1Institut de Pharmacologie et Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland; and 2Institut National de la Santé et de la Recherche Médicale, Unité 478, Faculté de Médecine Xavier Bichat, 75870 Paris Cedex 18, France
Submitted 19 February 2003 ; accepted in final form 7 May 2003
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ABSTRACT |
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collecting duct; PY motif; hypertension
The ENaCs in the apical membrane of the kidney connecting tubule and cortical collecting duct allow vectorial Na+ absorption from the tubule lumen (45). ENaC activity is under the control of aldosterone and vasopressin in response to stimuli such as volume contraction, salt depletion, or hyperkalemia.
Liddle's syndrome is a Mendelian form of low plasma renin hypertension due
to inappropriate Na+ absorption in the distal nephron
(19). Genetic linkage studies
in families of Liddle's syndrome patients permitted the identification of
pathogenic mutations in the - and
-subunits of ENaCs
(15,
16,
36,
43). These mutations, by
deleting or modifying a conserved PY motif (PPPxY sequence) in the cytoplasmic
COOH terminus of
-and
-ENaCs, cause an increase in ENaC activity
and channel stability at the surface of target cells
(31,
39). Proline-rich motifs are
generally involved in protein-protein interactions
(42). In the case of
-
and
-ENaC subunits, the PY motif can bind to the WW domain of the
ubiquitin protein ligase Nedd4-2
(18). In the Xenopus
oocyte expression system, this binding interaction leads to ENaC
ubiquitination, which tags the channel for endocytosis and degradation.
Consistent with these observations, mutations in the PY motif result in
deficient ubiquitination of ENaC and lead to retention at the cell surface
(41). A second model proposes
that the PY motif is part of an endocytic motif that is recognized by the
clathrin-adaptor protein (AP)-2 complex; this model does not preclude the
involvement of Nedd4-2 in regulation of channel activity at the cell surface
(35). Although the primary
mechanism of internalization and degradation of ENaC in the cell has not yet
been clearly established, mutagenesis experiments have demonstrated that the
PY motif in
- and
-ENaC subunits represents a critical
determinant of channel activity at the cell surface.
The stimulation of Na+ absorption in the distal nephron by
aldosterone is mainly due to an increase in the number of active ENaCs at the
cell surface (21,
26). This upregulation of ENaC
by aldosterone involves transcriptional and posttranscriptional events that
are initiated on the binding of aldosterone to its cytosolic receptor and
subsequent translocation of the complex to the nucleus
(45). Because the PY motif in
the COOH terminus of - and
-ENaC subunits seems to control ENaC
density at the cell surface, this regulatory domain represents a potential
target site for the ENaC response to aldosterone stimulation
(29). In support of this
hypothesis, recent experiments on the Xenopus oocyte have shown that
the aldosterone-induced serum and glucocorticoid-regulated kinase (SGK) can
modulate the interaction between Nedd4-2 and the PY motif of ENaC
(8). Vasopressin also
stimulates Na+ absorption in cultured epithelia by increasing the
density of active ENaCs at the cell surface
(24). The cAMP-mediated effect
of vasopressin might also involve the PY motif of ENaCs as the target site for
the modulation of channel stability at the cell surface
(37). According to these
recent data, ENaC mutants that cause Liddle's syndrome would be less
responsive or even unresponsive to stimulation by aldosterone and/or
vasopressin.
To address the question of the contribution of the PY motif of the -
and
-ENaC subunits in the ENaC response to aldosterone or vasopressin
stimulation, we have stably expressed mutants of the
- and
-ENaC
subunits in the mpkCCDcl4 cortical collecting duct cells, which is
a cell line that responds to aldosterone and vasopressin. We show that these
cells, which stably express
- and
-ENaC mutants of the PY motif,
exhibit a higher benzamil-sensitive Na+ current that is consistent
with the ENaC gain-of-function phenotype in Liddle's syndrome. The ENaC
channels with mutations in the PY motif retain the ability to mediate an
increased Na+ absorption in response to aldosterone or vasopressin
stimulation. These findings are consistent with early clinical observations
made by Liddle, who found that patients with pseudoaldosteronism (Liddle's
syndrome) responded to a challenge with aldosterone by a decrease in renal
Na+ excretion
(19).
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MATERIALS AND METHODS |
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Transfection of retrovirus producer cells. Virus producer cells (Phoenix cells) were grown in DMEM that contained 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. When transfected with the LZRS vector, Phoenix cells were capable of producing recombinant retroviruses after 48 h. Transfections of the retrovirus producer Phoenix cells with the LZRS vectors were done using a standard calcium phosphate transfection protocol. The Phoenix packaging cells transfected with retroviral vector (LZRS) were selected with puromycin (1 µg/ml). The recombinant retroviral particles produced by the packaging cells were harvested from the supernatant 4-5 days after transfection and were filtered through a 0.45-µm filter.
