Epithelial Na+ channel mutants causing Liddle's syndrome retain ability to respond to aldosterone and vasopressin

Muriel Auberson,1 Nicole Hoffmann-Pochon,1 A. Vandewalle,2 Stephan Kellenberger,1 and Laurent Schild1

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Liddle's syndrome is a monogenic form of hypertension caused by mutations in the PY motif of the COOH terminus of {beta}- and {gamma}-epithelial Na+ channel (ENaC) subunits. These mutations lead to retention of active channels at the cell surface. Because of the critical role of this PY motif in the stability of ENaCs at the cell surface, we have investigated its contribution to the ENaC response to aldosterone and vasopressin. Mutants of the PY motif in {beta}- and {gamma}-ENaC subunits ({beta}-Y618A, {beta}-P616L, {beta}-R564stop, and {gamma}-K570stop) were stably expressed by retroviral gene transfer in a renal cortical collecting duct cell line (mpkCCDcl4), and transepithelial Na+ transport was assessed by measurements of the benzamil-sensitive short-circuit current (Isc). Cells that express ENaC mutants of the PY motif showed a five- to sixfold higher basal Isc compared with control cells and responded to stimulation by aldosterone (10-6 M) or vasopressin (10-9 M) with a further increase in Isc. The rates of the initial increases in Isc after aldosterone or vasopressin stimulation were comparable in cells transduced with wild-type and mutant ENaCs, but reversal of the effects of aldosterone and vasopressin was slower in cells that expressed the ENaC mutants. The conserved sensitivity of ENaC mutants to stimulation by aldosterone and vasopressin together with the prolonged activity at the cell surface likely contribute to the increased Na+ absorption in the distal nephron of patients with Liddle's syndrome.

collecting duct; PY motif; hypertension


REGULATION OF NA+ AND WATER homeostasis by the distal nephron of the kidney is critical for the maintenance of normal arterial blood pressure. The recent identification of mutations in genes that encode the epithelial Na+ channel (ENaC), the mineralocorticoid receptor (MR), and the 11{beta}-hydroxysteroid dehydrogenase that causes monogenic forms of hypertension strongly supports this notion (13, 20, 36).

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 {beta}- and {gamma}-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 {beta}-and {gamma}-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 {beta}- and {gamma}-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 {beta}- and {gamma}-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 {beta}- and {gamma}-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 {beta}- and {gamma}-ENaC subunits in the ENaC response to aldosterone or vasopressin stimulation, we have stably expressed mutants of the {beta}- and {gamma}-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 {beta}- and {gamma}-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).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vector construction. The Epstein-Barr virus-based retroviral vector (LZRS) was kindly provided by G. Nolan and was used for transfection of the Phoenix retrovirus producer cells (27). This vector takes up semistable residence as episomes within the virus producer cell line. The rat {beta}- and {gamma}-subunits of ENaC cDNAs were subcloned into the LZRS vector. The {beta}-cDNA was modified by the addition of the {beta}-G525C mutation, which causes low affinity for amiloride block with or without different Liddle's mutations (Y618A, P616L, or R564stop; Ref. 32). In addition, the K570stop mutation was introduced in {gamma}-ENaCs (15).

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{Omega}) 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 {beta}-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-{beta} 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

where Isc(t) is Isc at time t; Isc,new is the rate of Isc increase due to insertion of newly active ENaCs (expressed in µA/min); and kremove is the rate constant of Isc decrease due to removal of ENaCs from the cell surface and is determined from the half-life of Isc decline after suppression of aldosterone stimulation (expressed in min-1; see Table 1). The Isc half-life at the cell surface is given as t1/2 = ln2/kremove.


View this table:
[in this window]
[in a new window]
 
Table 1. Effects of aldosterone on Isc in transduced mpkCCDc/4 cells

 

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

Data are expressed as means ± SE. Statistical differences between groups were calculated using Student's t-test.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Stable expression of ENaC mutants in mpkCCDcl4 cells. We have used mpkCCDcl4 cells, an immortalized mouse cortical collecting duct cell line, for the expression of the rat {beta}-ENaC subunit carrying mutations in the PY motif of the COOH terminus. These cells express an endogenous ENaC composed of the three {alpha}-, {beta}-, and {gamma}-subunits and exhibit amiloride-sensitive Na+ currents that respond to stimulation by aldosterone and vasopressin (3, 44). As a control, we have retrovirally transduced these cells with the rat {beta}-ENaC subunit carrying the {beta}-G525C mutation in the amiloride-binding domain that confers to the functional heteromeric {alpha},{beta},{gamma}-ENaC channel a resistance to amiloride (32). This mutation was introduced in the {beta}-ENaC sequence to enable us to functionally distinguish the channels that contained the transduced {beta}-G525C ENaC subunit from the endogenous ENaC expressed by the mpkCCDcl4 cells.

