYEL) in the extracellular loop of
-xENaC, as previously described (46). The
immunizing peptide was coupled to Sulfolink agarose (Pierce), and
anti-
-xENaC antibodies were affinity purified according
to the manufacturer's instructions. A chicken polyclonal antibody
raised against the COOH-terminal 33 residues of
-xENaC
and purified using a gamma yolk purification kit (Amersham Pharmacia
Biotech) was provided by Dr. J. Johnson (Univ. of Pittsburgh). The
characteristics of these antibodies have been previously described
(32).
Immunoblotting.
Sucrose gradient fractions (30 µg/lane) or biotinylated proteins were
separated by 7.5% SDS-PAGE and transferred to Immobilon PVDF paper
(Millipore) as described (38). Blots of sucrose gradient factions were probed with rabbit anti--xENaC (15 µg/ml)
antibody, followed by the appropriate secondary antibody coupled to
biotin. Bound antibodies were detected by using a Western-Lite Plus
chemiluminescent detection system (Tropix, Bedford, MA). Controls
consisted of preincubation of anti-
-xENaC antibodies with
excess-free peptide (100 µg/ml). Blots of biotinylated proteins were
either probed with the rabbit anti-
-xENaC (15 µg/ml)
antibody followed by the appropriate horseradish peroxidase-conjugated
secondary antibody or with streptavidin-alkaline phosphatase conjugate
followed by chemiluminescent detection (Tropix). A chicken
anti-
-xENaC antibody directed against the COOH terminus
was used to probe blots as previously described (32). The
blots were scanned and analyzed using the IPLab Gel densitometry
program (Scanalytics, Vienna, VA). The half-life of ENaC recovered from
the cell surface was calculated using the equation
T0.5 =
0.693 t/ln
(A/A0), where t = time, A = recovery
of ENaC at time t, and A0 = recovery of ENaC at time 0. Recovery of
-xENaC at 24 and
48 h was normalized to
-xENaC recovered from the
apical membrane at time 0, which is defined as 100%. The
linear range of the densitometer was determined by scanning film
exposed to slot blot of increasing amounts of biotinylated A6 cell
extract probed with streptavidin-alkaline phosphatase. For experiments
using anti-
-xENaC antibodies to probe blots of
streptavidin-precipitated surface proteins, the scanned densities of
the bands were within the linear range of the densitometer.
Measurement of Isc and transepithelial potential difference. A6 monolayers grown on Costar filter inserts were placed in a modified Ussing chamber, and the transepithelial potential and Isc were measured using a DVC-1000 voltage clamp (World Precision Instruments, Sarasota, FL), as previously described (18).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We previously raised and characterized an antibody is directed
against residues 107-125 within the extracellular
domain of -xENaC. This antibody specifically
recognized in vitro translated
-xENaC, and it did not
recognize in vitro translated
- or
-xENaC (46). Three distinct polypeptides (relative molecular mass
of ~70, ~150, and ~180 kDa) were specifically recognized when an immunoblot of A6 cell Triton X-100 extract was probed with the anti-
-xENaC antibody (46). The 70-kDa
polypeptide is consistent with the predicted size of nonglycoslyated
-xENaC (30). We have previously demonstrated
that the 180-kDa polypeptide was recognized by antibodies directed
against two distinct domains within
-xENaC: the
antibody directed against the extracellular domain of
-xENaC and an antibody directed against the COOH-terminal intracellular domain of
-xENaC (46).
In addition,
-xENaC was shown to
coimmunoprecipitate with the 180-kDa polypeptide (46).
We previously reported that the distributions of the 70-, 150-, and
180-kDa polypeptides recognized by the anti--xENaC
antibody peaked in distinct fractions after centrifugation of Triton
X-100 extracts of A6 cell monolayers on a 5-20% linear sucrose
density gradient (46). In the present study, we determined
which of these polypeptides is expressed at the cell surface of A6
cells. To first confirm that 180- and 150-kDa
-xENaC are
present within distinct fractions after sedimentation through the
sucrose gradient, peak fractions containing 180- (fractions
4-8) and 150-kDa
-xENaC (fractions 12 and
13), as well as fractions 1-3 (control), were subjected to SDS-PAGE and transferred to PVDF paper. The blots were
probed with the anti-
-xENaC antibody directed against the ectodomain of
-xENaC in the presence or absence of a
peptide immunogen. As shown in Fig. 1,
180- and 150-kDa
-xENaC were in distinct fractions and
were specifically recognized by the anti-
-xENaC antibody.