Cell infection. We used mpkCCDcl4 cells, which are a clone of principal cells that have been derived from microdissected cortical collecting ducts of a transgenic mouse (3). Cells were routinely grown on plastic tissue-culture flasks in a modified Ham's F-12 medium (Life Technologies) supplemented with 60 nM sodium selenate, 5 µg/ml transferrin, 2.5 nM dexamethasone, 1 nM triiodothyronine, 10 ng/ml epidermal growth factor (EGF), 5 µg/ml insulin, 11 mM D-glucose, 2% FSC, 10 mM HEPES, pH 7.4, 100 U/ml penicillin, and 100 µg/ml streptomycin. The recombinant viruses harvested from the Phoenix producer cells were used immediately for transduction of the mpkCCDcl4 cells. After 24 h, the medium containing the viruses was replaced with fresh modified Ham's F-12 medium. Delivery of more than one ENaC-subunit gene in the mpkCCDcl4 cells was obtained by performing sequential infections. Expression of the exogenous ENaC subunit proteins remained stable in the transduced cells during >12 passages after infection. Cells were studied between passages 32 and 42.
Electrophysiological studies. Electrophysiological studies were
performed on confluent cell monolayers grown on collagen-coated filters
(Transwell 0.4-µm pore, 4.7 cm2 or Snap-well 0.4-µm pore, 1
cm2; Corning Costar, Cambridge, MA). Cells were maintained for 5
days in the modified Ham's F-12 medium described above and then transferred in
a medium of identical composition but deprived of EGF, transferrin, and FSC.
Before measurements, filters were maintained overnight in Ham's F-12 medium
supplemented with 11 mM D-glucose, 100 U/ml penicillin, and 100
µg/ml streptomycin. Transepithelial short-circuit currents
(Isc) were recorded on confluent mpkCCDcl4
cells grown on filters and mounted in Ussing chambers. The epithelium was
maintained under a current-clamp condition instead of a voltage-clamp
condition to avoid high transepithelial Na+ flux, which could
saturate the transport capacities of the cells. The Isc
(µA/cm2) and the transepithelial resistance (k) were
calculated from ±10-µA pulses of 20-ms duration elicited by a
computer-controlled voltage-clamp apparatus (Physiological Instruments, San
Diego, CA). The amiloride-sensitive Isc defines the
Isc sensitive to 10 µM amiloride carried by
Na+ ions through endogenously expressed ENaC channels. The
amiloride-resistant Isc defines the
Isc sensitive to 500 µM benzamil but resistant to 10
µM amiloride and represents Isc carried by
Na+ ions through ENaC with the
-G525C mutation.
Electrophysiological recordings were performed in symmetrical solutions that
contained (in mM) 120 NaCl, 5 KCl, 25 NaHCO3, 1 sodium pyruvate,
0.9 sodium phosphate, 10 glucose, and 1 MgCl2 or in Ham's F-12
medium supplemented with 11 mM D-glucose, 100 U/ml penicillin, and
100 µg/ml streptomycin, both bubbled with 95% O2-5%
CO2.
The patch-clamp technique in the outside-out configuration was used to measure ENaC activity from confluent mpkCCDcl4 cells grown on transparent filters. The extracellular (bath) solution contained (in mM) 135 lithium or sodium methanesulfonate, 2 CaCl2, 1 MgCl2, 5 BaCl2, 10 HEPES, and 2 glucose, pH 7.4. The pipette solution contained (in mM) 103 potassium aspartate, 7 KCl, 20 CsOH, 20 tetraethylammonium chloride, 5 EGTA, and 10 HEPES, pH 7.4 (with KOH).
Immunoprecipitation studies. Immunoprecipitation studies were done
using polyclonal-specific anti- antibodies
(9). Cells were grown on
filters for 5 days in modified Ham's F-12 and 5 days in the same medium but
deprived of EGF, transferrin, and FCS. Filters were rinsed three times in a
methionine-free medium and pulsed at 37°C for 30 min with 200 µl of
methionine-free medium that contained 1 mCi/ml [35S]methionine
added to the basal side of inverted filters. Cells were washed two times with
ice-cold 1x PBS, scraped on ice in 250 µl of lysis buffer (50 mM
HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol,
1% Triton X-100, 1 mM PMSF, and 10 µg/ml each of leupeptin, pepstatin A,
and aprotinin), and centrifuged (13,000 g, 10 min) at 4°C. The
amount of incorporated [35S]methionine was determined by
trichloroacetic acid (10%) precipitation and similar counts per minute were
submitted to immunoprecipitation. The samples were incubated with 10 µl of
the specific antibody and 25 µl of protein A-Sepharose beads overnight at
4°C. The beads were centrifuged and washed five times with lysis buffer.
The immunoprecipitated proteins were recovered in Laemmli sample buffer. The
samples were boiled at 95°C for 5 min and then loaded on 5-10%
SDS-polyacrylamide gradient gel. After electrophoresis, gels were fixed and
treated with sodium salicylate and exposed on X-Omat S films (Eastman Kodak)
at -70°C for 24 h to 3 days.
Data analyses. The changes in Isc induced by aldosterone were fitted to a simple model in which changes in Isc result from both the addition of newly active ENaCs at the cell surface and the rate of ENaC removal (kremove) from the cell surface. We assume that new channels are added at a constant rate that corresponds to a constant addition of current (Isc,new).
At any given time, Isc is given by the equation
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Knowing the kremove value, it was possible to determine
Isc,new from the equilibrium baseline value of
Isc considering that at the steady state, the number of
new ENaCs added is equal to the number of ENaCs removed
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Data are expressed as means ± SE. Statistical differences between groups were calculated using Student's t-test.