The missense mutation Y618H in the PY motif {beta}-ENaC is associated with Liddle's syndrome (43). We have introduced the {beta}-Y618A mutation in the {beta}-G525C mutant background, and cells were transduced with this {beta}-G525C + Y618A ENaC construct (31). The mpkCCDcl4 cells that express {beta}-G525C + Y618A ENaCs were compared with control cells transduced with the {beta}-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-{beta}-ENaC antibodies could not clearly recognize the endogenous {beta}-ENaC subunit in mpkCCDcl4 transduced with GFP due to the low level of {beta}-ENaC expression. In cells transduced with the exogenous {beta}-G525C subunit, a clear band is detected at 94 kDa that corresponds to the apparent molecular mass of {beta}-ENaCs (14). Cells transduced with {beta}-G525C or {beta}-G525C + Y618A exhibit a higher level of expression of subunit protein compared to endogenous {beta}-ENaCs. Expression of the exogenous {beta}-G525C ENaC protein carrying the mutation {beta}-Y618A was slightly lower than the expression of the control {beta}-G525C ENaC subunit.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1. Expression of exogenous {beta}-G525C endothelial Na+ channel (ENaC) subunits in transduced mpkCCDcl4 cells. A: confluent cells transduced with {beta}-G525C ENaCs, {beta}-G525C + Y618A ENaCs, or with green fluorescent protein (GFP) as control were grown on filters and deprived of steroid hormones the day before the experiments. {beta}-ENaC subunit was immunoprecipitated with anti-{beta}-ENaC antibody, and preimmune serum (PIS) was used as control. B: sensitivity of short-circuit current (Isc) to amiloride was measured in the presence (open symbols) or absence (closed symbols) of aldosterone in confluent mpkCCDcl4 cells transduced with GFP ({blacktriangleup}, {triangleup}), {beta}-G525C ENaCs ({bullet},{circ}), or {beta}-G525C + Y618A ENaCs ({blacksquare},{square}). Fit of data ({blacktriangleup}) to Langmuir isotherms yielded an amiloride IC50 of 0.2 µM; data ({bullet}) and ({blacksquare}) were fitted assuming a biphasic dose-response relationship and yielded an IC50 of 0.2 µM for the high-affinity component of amiloride block and 250 µM for the low-affinity component. Each point represents 5-7 different filters. C: single-channel recording in mpkCCDcl4 cells transduced with {beta}-G525C + Y618A ENaCs. Patches were obtained from the apical membrane in the outside-out configuration. Recording was performed at a voltage (Vpipet) of -100 mV with Li+ ions in the extracellular bath as charge carrier. Channel openings correspond to downward deflections. Dark line above the trace indicates the period when benzamil (500 µM) was added to the external side of the channel.

 

The {beta}-ENaC subunits cannot form functional homomeric channels at the cell surface (12). To confer amiloride resistance to functional ENaCs, the {beta}-G525C ENaC subunits have to coassemble with the endogenous {alpha}- and {gamma}-ENaC subunits. Only ENaC channels that contain an exogenous {beta}-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 {beta}-G525C or {beta}-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 {beta}-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 {beta}-G525C or the {beta}-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 {beta}-ENaC subunit. The titration curves of the Isc carried by the {beta}-G525C or the {beta}-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 {beta}-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 {beta}-G525C or {beta}-G525C + Y618A subunits relative to the endogenous ENaCs is identical.