Polypeptides with apparent molecular masses of 120 and 66 kDa were
recognized in a nonspecific manner. A polypeptide with an apparent
molecular mass of 50 kDa, present in fractions 1-3, was
specifically recognized by the anti-
-xENaC antibody and
may represent a degradation product. Several groups have suggested that
ENaC has a subunit stoichiometry of
2,
1,
1, (10,
19), although conflicting results have been reported
(35). We previously speculated that 180-kDa
-xENaC represents a dimer of mature
-subunits, whereas
150-kDa
-xENaC represents a dimer of immature
-subunit (46).
|
To determine whether 180-kDa -xENaC is expressed at the
cell surface, A6 cell monolayers grown on Millipore HAWP filters were
labeled on their apical surfaces with biotin-LC-hydrazide and extracted
with Triton X-100. The extracts were subjected to 5-20% linear
sucrose density centrifugation. Gradient fractions 4-8
were pooled, and biotinylated proteins were captured by passing the
pooled fractions over an avidin column. Biotinylated proteins were
subsequently eluted, subjected to SDS-PAGE, and transferred to PVDF
paper. The resulting blots were probed with the
anti-
-xENaC antibody in the presence or absence of excess
free peptide immunogen. As shown in Fig.
2A, 180-kDa
-xENaC was specifically recognized by the
anti-
-xENaC antibody. To further confirm that 180-kDa
-xENaC is expressed at the cell surface, A6 cell
monolayers were grown on Costar supports, and apical plasma membrane
proteins were labeled with sulfo-NHS-biotin. Cells were then
solubilized and subjected to immunoprecipitation with the
anti-
-xENaC antibody in the absence or presence of free
peptide immunogen. The immunoprecipitated proteins were separated by
SDS-PAGE, transferred to PVDF paper and then probed with alkaline
phosphatase-conjugated streptavidin. As shown in Fig. 2B,
biotinylated 180-kDa
-xENaC was specifically immunoprecipitated by the anti-
-xENaC antibody, but
150-kDa
-xENaC was not detected.
|
To provide additional evidence that 180-kDa
-xENaC is expressed at the cell surface,
we used an antibody raised against the COOH-terminal 33 residues of
-xENaC (32). A6 apical cell surface proteins
were biotin labeled and precipitated with streptavidin-agarose. The
precipitated proteins were subjected to SDS-PAGE and transferred to
PVDF paper. The blot was probed with either the antibody directed against the COOH terminus or the antibody against an extracellular domain of
-xENaC. Both antibodies recognized
180-kDa
-xENaC (Fig. 2C). Furthermore, a
blot probed with the anti-
-xENaC antibody directed
against its COOH-terminal domain in the presence or absence of peptide
immunogen confirmed that this antibody specifically recognized 180-kDa
-xENaC (Fig. 2D). Taken together, these data provide convincing evidence that 180-kDa
-xENaC is
expressed at the apical plasma membrane of A6 epithelial cells.
Previous studies have suggested that ENaC is rapidly degraded after its
biosynthesis, and that its half-life may be on the order of hours
(24, 40, 45). However, these studies that examined ENaC
biosynthesis and maturation using pulse-chase metabolic labeling
protocols in conjunction with immunoprecipitation identified immature
subunits, as it was apparently difficult to detect mature subunits
(24, 40). We used cell surface biotinylation as an alternative pulse-chase protocol to examine the turnover of cell surface-expressed -xENaC. A6 epithelia were grown on
permeable supports, and apical membrane proteins were labeled with
NHS-biotin. Cells were then solubilized or, alternatively, they were
placed back in an incubator for 24 or 48 h before solubilization.
-xENaC present at the apical surface at the time of
biotinylation was detected by immunoprecipitation with the anti-
ENaC
antibody directed against the extracellular domain followed by probing
protein blots with alkaline phosphatase-conjugated streptavidin (Fig.
3A). The recovery of
biotinylated
-ENaC decreased as cells remained in culture for
24-48 h after surface labeling (Fig.