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RESULTS |
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The missense mutation Y618H in the PY motif -ENaC is associated with
Liddle's syndrome (43). We
have introduced the
-Y618A mutation in the
-G525C mutant
background, and cells were transduced with this
-G525C + Y618A ENaC
construct (31). The
mpkCCDcl4 cells that express
-G525C + Y618A ENaCs were
compared with control cells transduced with the
-G525C ENaC or the green
fluorescent protein (GFP, thus with ENaCs containing only endogenous
subunits). Immunoprecipitation experiments in
Fig. 1A show that in
the absence of aldosterone, anti-
-ENaC antibodies could not clearly
recognize the endogenous
-ENaC subunit in mpkCCDcl4
transduced with GFP due to the low level of
-ENaC expression. In cells
transduced with the exogenous
-G525C subunit, a clear band is detected
at 94 kDa that corresponds to the apparent molecular mass of
-ENaCs
(14). Cells transduced with
-G525C or
-G525C + Y618A exhibit a higher level of expression of
subunit protein compared to endogenous
-ENaCs. Expression of the
exogenous
-G525C ENaC protein carrying the mutation
-Y618A was
slightly lower than the expression of the control
-G525C ENaC
subunit.
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The -ENaC subunits cannot form functional homomeric channels at the
cell surface (12). To confer
amiloride resistance to functional ENaCs, the
-G525C ENaC subunits have
to coassemble with the endogenous
- and
-ENaC subunits. Only
ENaC channels that contain an exogenous
-G525C ENaC subunit remain
active in the presence of inhibitory concentrations of amiloride (10 µM).
Figure 1B shows the
dose-dependent amiloride inhibition curve of the Isc in
transduced mpkCCDcl4 cells. The cells transduced with GFP exhibit
an amiloride-sensitive Isc with an inhibitory constant
(IC50) of 0.2 µM, whereas the cells that express the exogenous
-G525C or
-G525C + Y618A ENaC subunits show a biphasic inhibition
curve with a large amiloride-resistant component of Isc.
In these latter cells, only 15% of the Isc was sensitive
to 10 µM amiloride, a concentration that maximally inhibited
Isc carried by endogenous ENaCs in cells transduced with
GFP (Fig. 1A). The
large amiloride-resistant component of Isc
(IC50 = 250 µM) is carried by channels that have incorporated
the
-G525C ENaC subunit; this amiloride-resistant
Isc component is completely inhibited by 500 µM
benzamil, which is a more potent analog of amiloride. In both cell lines that
express the
-G525C or the
-G525C + Y618A ENaC subunits, the
fraction of the amiloride-sensitive Isc represents <15%
of the overall benzamil-blockable Isc, which indicates
that >85% of the channels expressed have incorporated the exogenous
-ENaC subunit. The titration curves of the Isc
carried by the
-G525C or the
-G525C + Y618A ENaCs were similar in
the presence or absence of aldosterone, which indicates that the fraction of
active endogenous ENaCs relative to ENaCs with an exogenous
-subunit
does not vary after aldosterone stimulation. These data also show that in the
transduced mpkCCDcl4 cells, the ratio of functional expression of
ENaCs containing
-G525C or
-G525C + Y618A subunits relative to the
endogenous ENaCs is identical.
The single-channel characteristics of the -G525C + Y618A ENaC mutant
were verified by the patch-clamp technique in transduced cells grown on a
filter. As shown in Fig.
1C, the ENaC activity was detected in outside-out patches
from the apical membrane, and channel activity was determined by the product
of the number of channels (N) times the open probability
(Po). The channel activity in this patch (N
x Po = 0.453) was not affected by benzamil at a
concentration of 0.5 µM (N x Po = 0.437,
data not shown) that normally completely inhibits endogenous wild-type ENaC.
ENaC activity was largely inhibited (N x Po
= 0.009) by high doses (500 µM) of benzamil, which is consistent with the
channel resistance to amiloride (Fig.
1C); in addition, the single-channel conductance
(G) in the presence of Na+ or Li+ ions was
slightly lower (GNa = 2.1 and GLi =
4.2 pS) than the wild-type ENaCs expressed in Xenopus oocytes, which
is another functional signature of the
,
-G525C
-ENaC
channel (32). Taken together,
these experiments demonstrate that exogenous
-G525C mutant subunits
expressed in mpkCCDcl4 cells can assemble with endogenous ENaC
subunits to form functional channels at the cell surface with the expected
functional characteristics.
Transduced -ENaC mutants respond to aldosterone. To
test whether the functional ENaC that contains transduced
-ENaC subunits
responds to aldosterone, mpkCCDcl4 cells transduced with GFP,
-G525C, or
-G525C + Y618A ENaC mutants were grown on filters.
After formation of a confluent monolayer, they were challenged with
10-6 M aldosterone for 4-5 h. This high concentration of
aldosterone was used to induce a maximal effect on Na+ transport by
occupation of both types of MR receptors by the ligand.
Figure 2 shows that in cells
transduced with GFP as a control, the benzamil (500 µM)-inhibitable
Isc was entirely sensitive to a low concentration of
amiloride (10 µM), which is expected for cells that exclusively express
endogenous ENaC. This amiloride-sensitive current increased by about threefold
after 4 h of incubation with aldosterone. Overexpression of the
-G525C
ENaC subunit did not significantly change the level of the basal
Isc; the Isc was mainly resistant to
10 µM amiloride but sensitive to 500 µM benzamil, which is consistent
with a majority of channels made of the amiloride-resistant ENaC that contains
the exogenous
-G525C subunit. Aldosterone stimulated both
amiloride-sensitive and amiloride-resistant fractions of
Isc by about sixfold, thereby indicating that ENaC
channels that contain either endogenous
-ENaC or exogenous
-G525C
ENaC subunits respond to aldosterone. Cells that express the Liddle
-G525C + Y618A ENaC mutant subunit exhibited a higher basal
Isc that was almost completely resistant to 10 µM
amiloride, which is consistent with the previously reported hyperactivity of
the
-Y618A ENaC mutant at the cell surface of Xenopus oocytes.
In these cells, both amiloride-sensitive (10 µM) and amiloride-resistant
fractions of Isc increased on aldosterone stimulation,
thereby indicating that the
-G525C + Y618A mutant retains its ability to
respond to aldosterone.
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We have generated different cell lines that express -or
-ENaC
mutants and cause Liddle's syndrome: the missense mutation
-P616L,
-ENaC with the COOH-terminal truncation mutation at position
-R564
(
-R564stop), and the corresponding truncated K570stop in the
-ENaC subunit that also deletes the PY motif
(15,
16,
36). For the generation of
these cell lines, we choose mpkCCDcl4 cells with a low level of
baseline Isc to avoid possible saturation of the
transcellular Na+ transport capacity in cells that express ENaC
with activating mutations. In Table
1, we compared Isc measured in the presence of
10 µM amiloride in cells transduced with these Liddle's ENaC mutants, with
parental cells transduced with their respective control
-G525C ENaC
construct. In these experiments, the amiloride-sensitive component of
Isc that reflects the activity of endogenous ENaCs
represented 15-25% of the 500 µM benzamil-inhibitable current carried by
the ensemble of the functional ENaCs. The results obtained with the
mpkCCDcl4 cells that express ENaCs with missense mutations or
deletions in the PY motif of
-ENaCs all show a higher baseline
Isc than the respective control mpkCCDcl4
cells. The aldosterone-induced absolute increase in Isc
(
Isc) after 5 h (early response to aldosterone) was
similar in magnitude in cells transduced with ENaC Liddle's mutants and in
control cells, which indicates identical responses to the hormone. Similar
results were obtained with mpkCCDcl4 cells that express both
- and
-ENaC truncation mutants in the COOH terminus. According to
functional and biochemical studies, these mutations in the PY motif of
-
and
-ENaCs are sufficient to nearly abolish the interaction between
ENaCs and WW domains of rNedd4-1
(30,
31,
40).
A large body of evidence indicates that aldosterone stimulation of the
transepithelial Na+ transport in tight epithelia results from the
appearance of newly active ENaCs at the cell surface
(45). We have measured the
kinetics of appearance of newly active ENaC mutants at the cell surface. An
experiment is shown in Fig.
3A with the recording of the time course of
Isc changes after the addition of aldosterone to cells
that express -G525C and
-G525C +Y618A ENaCs. The recording starts
after the addition of aldosterone, and Isc measurements in
the presence of 10 µM amiloride were taken every 20 s over a >4-h time
period. Typically, the cells that express the
-Y618A mutant have a
higher baseline Isc value than the control cells. After a
40-min period of latency, the Isc starts to increase in
parallel in both cell lines and reaches a peak after 3-4 h. As shown in
Table 1, in every cell line
transduced with exogenous
-ENaC mutants, the rate of
Isc increase
(
Isc/
t, in µA/min) was
comparable to that in its respective control cells transduced with
-G525C ENaCs. These results indicate that aldosterone has similar
effects during the early phase of the response on the magnitude of the
ENaC-dependent Na+ transport in cells that express ENaC controls
and in cells transduced with
-and
-ENaC mutant subunits lacking
the PY motif.
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We also measured the recovery of the Isc from
aldosterone stimulation in the different mpkCCDcl4 cell lines that
we generated. As shown in Fig.
3B, 40 min after aldosterone removal,
Isc started to decline faster in control cells transduced
with -G525C ENaCs than in cells that expressed the
-G525C + Y618A
mutant. Similar results were obtained with addition of the protein synthesis
inhibitor cycloheximide (0.5 mg/ml) while aldosterone was maintained in the
bath. The half-time for Isc decline in control
mpkCCDcl4 cells after aldosterone removal ranged between 50 and 60
min (see Table 1) and was
comparable to the Isc decline measured after cycloheximide
treatment (56.4 ± 8.7 min, n = 4). These values are consistent
with the half-life of ENaCs at the cell surface that was recently reported in
ENaC-transfected Madin-Darby canine kidney cells
(17). The rate of
Isc decline in mpkCCDcl4 cells that expressed
the different ENaC Liddle's mutants was significantly slower (see
Table 1). This observation is
consistent with a longer half-life of activity for ENaC Liddle's mutants at
the cell surface compared with control ENaCs.
Compared with mpkCCDcl4 cells that express wild-type ENaCs,
those that express ENaCs with mutations in the PY motif exhibit two main
features. First, these cells have a longer half-life of channel activity at
the cell surface that can account for a larger baseline
Isc. Second, they respond to aldosterone with a similar
increase in Na+ transport during the first hours of stimulation.