4A). When compared with the
recovery of cell surface (i.e., biotinylated)
-xENaC immediately after surface biotinylation, recovery of biotinylated
-xENaC after 24 h was 61 ± 15, and was 28 ± 8% after 48 h (Fig. 3B). The half-life of
biotinylated
-xENaC (i.e., expressed at the apical
surface at t = 0) was 30 h.
|
|
To confirm these observations, A6 cells were detergent solubilized 0 or
24 h after apical surface biotinylation. A6 apical cell surface
proteins were precipitated with streptavidin-agarose, subjected to
SDS-PAGE and transferred to PVDF paper. The blots were probed with the
anti--xENaC antibody directed against the extracellular
domain. The recovery of biotinylated
-xENaC after a 24-h
chase period was 52 ± 15% of the amount of biotinylated
-xENaC recovered immediately after surface biotinylation
(Fig. 3C), consistent with a half-life of biotinylated
-xENaC of 24 to 30 h.
Several recent studies suggest that expression of -ENaC mRNA or
protein is increased in response to aldosterone (4, 9, 23-24). An increase in immunoreactive
-ENaC localized to
the region of the apical plasma membrane was observed in rat or mouse
connecting tubules or cortical collecting tubules in response to
dietary NaCl restriction (21, 23). However, conflicting
data regarding changes in ENaC surface expression in A6 cells in
response to aldosterone have been reported (15-16,
45). We examined whether apical plasma membrane expression of
-xENaC was altered by long-term (24 h) exposure of A6
cells to aldosterone. Cells were grown on permeable supports in
serum-free media for >72 h to deplete cells of steroid hormones. Cells
were then placed in serum-free media supplemented with 1 µM
aldosterone, or maintained in serum-free medium in the absence of
aldosterone. A6 monolayers exposed to aldosterone for 24 h clearly
showed activation of transepithelial Na+ transport,
compared with monolayers that were not exposed to aldosterone.
Aldosterone treatment resulted in a 5.3-fold increase in
Isc (P < 0.0001 aldosterone vs.
control) and a 6.6-fold increase in transepithelial potential
difference (P < 0.0001 aldosterone vs. control). To
examine whether aldosterone treatment led to changes in apical plasma
membrane expression of the
-subunit, A6 cell monolayers were
apically labeled with NHS-biotin, lysed, and
-xENaC was
immunoprecipitated from cell lysates using the antibody directed
against the extracellular domain of
-xENaC. The surface
pool of
-xENaC was detected by probing blots of the immunoprecipitates with alkaline phosphatase-conjugated streptavidin. Cell surface expression of
-xENaC was modestly (1.4-fold)
but not significantly increased when cells were exposed to aldosterone, compared with nonaldosterone-treated controls (n = 22, P > 0.05, aldosterone vs. control; Table
1, Fig. 4A). To confirm these observations, A6 apical cell surface proteins were precipitated with
streptavidin-agarose after surface biotinylation. The precipitated proteins were subjected to SDS-PAGE and transferred to PVDF paper, and
the blots were probed with the anti-
-xENaC antibody
directed against the extracellular domain. Cell surface expression of
-xENaC was unchanged in response to 24-h aldosterone
exposure (n = 5, P > 0.05, aldosterone
vs. control; Table 1, Fig. 4B).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous work examining ENaC biosynthesis in A6 cells using a
metabolic labeling, pulse-chase protocol suggested that
-xENaC has an apparent molecular mass of 70 kDa and has a
relatively short half-life (~1 to 2 h) that is not altered by
aldosterone (24). Although the 70-kDa polypeptide was
shown to coimmunoprecipitate with
- and
-xENaC, it was
unclear whether this polypeptide represented mature
-xENaC (24). Weisz and co-workers
(45) have recently confirmed the rapid turnover of whole
cell
-,
-, and
-xENaC in A6 cells. Valentijn and
co-workers (40) also examined ENaC biosynthesis in
Xenopus oocytes. They demonstrated that ENaC was synthesized
in the endoplasmic reticulum and that the vast majority of newly
synthesized ENaC remained in pre-Golgi compartments and were degraded
via proteasomes. Only a minor fraction of newly synthesized ENaC had
complex oligosaccharides and were expressed at the cell surface. Kosari
and co-workers (19) and Firsov and co-workers
(10) have suggested that ENaC has a subunit stoichiometry of
2,
1, and
1. We speculate that 180-kDa
-xENaC