These observations are illustrated in Fig.
4 with the mean Isc changes before and after
aldosterone administration in mpkCCDcl4 cells that express the ENaC
Liddle's mutants -G525C + Y618A and the
- and
-truncated
ENaC subunits together with their respective controls. The question remains
whether a higher stimulation of Na+ transport by aldosterone should
be expected in cells that express ENaCs with a longer half-life of activity at
the cell surface. We addressed this question using a simple mathematical
model, assuming that aldosterone stimulates Na+ transport simply by
increasing the rate of appearance of newly active ENaCs at the cell surface
(see MATERIALS AND METHODS). Such a model for aldosterone
stimulation can be justified by evidence that shows that aldosterone indeed
increases the number of active ENaCs at the cell surface
(21,
26). In our model, the
steady-state Isc basically results from an equilibrium
between the rates of insertion and retrieval of active ENaCs at the cell
surface. We have used this model to predict the Isc
response to aldosterone in cells that express control and Liddle's mutants
that differ in their respective activity half-lives. The fit of our data to
such a model reveals that the differences in the baseline
Isc values between cells that express control and Liddle's
mutant ENaCs can account for an ENaC half-life at the cell surface of 60 min
for the wild-type ENaC, 410 min for the
-G525C + Y618A mutant, and 650
min for the
-and
-COOH-terminal truncated ENaC mutants. Our model
also predicts that the rate of Na+ transport
(Isc) increase is basically the same during the first 2-3
h of aldosterone stimulation in cells transduced with ENaC control and ENaC
Liddle's mutants and that we should not expect large differences in the early
aldosterone response.
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During the late phase of the aldosterone response, the ENaC Liddle's
mutants are likely to continue to accumulate with time at the plasma membrane
due to their longer half-life at the cell surface, while the
Isc in cells transduced with control ENaCs will have
already reached a steady-state level due to the equilibrium between the rate
of channel insertion and removal from the cell surface. Thus we expected to
see, after several hours of stimulation, a larger aldosterone-induced
Isc in mpkCCDcl4 cells that express ENaC
Liddle's mutants. In these experiments investigating the long-term effects of
aldosterone, mpkCCDcl4 cells that express control and Liddle's
mutant ENaCs were maintained during the time of aldosterone stimulation (16 h)
in an apical low-Na+ medium that contained amiloride to inhibit
apical Na+ entry. The Isc was then measured 4
and 16 h after addition of aldosterone in the presence of a normal apical
Na+ medium without amiloride. The experiments in
Fig. 5 show that in both
mpkCCDcl4 cells that express wild-type ENaC and ENaCs carrying the
Y618A mutation, aldosterone did not induce a sustained Isc
stimulation for a 16-h time period. Rather, Isc reached a
peak 4 h after the addition of aldosterone and then slowly decreased, thereby
showing no clear late response of aldosterone in both mpkCCDcl4
cell lines. Under conditions of low baseline Na+ transport using
amiloride and low-apical Na+ medium during incubation, the
instantaneous measurement of Isc revealed that after 4 h,
the aldosterone-induced Isc was already higher and after
16 h, it was still measurable in mpkCCDcl4 cells that express the
-Y618A mutant, whereas in control cells, Isc
returned to baseline values. This stronger and prolonged response to
aldosterone by the Liddle's
-Y618A mutant ENaCs compared with control
ENaCs further supports a longer half-life and the retention of the active ENaC
mutants at the cell surface. Thus we can conclude that mpkCCDcl4
cells that express ENaCs with mutations in the PY motif respond to aldosterone
during the early stimulatory phase with changes in Na+ transport
that are similar in magnitude and kinetics of appearance as in control cells.
The surface retention of active Liddle's mutant ENaCs can account for a
prolonged and even stronger effect of aldosterone on Na+ absorption
after 4-5 h of aldosterone stimulation.
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Effects of vasopressin on Na+ absorption in cells
that express ENaC mutants. Vasopressin and cAMP both stimulate
Na+ transport in the cortical collecting duct and in cultured
mpkCCDcl4 cells (3,
6). In mpkCCDcl4
cells transfected with GFP, the 500 µM benzamilinhibitable
Isc was completely blocked by 10 µM amiloride in the
absence or after stimulation by 10-9 M vasopressin. In
mpkCCDcl4 cells that express -G525C ENaCs, the largest
fraction of the benzamil-inhibitable Isc was insensitive
to 10 µM amiloride. The addition of 10-9 M
vasopressin for 2 h stimulated Isc mediated by both
endogenous amiloride-sensitive ENaCs and amiloride-resistant ENaCs that
contained the
-G525C subunit (Fig.
6). In cells that express the
-G525C + Y618A mutant, the
basal Isc was higher than in control cells, as expected
for a Liddle's mutation, and in the presence of vasopressin, both
amiloride-sensitive and -resistant components of Isc
increased. These experiments indicate that the
-G525C + Y618A mutant
ENaCs respond to stimulation by vasopressin.