specifically recognized by the two distinct
anti-
-xENaC antibodies used in the present study
represent a dimer of
-xENaC subunits that is expressed at
the apical surface of A6 cells. Using our anti-
-xENaC antibody directed against an extracellular epitope, Weisz and co-workers (45) also observed the 180-kDa
-xENaC expressed at the apical plasma membrane. The
180-kDa
-xENaC may represent a terminally glycosylated
dimer of
-subunits, and the 150-kDa
-xENaC may
represent a dimer of core or nonglycosylated
-subunits. However,
Weisz et al. (45) observed that the 180-kDa polypeptide was resistant to treatment with either N-glycanase or
endoglycosidase H, and Prince and Welsh (29) suggested
that ENaC expressed at the cell surface of Chinese hamster ovary cells
is deglycosylated. Our previous observations (46) suggest
that 180-kDa
-xENaC is not an
and/or
heterodimer,
and Weisz et al. (45) suggest that it is not an
-
and/or a
-heterodimer. We have not been successful in dissociating
these putative
-subunit dimers, despite treatment with
dithiothreitol (100 mM), urea (8 M), and SDS (3%). It was surprising
that the putative
-xENaC dimers resisted dissociation into monomers, particularly in light of published observations (4, 23) indicating that mouse, rat, and human
-ENaC migrate with an apparent Mr of ~90 to
100 kDa. However, there are examples in the literature of oligomeric
integral membrane proteins, including ion channels, that are resistant
to disruption by conventional methods used to dissociate protein
oligomers. For example, the K+ channel of
Streptomyces lividans is a stable tetramer in the presence
of SDS and
-mercaptoethanol (14). Glycophorin A, the major integral membrane protein of the erythrocyte, is a dimer that is
resistant to disruption by heat and SDS (20). Bacterial outer membrane porins are stable trimers in the presence of SDS and 8 M
urea but dissociate into monomers when heated (39). The
stability of these multimers may be conferred through secondary structural interactions (i.e.,
-helices or
-sheets). Secondary structural interactions between
-xENaC monomers might
have a role in conferring stability to the 180-kDa
-xENaC.
Given the difficulties in identifying the pool of cell
surface-expressed ENaC in epithelia, limited information exists
regarding the turnover of mature ENaC. A half-life of 3.6 h has
been reported for functional Na+ channels expressed at the
plasma membrane of Xenopus oocytes (40). The
functional half-life of ENaC increases to 30 h when channels
express a Liddle's mutation (40). However, these studies did not address the turnover of the biochemical pool of channels, which
may differ from the measured half-life of functional channels if the
channels recycle between endosomal and plasma membrane compartments.
Data from a recent study examining Fischer rat thyroid cells
transiently transfected with -,
-, and
-ENaC have suggested that ENaC is inserted and retrieved from the plasma membrane in a
regulated manner (34) and support the notion that ENaC
recycles between endosomal and plasma membrane compartments. Our data
suggest that the half-life of mature
-xENaC is ~24 to
30 h. Weisz and co-workers (45) examined the turnover
of cell surface-expressed
-xENaC in A6 cells. Their data
indicated that the recovery of biotinylated (i.e., cell
surface-expressed)
-xENaC was unchanged over a 24-h
period, suggesting a half-life of considerably longer than 24 h.
Taken together, these data are consistent with the notion that once
ENaC is expressed at the plasma membrane, it has a relatively slow
turnover rate. As mentioned above, this clearly differs from the
reported half-life of ENaC subunits in A6 epithelia determined by
metabolic labeling and pulse-chase analyses, which was on the order of
1-2 h (24, 45). It also differs from the biochemical
half-life of 9.5 to 11 h observed in Xenopus oocytes by
Valentjin and co-workers (40). We agree with their
findings, which suggested that studies using metabolic labeling and
pulse-chase protocols to examine ENaC turnover in Xenopus
oocytes are limited to analyses of immature subunits and that the
assembly and processing of ENaC to a mature form are inefficient.
However, once ENaC matures and reaches the plasma membrane, our data
suggest that the turnover is slow and is similar to that reported for
"mature" cystic fibrosis transmembrane conductance regulator (CFTR)
(22, 44).