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Recent evidence shows that vasopressin promotes the appearance of newly
active channels at the cell surface. We asked whether the rate of appearance
of newly active channels is similar in mpkCCDcl4 cells that express
control -G525C and
-G525C + Y618A mutant ENaCs. As shown in
Fig. 7A, in both cell
lines, vasopressin induced a rapid increase in Isc during
the first 10 min followed by a slower rate of Isc increase
that lasted 1 h. Then, Isc started to decline despite the
presence of vasopressin in the bathing solution. The vasopressin-induced
increase in Isc measured in the presence of 10 µM
amiloride to block endogenous ENaCs was inhibited by the addition of 500 µM
benzamil at the apical surface. Similarly, forskolin, a nonspecific agonist
for adenylyl cyclase, was also able to stimulate Isc
through the amiloride-resistant ENaCs with and without the Y618A Liddle's
mutation (Fig. 7B).
The absolute magnitude and the rate of changes of Isc were
comparable in mpkCCDcl4 cells that expressed control and mutant
ENaCs. Table 2 summarizes
characteristics of the vasopressin response in cells that express control
ENaCs, the
-G525C + Y618A mutant, or the double-
- and
-ENaC
COOH-terminal truncated mutants. All three cell lines responded to vasopressin
and exhibited a similar increase in the magnitude of Isc
(
Isc). In the mpkCCDcl4 cells that were
transduced with GFP or that express control
-G525C and
-G525C +
Y618A mutant ENaCs, the rate of Isc increase was
comparable. A slightly but significantly lower rate of Isc
increase was measured in cells transduced with both
- and
-ENaC
COOH-terminal truncated mutants. Considering the variations in the rate of
vasopressin-induced increase in Isc in both control cells
transduced with GFP or
-G525C ENaCs, the
-Y618A ENaC mutation does
not seem to greatly affect the ENaC response to vasopressin. Therefore, the PY
motif in the COOH terminus of
- and
-ENaCs does not seem to be
required for ENaC stimulation by vasopressin. However, in cells that express
the double-
- and -
ENaC COOH-terminal truncated mutants, the
slightly lower rate of Isc increase could be related to
the deletion of sequences in the COOH terminus of
- or
-ENaCs
other than the PY motif.
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The vasopressin effect of Na+ transport was rapidly reversible
on removal of vasopressin from the bath: Isc decreased
with a slower rate in cells that express the -G525C + Y618A mutant
compared with control cells (half-times, 245.3 ± 45.3 vs. 35.4 ±
9 min, respectively; Fig.
7C). This suggests that the recovery from vasopressin
stimulation likely involves retrieval of ENaCs from the cell surface and
requires functional PY motifs. In summary, these data indicate that mutation
of the Tyr within the PY motif of
-ENaCs does not affect the magnitude
of the response to vasopressin of the cortical collecting duct cells that
express the ENaCs; we cannot exclude that sites other than the PY motif in the
COOH terminus of
- or
-ENaCs might contribute the vasopressin
response.
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DISCUSSION |
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Transduced mpkCCDcl4 cells that express
amiloride-resistant ENaC mutants. The exogenous rat -G525C mutant
ENaC subunit that confers to the functional ENaC a resistance to amiloride was
retrovirally transduced in the mpkCCDcl4 cells under the control of
a viral promoter. This exogenous
-G525C ENaC subunit was therefore
constitutively expressed and not transcriptionally regulated by aldosterone in
the mpkCCDcl4 cells as in the endogenous
-ENaCs. The cells
transduced with the exogenous
-G525C ENaCs showed a higher expression of
-ENaC subunits at the protein level without a corresponding change in
the basal level of Isc. The reason is that
-ENaC
subunits alone do not form homomeric functional channels at the cell surface;
only the heteromeric
,
,
-ENaC is functional
(5). Thus it is likely that in
mpkCCDcl4 cells transduced with and overexpressing the
-G525C
subunits,
- and
-ENaC subunits become limiting for expression of
ENaC activity at the cell surface. Accordingly, overexpression of
-ENaCs
only will not increase the level of baseline Isc.
Despite the fact that the exogenous -G525C ENaC subunit is
constitutively expressed in the mpkCCDcl4 cells, the channels made
of the transduced
-ENaC subunit respond to aldosterone. In target
epithelia, the stimulation of Na+ transport by aldosterone requires
de novo protein synthesis of
-ENaCs as well as other
aldosterone-induced proteins that regulate ENaC activity
(23,
28). In the intact distal
nephron, aldosterone induces the expression of
-ENaC, whereas
-
and
-ENaCs are constitutively expressed
(11). Thus in the
mpkCCDcl4 cells transduced with the exogenous
-G525C subunit,
the newly synthesized
-ENaC subunits in response to aldosterone
stimulation likely assemble with the available
- and
-ENaC
subunits; in the case of cells that overexpress the
-G525C ENaC subunit,
channel assembly will occur preferentially with the exogenous
-G525C
subunit because of its higher level of protein expression (see
Fig. 1A). This
preferential assembly results in a majority of active but amiloride-resistant
ENaCs at the cell surface due to the
-G525C mutation in the subunit.
Results of the Isc sensitivity to amiloride indicate that
>85% of the ENaCs at the surface of cells transfected with
-G525C
ENaCs have incorporated an exogenous mutant
-ENaC subunit and responded
to aldosterone.
mpkCCDcl4 cells that express
-ENaC mutants causing Liddle's syndrome. Gain-of-function
mutations that cause Liddle's syndrome have been identified in the conserved
PY motif in the COOH-terminal end of
-and
-ENaC subunits.