Several groups have examined mechanisms of regulation of ENaC by aldosterone. Aldosterone dramatically increases Na+ transport in the mammalian collecting duct and distal colon (13, 41) and increases transepithelial Na+ transport across epithelial cell lines derived from amphibian and mammalian distal nephron (4, 9, 16, 18). Previous work in cultured renal cell lines and in rat kidney and colon suggests that aldosterone regulates Na+ channels by two distinct mechanisms: 1) increases in Na+ channel Po (16) and 2) increases in ENaC protein expression at the plasma membrane (4, 9, 23, 31). Aldosterone also enhances transcription and translation of Na+-K+-ATPase, and increases in basolateral Na+-K+-ATPase expression also have an important role in the activation of transepithelial Na+ transport by aldosterone (42-43). A serum and growth factor-regulated kinase (sgk) was recently identified as an aldosterone-regulated gene product that activates ENaC by increasing the number of channels expressed at the cell surface (2, 6, 25). Consistent with this notion, rats maintained on a low-NaCl diet to induce extracellular volume depletion and stimulate aldosterone secretion were found to have an increase in the expression of Na+ channels in the apical region of the collecting duct principal cells (27). Mechanisms by which aldosterone regulates Na+ channel Po are poorly understood, although several regulatory pathways that likely participate in this process have been identified, including methylation, activation of the ras signaling pathway, and activation of phosphoinositide 3-kinase, (3, 5, 36-37, 41).
Patch-clamp analyses of Na+ channels in A6 epithelia
suggested that both the early and late phases of ENaC activation by
aldosterone are associated with an increase in channel
Po (16). Consistent with the notion
that aldosterone activates ENaC in A6 epithelia by increasing
Po, we previously observed that long-term (24 h) treatment of A6 cells with aldosterone did not result in large changes
in the cell surface pool of putative Na+ channels, when
compared with nonaldosterone-treated cells or with cells that were
treated with the aldosterone antagonist spironolactone (18). In this previous study, we used an anti-idiotypic
antibody that recognized a putative amiloride-binding domain within the -subunit of ENaC to examine whether aldosterone treatment altered plasma membrane expression of ENaC (17). In the present
study, no significant changes in the plasma membrane expression of
-xENaC were observed in response to 24-h aldosterone
exposure (Table 1 and Fig. 4), suggesting that the 5.3-fold increase in
Isc observed with aldosterone primarily reflects
an increase in ENaC Po (16). Also
consistent with this notion, levels of expression of
-,
-, and
-xENaC are, at best, only modestly altered by aldosterone in A6 epithelia (Middleton P, Al-Khalili O, Yue G, Zuckerman J, Kleyman
TR, and Eaton DC, unpublished observations). A6 epithelia express the
kinase sgk in a dexamethasone-regulated manner; however, a
time course of sgk protein expression in response to
dexamethasone suggests that sgk expression increases early
(0.5 to 6 h) but falls after 24 h (6). Given
these observations, we were not surprised that the plasma membrane pool
of
-xENaC was essentially unchanged following a 24-h
treatment with aldosterone. Weisz and co-workers (45) also
observed that cell surface expression of
-xENaC was
unchanged after a 3- or 18-h exposure to aldosterone. Cell surface
expression of
-xENaC was unchanged as well in response to
aldosterone; however, surface expression of
-xENaC
increased by ~60% after an 18-h exposure to aldosterone, raising the
possibility that aldosterone might alter ENaC subunit stoichiometry.
ENaCs are heterotetrameric channels (10, 19). Assuming
that
-,
-, and
-channels as well as
- or
-channels
have an
-subunit stoichiometry of two, our results and the
observations reported by Weisz et al. (45) suggest that
exposure of A6 cells to aldosterone for up to 24 h does not alter
the number of channels expressed at the plasma membrane.
In summary, mature -xENaC exists at the apical surface of
A6 cells as a 180-kDa polypeptide. The half-life of mature
-xENaC was found to be 24 to 30 h, suggesting that
once
-xENaC is expressed at the plasma membrane, its
turnover rate is similar to that reported for mature CFTR. No
significant change in apical surface expression of
-xENaC
was observed after a 24-h exposure to aldosterone, despite a 5.3-fold
increase in Isc. These data support the notion that the increase in Isc in A6 epithelial cells
in response to aldosterone is mediated via regulatory pathways that
alter
-xENaC Po (3,
37). The increases in apical membrane expression of ENaC in rat
and mouse cortical collecting ducts observed in response to long-term
dietary Na+ deprivation, and the increases in surface
expression of ENaC in response to the kinase sgk, suggest
that aldosterone-mediated regulation of ENaC involves regulation of
both the surface pool of channels as well as channel
Po. The predominant effect likely depends on the
cell type as well as the duration of exposure to aldosterone.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-51391 (to T. R. Kleyman), DK-50268 (to D. C. Eaton), and DK-56596 (to P. R. Smith). J. B. Zuckerman and B. Hu were supported by postdoctoral fellowships from the Cystic Fibrosis Foundation.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: T. R. Kleyman, Renal-Electrolyte Div., A919 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261 (E-mail: kleyman{at}pitt.edu).
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.
Received 22 January 2001; accepted in final form 23 March 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alvarez de la Rosa, D,
Canessa CM,
Fyfe GK,
and
Zhang P.
Structure and regulation of amiloride-sensitive sodium channels.
Annu Rev Physiol
62:
573-594,
2000[ISI][Medline].
2.
Alvarez de la Rosa, D,
Zhang P,
Naray-Fejes-Toth A,
Fejes-Toth G,
and
Canessa CM.
The serum and glucocorticoid kinase sgk increases the abundance of epithelial sodium channels in the plasma membrane of Xenopus oocytes.
J Biol Chem
274:
37834-37839,
1999
3.
Becchetti, A,
Kemendy AE,
Stockand JD,
Sariban-Sohraby S,
and
Eaton DC.
Methylation increases the open probability of the epithelial sodium channel in A6 epithelia.
J Biol Chem
275:
16550-16559,
2000
4.
Bens, M,
Vallet V,
Cluzeaud F,
Pascual-Letallec L,
Kahn A,
Rafestin-Oblin ME,
Rossier BC,
and
Vandewalle A.
Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line.
J Am Soc Nephrol
10:
923-934,
1999
5.
Blazer-Yost, BL,
Punescu TG,
Helman SI,
Lee KD,
and
Vlahos CJ.
Phosphoinositide 3-kinase is required for aldosterone-regulated sodium reabsorption.
Am J Physiol Cell Physiol
277:
C531-C536,
1999
6.
Chen, SY,
Bhargava A,
Mastroberardino L,
Meijer OC,
Wang J,
Buse P,
Firestone GL,
Verrey F,
and
Pearce D.
Epithelial sodium channel regulated by aldosterone-induced protein sgk.
Proc Natl Acad Sci USA
96:
2514-2519,
1999
7.
Coupaye-Gerard, B,
Bookstein C,
Duncan P,
Chen XY,
Smith PR,
Musch M,
Ernst SA,
Chang EB,
and
Kleyman TR.
Biosynthesis and cell surface delivery of the NHE1 isoform of Na+/H+ exchanger.
Am J Physiol Cell Physiol
271:
C1639-C1645,
1996
8.
Coupaye-Gerard, B,
Zuckerman JB,
Duncan P,
Bortnik A,
Avery DI,
Ernst SA,
and
Kleyman TR.
Delivery of newly synthesized Na+-K+-ATPase to the plasma membrane of A6 epithelia.
Am J Physiol Cell Physiol
272:
C1781-C1789,
1997
9.
Dijkink, L,
Hartog A,
Deen PM,
van Os CH,
and
Bindels RJ.
Time-dependent regulation by aldosterone of the amiloride-sensitive Na+ channel in rabbit kidney.
Pflügers Arch
438:
354-360,
1999[ISI][Medline].
10.
Firsov, D,
Gautschi I,
Merillat AM,
Rossier BC,
and
Schild L.
The heterotetrameric architecture of the epithelial sodium channel (ENaC).
EMBO J
17:
344-352,
1998
11.
Frindt, G,
Masilamani S,
Knepper MA,
and
Palmer LG.
Activation of epithelial Na channels during short-term Na deprivation.
Am J Physiol Renal Physiol
280:
F112-F118,
2001
12.
Garty, H.
Regulation of the epithelial Na+ channel by aldosterone: open questions and emerging answers.
Kidney Int
57:
1270-1276,
2000[ISI][Medline].
13.
Garty, H,
and
Palmer LG.
Epithelial sodium channels: function, structure, and regulation.
Physiol Rev
77:
359-396,
1997
14.
Heginbotham, L,
Odessey E,
and
Miller C.
Tetrameric stoichiometry of a prokaryotic K+ channel.