The PY motifs of - or
-ENaCs are involved in ENaC
internalization and/or its degradation as shown by mutations of this motif
that increase ENaC activity at the cell surface
(29). In mpkCCDcl4
cells, the expression of different mutations or truncations of the PY motif of
- and
-ENaCs resulted in a five- to sixfold increase in the basal
Isc compared with control cells. This higher baseline
Isc was observed for comparable levels of expression of
-G525C or
-G525C + Y618A ENaC subunits. Recovery of
Isc from aldosterone stimulation showed that the active
-G525C + Y618A ENaC mutant is more stable at the cell surface than the
wild-type ENaC with a longer half-life of activity. Taken together, these
observations are consistent with experimental evidence obtained from
Xenopus oocytes and suggest the possibility that the increased
activity at the cell surface of ENaC mutants that cause Liddle's syndrome
might be due to a deficient binding interaction with its partner such as
Nedd4-2. Mutations in the PY motif of
-ENaCs have only little effects
on ENaC function in Xenopus oocytes, and to date no mutations that
cause the syndrome have been found in the PY motif of
-ENaCs
(30,
31). This relatively small
effect of
-ENaC mutations correlates with the reduced ability of the PY
motif of
-ENaCs to interact with WW domains Nedd4-1, whereas mutations
in the PY motif of
- or
-ENaCs impair most of the ability to bind
this Nedd4 protein (40).
Aldosterone response of Liddle's mutant ENaCs. In the cells that
express - and
-ENaCs with mutations in the PY motif, aldosterone
induced a significant increase in Isc that after 4-5 h was
comparable in absolute magnitude to the response of the cells that express
control ENaCs. The kinetics of activation of the amiloride-sensitive
Isc during the early phase of aldosterone stimulation were
comparable in cells transduced with control or Liddle's mutant ENaCs. We can
conclude that the early response of the mpkCCDcl4 cells to
aldosterone is maintained and not affected by mutations in the PY motif of
ENaCs. These results are expected if aldosterone acts on cortical collecting
duct cells primarily by promoting the appearance of newly active ENaCs at the
cell surface. This is supported by recent immunochemical experiments that have
evidenced a rapid translocation of ENaC subunits from a cytoplasmic pool to
the apical membrane 2-4 h after aldosterone stimulation
(21,
45). The rapid insertion of
newly active ENaCs at the cell surface together with an inhibition of their
internalization could theoretically be a very efficient way for aldosterone to
increase Na+ transport. In our experiments comparing the
aldosterone response of cells that express wild-type ENaCs and mutants that
differ in their half-life of channel activity at the cell surface, we have not
been able to show a difference in the early stimulation of Na+
transport between these cells. This suggests that the modulation of ENaC
stability at the cell surface does not play a major role in the aldosterone
response. Recent biochemical evidence indicates that aldosterone does not
change the half-life of ENaCs in the cell and at the cell surface
(2,
23).
However, the question remains as to whether the longer half-life of the
Liddle's mutant ENaCs could have functional consequences on transepithelial
Na+ transport during the late aldosterone response. One could
expect that by promoting the insertion at the apical membrane of ENaC mutants
with longer half-lives, aldosterone should ultimately induce a higher
Isc in cells that express Liddle's mutant compared with
control ENaCs. We have measured amiloride-sensitive Isc up
to 16 h of aldosterone stimulation in mpkCCDcl4 cells and observed
that despite the continuous presence of the hormone, the
Isc declines after 4-5 h. Nevertheless, after 16 h of
stimulation, a significant higher aldosterone-induced Isc
was measured in mpkCCDcl4 cells that express the Liddle's mutant,
whereas in the controls cells Isc returned to the baseline
values. This observation is consistent with retention of active ENaC mutants
at the cell surface. It is interesting to note that the inhibition of ENaC
transport activity during early aldosterone stimulation seems to enhance the
Isc response in mpkCCDcl4 cells that express
the ENaC -Y618A mutant (see Figs.
2 and
5 for comparison). This
observation further supports the ability of the Liddle's mutant ENaCs to
respond to aldosterone and suggests that the high transcellular Na+
transport in mpkCCDcl4 cells that express the mutants ENaCs may
induce feedback-inhibitory effects on the aldosterone response.
The aldosterone-signaling pathway initiated by the binding of the ligand to the mineralocorticoid receptor and ending with the activation of ENaCs at the cell surface remains to be deciphered. In the aldosterone-sensitive distal nephron, the SGK is an early aldosterone-induced protein, and its intracellular accumulation seems to precede the apical translocation of ENaCs (21). In addition, coexpression of ENaCs and SGK in Xenopus oocytes increases cell surface expression of active ENaCs. Together, these experiments strongly suggest that SGK mediates at least in part the aldosterone-induced translocation of active ENaCs at the cell surface (1, 7, 21, 25). Recently, it was shown in Xenopus oocytes that SGK can phosphorylate Nedd4-2, which leads to an inhibition of the binding interaction between Nedd4-2 and ENaCs and a retention of ENaCs at the cell surface (8, 38). The physiological relevance of this signaling pathway involving SGK and Nedd4-2 phosphorylation has not yet been established in aldosterone-responding epithelia. Our results obtained from cortical collecting duct cells that express mutants ENaCs show that the PY motif is not essential for the early response to aldosterone stimulation.