Biochemistry
36:
10335-10342,
1997[ISI][Medline].
15.
Helman, SI,
Liu X,
Baldwin K,
Blazer-Yost BL,
and
Els WJ.
Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia.
Am J Physiol Cell Physiol
274:
C947-C957,
1998
16.
Kemendy, AE,
Kleyman TR,
and
Eaton DC.
Aldosterone alters the open probability of amiloride-blockable sodium channels in A6 epithelia.
Am J Physiol Cell Physiol
263:
C825-C837,
1992
17.
Kieber-Emmons, T,
Lin C,
Foster MH,
and
Kleyman TR.
Antiidiotypic antibody recognizes an amiloride binding domain within the subunit of the epithelial Na+ channel.
J Biol Chem
274:
9648-9655,
1999
18.
Kleyman, TR,
Coupaye-Gerard B,
and
Ernst SA.
Aldosterone does not alter apical cell-surface expression of epithelial Na+ channels in the amphibian cell line A6.
J Biol Chem
267:
9622-9628,
1992
19.
Kosari, F,
Sheng S,
Li J,
Mak DO,
Foskett JK,
and
Kleyman TR.
Subunit stoichiometry of the epithelial sodium channel.
J Biol Chem
273:
13469-13474,
1998
20.
Lemmon, MA,
Flanagan JM,
Hunt JF,
Adair BD,
Bormann BJ,
Dempsey CE,
and
Engelman DM.
Glycophorin A dimerization is driven by specific interactions between transmembrane -helices.
J Biol Chem
267:
7683-7689,
1992
21.
Loffing, J,
Pietri L,
Aregger F,
Bloch-Faure M,
Ziegler U,
Meneton P,
Rossier BC,
and
Kaissling B.
Differential subcellular localization of ENaC subunits in mouse kidney in response to high- and low-Na diets.
Am J Physiol Renal Physiol
279:
F252-F258,
2000
22.
Lukacs, GL,
Chang XB,
Bear C,
Kartner N,
Mohamed A,
Riordan JR,
and
Grinstein S.
The delta F508 mutation decreases the stability of cystic fibrosis transmembrane conductance regulator in the plasma membrane. Determination of functional half-lives on transfected cells.
J Biol Chem
268:
21592-21598,
1993
23.
Masilamani, S,
Kim GH,
Mitchell C,
Wade JB,
and
Knepper MA.
Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney.
J Clin Invest
104:
R19-R23,
1999
24.
May, A,
Puoti A,
Gaeggeler HP,
Horisberger JD,
and
Rossier BC.
Early effect of aldosterone on the rate of synthesis of the epithelial sodium channel subunit in A6 renal cells.
J Am Soc Nephrol
8:
1813-1822,
1997[Abstract].
25.
Naray-Fejes-Toth, A,
Canessa C,
Cleaveland ES,
Aldrich G,
and
Fejes-Toth G.
Sgk is an aldosterone-induced kinase in the renal collecting duct. Effects on epithelial Na+ channels.
J Biol Chem
274:
16973-16978,
1999
26.
Nelson, WJ,
Wilson R,
and
Mays RW.
Biochemical methods for studying supramolecular complexes involving cell adhesion molecules, integral membrane proteins, and the cytoskeleton.
In: Cell-Cell Interactions: A Practical Approach, edited by Stevenson BR,
Gallin WJ,
and Paul DL.. New York: IRL, 1992, p. 227-255.
27.
Pacha, J,
Frindt G,
Antonian L,
Silver RB,
and
Palmer LG.
Regulation of Na channels of the rat cortical collecting tubule by aldosterone.
J Gen Physiol
102:
25-42,
1993[Abstract].
28.
Prince, LS,
Tousson A,
and
Marchase RB.
Cell surface labeling of CFTR in T84 cells.
Am J Physiol Cell Physiol
264:
C491-C498,
1993
29.
Prince, LS,
and
Welsh MJ.
Cell surface expression and biosynthesis of epithelial Na+ channels.
Biochem J
336:
705-710,
1998[ISI][Medline].
30.
Puoti, A,
May A,
Canessa CM,
Horisberger JD,
Schild L,
and
Rossier BC.
The highly selective low-conductance epithelial Na channel of Xenopus laevis A6 kidney cells.
Am J Physiol Cell Physiol
269:
C188-C197,
1995
31.
Renard, S,
Voilley N,
Bassilana F,
Lazdunski M,
and
Barbry P.
Localization and regulation by steroids of the ,
, and
subunits of the amiloride-sensitive Na+ channel in colon, lung and kidney.
Pflügers Arch
430:
299-307,
1995[ISI][Medline].
32.
Rokaw, MD,
Wang JM,
Edinger RS,
Weisz OA,
Hui D,
Middleton P,
Shlyonsky V,
Berdiev BK,
Ismailov I,
Eaton DC,
Benos DJ,
and
Johnson JP.
Carboxylmethylation of the subunit of xENaC regulates channel activity.
J Biol Chem
273:
28746-28751,
1998
33.
Schmidt, TJ,
Husted RF,
and
Stokes JB.
Steroid hormone stimulation of Na+ transport in A6 cells is mediated via glucocorticoid receptors.
Am J Physiol Cell Physiol
264:
C875-C884,
1993
34.
Snyder, PM.
Liddle's syndrome mutations disrupt cAMP-mediated translocation of the epithelial Na+ channel to the cell surface.
J Clin Invest
105:
45-53,
2000
35.
Snyder, PM,
Cheng C,
Prince LS,
Rogers JC,
and
Welsh MJ.
Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits.
J Biol Chem
273:
681-684,
1998
36.
Spindler, B,
Mastroberardino L,
Custer M,
and
Verrey F.
Characterization of early aldosterone-induced RNAs identified in A6 kidney epithelia.
Pflügers Arch
434:
323-331,
1997[ISI][Medline].
37.
Stockand, JD,
Spier BJ,
Worrell RT,
Yue G,
Al-Baldawi N,
and
Eaton DC.
Regulation of Na(+) reabsorption by the aldosterone-induced small G protein K-Ras2A.
J Biol Chem
274:
35449-35454,
1999
38.
Stoner, LC,
Engbretson BG,
Viggiano SC,
Benos DJ,
and
Smith PR.
Amiloride-sensitive apical membrane sodium channels of everted Ambystoma collecting tubule.
J Membr Biol
144:
147-156,
1995[ISI][Medline].
39.
Tokunaga, M,
Tokunaga H,
Okajima Y,
and
Nakae T.
Characterization of porins from the outer membrane of Salmonella typhimurium. 2. Physical properties of the functional oligomeric aggregates.
Eur J Biochem
95:
441-448,
1979[Abstract].
40.
Valentijn, JA,
Fyfe GK,
and
Canessa CM.
Biosynthesis and processing of epithelial sodium channels in Xenopus oocytes.
J Biol Chem
273:
30344-30351,
1998
41.
Verrey, F.
Early aldosterone action: toward filling the gap between transcription and transport.
Am J Physiol Renal Physiol
277:
F319-F327,
1999
42.
Verrey, F,
Kraehenbuhl JP,
and
Rossier BC.
Aldosterone induces a rapid increase in the rate of Na-K-ATPase gene transcription in cultured kidney cells.
Mol Endocrinol
3:
1369-1376,
1989[Abstract].
43.
Verrey F, Schaerer E, Fuentes P, Kraehenbuhl JP, and Rossier BC.
Effects of aldosterone on Na-K-ATPase transcription, mRNAs, and protein
synthesis, and on transepithelial Na+ transport in A6
cells. Prog Clin Biol Res: 463-468, 1988.
44.
Wagner, JA,
McDonald TV,
Nghiem PT,
Lowe AW,
Schulman H,
Gruenert DC,
Stryer L,
and
Gardner P.
Antisense oligodeoxynucleotides to the cystic fibrosis transmembrane conductance regulator inhibit cAMP-activated but not calcium-activated chloride currents.
Proc Natl Acad Sci USA
89:
6785-6789,
1992[Abstract].
45.
Weisz, OA,
Wang JM,
Edinger RS,
and
Johnson JP.
Non-coordinate regulation of endogenous epithelial sodium channel (ENaC) subunit expression at the apical membrane of A6 cells in response to various transporting conditions.
J Biol Chem
275:
39886-39893,
2000
46.
Zuckerman, JB,
Chen X,
Jacobs JD,
Hu B,
Kleyman TR,
and
Smith PR.
Association of the epithelial sodium channel with Apx and -spectrin in A6 renal epithelial cells.
J Biol Chem
274:
23286-23295,
1999