Insulin and aldosterone have synergetic actions on Na+
transport. The signaling pathway for both hormones requires
phosphatidylinositol 3-kinase
(46). Recently it was shown in
A6 kidney cells that phosphatidylinositol 3-kinase is necessary for the
aldosterone effect on Na+ transport and on the phosphorylation of
SGK. Considering that insulin also increases SGK phosphorylation, these
observations suggest that SGK represents a critical point of convergence
between the aldosterone- and the insulin-signaling pathways. We found that
cells that express the -Y618A mutant respond to insulin in a way similar
to the cells that express control ENaCs (data not shown). This further
indicates that the target sequence crucial for the SGK effect on ENaC
regulation is likely not the PY motif in the COOH-terminal end of ENaCs.
Our data indicate that neither the PY motif nor the COOH terminus of
- and
-ENaCs is required for the ability of the channel to
respond to aldosterone. Therefore, the phosphorylation of the COOH terminus of
-and
-ENaC cells and its modulation observed in Madin-Darby
canine kidney cells do not seem to be directly related to the early response
of the channel to aldosterone
(34). The contribution of
other potential phosphorylation sites, in particular in the NH2
terminus of the
,
,
-ENaC subunits in the channel response
to aldosterone, remains to be investigated.
Vasopressin response. The increased Na+ absorption in response to vasopressin is due to a stimulation of ENaCs at the cell surface (10). The vasopressin effect is mediated by the binding of vasopressin to the V2 receptor, which leads to an increase in intracellular cAMP content. Patch-clamp experiments in A6 cells showed that vasopressin and cAMP increase the number of active ENaCs per patch (22). More recently, surface labeling of ENaCs showed that vasopressin inserts newly active ENaC molecules at the cell surface, which can fully account for the increase in Na+ current (24).
Our results demonstrate that cells that express the Y618A ENaC mutant
retain the ability to respond to vasopressin with a comparable increase in the
absolute magnitude of Isc. Furthermore, the rate of the
initial increase in Isc was comparable in
mpkCCDcl4 cells transduced with control ENaCs and with the
-G525C + Y618A Liddle's mutant, which suggests similar rates of
activation for ENaCs at the cell surface. Thus cortical collecting duct cells
that express either wild-type or
-Y618A mutant ENaCs have similar
responses to vasopressin stimulation. This response is dependent on cAMP,
since forskolin induces similar stimulation of Isc in both
cells that express wild-type and mutant ENaC. Recent experiments in rat
thyroid cells transfected with ENaCs showed that the
-Y618A mutation
disrupts the cAMP-induced insertion of ENaC at the cell surface
(37). The discrepancies
between this report and our results remain unclear and could be due to
differences in the cellular environment of ENaCs in thyroid cells that do not
physiologically respond to vasopressin or express ENaCs. The
mpkCCDcl4 cells that express the double COOH-terminal truncated
- and
-ENaC mutant show a slightly attenuated response to
vasopressin, which suggests the possibility that sites in the COOH termini of
- and
-ENaC other than the PY motif could contribute to the
stimulation of ENaCs by vasopressin. It has recently been reported that the
COOH termini of
- and
-ENaCs can be phosphorylated in vitro in
the vicinity of the PY motif, but the physiological role of these
phosphorylation sites remains to be established
(33,
34).
Physiological relevance. We have studied the aldosterone and vasopressin response in transduced mpkCCDc14 cells that overexpress ENaC mutants causing Liddle's syndrome. It is possible that the ENaC biology in these engineered epithelial cells as in any other cell expression system does not necessarily reflect the precise biology of ENaCs in the native tissue. Thus how can we consider our results in the context of the early clinical observation made by Liddle in his patients with pseudoaldosteronism (19)? Typically, patients with pseudoaldosteronism have a low plasma level of aldosterone and low plasma renin activity (4). Interestingly, Liddle observed in one of his patients maintained on a 30-meq Na+/day diet that treatment with exogenous aldosterone decreased the urinary Na+ excretion to <1 meq/day (19). Together with our results, this observation strongly suggests that Liddle's patients responded efficiently to aldosterone stimulation because of the conserved responsiveness of the ENaC mutant to the hormone.
Does this conserved response together with a prolonged activity at the cell surface of the ENaC mutants to aldosterone have a pathophysiological relevance in patients with Liddle's syndrome, considering that their plasma levels of aldosterone are chronically low? Hypertension in patients with Liddle's syndrome usually starts at an early age. The sequence of pathophysiological events in these patients that starts from an abnormally high distal Na+ absorption and leads ultimately to the development of hypertension remains unclear. It is conceivable that before the development of volume expansion and hypokalemia, the plasma aldosterone levels in these patients remain in the normal range. At this preclinical stage, it is possible that a prolonged response of the distal nephron to aldosterone and/or vasopressin, due to retention of active ENaCs at the apical membrane, may represent a key event in the development of volume expansion, renal injuries, and high blood pressure in patients with Liddle's syndrome.
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ACKNOWLEDGMENTS |
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This work was supported by Grant 31-59,217.99 from the Swiss National Science Foundation to L. Schild. S. Kellenberger was supported by a National Institutes of Health Specialized Center of Research grant for hypertension, and M. Auberson was supported by Human Frontier Science Program Grant RG00261/2000.
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FOOTNOTES |
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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.
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REFERENCES |
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