The single-channel characteristics of the {beta}-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 {alpha},{beta}-G525C {gamma}-ENaC channel (32). Taken together, these experiments demonstrate that exogenous {beta}-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 {beta}-ENaC mutants respond to aldosterone. To test whether the functional ENaC that contains transduced {beta}-ENaC subunits responds to aldosterone, mpkCCDcl4 cells transduced with GFP, {beta}-G525C, or {beta}-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 {beta}-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 {beta}-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 {beta}-ENaC or exogenous {beta}-G525C ENaC subunits respond to aldosterone. Cells that express the Liddle {beta}-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 {beta}-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 {beta}-G525C + Y618A mutant retains its ability to respond to aldosterone.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2. ENaC mutants transduced in mpkCCDcl4 cells respond to aldosterone. Benzamil (500 µM)-sensitive Isc (Isc benz) was measured in the absence and 5 h after addition of aldosterone (10-6 M) in mpkCCDcl4 cells transduced with GFP, {beta}-G525C ENaCs, and {beta}-G525C + Y618A ENaCs. Hatched portions of bars represent the fraction of Isc benz due to the endogenous channels that was sensitive to amiloride (10 µM). Transepithelial voltages (in mV) in the absence and presence of aldosterone were, respectively, -18.6 ± 7 and -55.2 ± 5.4 for GFP, -18.7 ± 6.4 and -88.2 ± 13.5 for {beta}-G525C, and -51.9 ± 8.5 and -86 ± 15 for {beta}-G525C + Y618A. Transepithelial resistance (in k{Omega}) did not significantly change with aldosterone and ranged from 1.3 ± 0.1 to 3.4 ± 0.8 in the different experiments. Bars represent the means ± SE of 9 different filters; *P < 0.05 vs. untreated cells.

 

We have generated different cell lines that express {beta}-or {gamma}-ENaC mutants and cause Liddle's syndrome: the missense mutation {beta}-P616L, {beta}-ENaC with the COOH-terminal truncation mutation at position {beta}-R564 ({beta}-R564stop), and the corresponding truncated K570stop in the {gamma}-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 {beta}-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 {beta}-ENaCs all show a higher baseline Isc than the respective control mpkCCDcl4 cells. The aldosterone-induced absolute increase in Isc ({Delta}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 {beta}- and {gamma}-ENaC truncation mutants in the COOH terminus. According to functional and biochemical studies, these mutations in the PY motif of {beta}- and {gamma}-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 {beta}-G525C and {beta}-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 {beta}-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 {beta}-ENaC mutants, the rate of Isc increase ({Delta}Isc/{Delta}t, in µA/min) was comparable to that in its respective control cells transduced with {beta}-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 {beta}-and {gamma}-ENaC mutant subunits lacking the PY motif.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3. Effects of aldosterone and recovery from stimulation in mpkCCDcl4 cells that express {beta}-G525C or {beta}-G525C + Y618A ENaC subunits. A: representative traces of Isc recordings performed on cells transfected with {beta}-G525C ENaCs (solid black line, left axis) and {beta}-G525C + Y618A ENaCs (solid gray line, right axis) after the addition of aldosterone (10-6 M) at time = 0. Isc was recorded in the presence of amiloride (10 µM). Fraction of the Isc sensitive to amiloride (10 µM) corresponded to 10 and 15% of the benzamil-inhibitable Isc for {beta}-G525C and {beta}-G525C + Y618A ENaCs, respectively (not shown). Time when benzamil (500 µM) was added is indicated (arrow). Isc in cells transfected with {beta}-G525C and {beta}-G525C + Y618A ENaCs in the absence of aldosterone are indicated by dotted black and gray lines, respectively. B: time course of Isc changes after aldosterone removal (circles) or after the addition (arrow, time = 0) of 0.5 mg/ml cycloheximide (triangles) to mpkCCDcl4 cells transduced with {beta}-G525C (closed symbols) or {beta}-G525C + Y618A ENaCs (open symbols) constructs. Isc was recorded in the presence of amiloride (10 µM). Values are means ± SE from 4-9 separate experiments.

 

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 {beta}-G525C ENaCs than in cells that expressed the {beta}-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 {beta}-G525C + Y618A and the {beta}- and {gamma}-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 {beta}-G525C + Y618A mutant, and 650 min for the {beta}-and {gamma}-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.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 4. Isc recordings during the early aldosterone response in mpkCCDcl4 cells that express control and Liddle's mutant ENaCs. A: mean Isc values in cells that express {beta}-G525C + Y618A ENaCs (top trace, {blacksquare}, n = 6) or control {beta}-G525C ENaC cells (bottom trace, {bullet}, n = 4) were determined in the presence of amiloride (10 µM) and after stimulation by aldosterone (10-6 M) at time = 0. Fit of data (thin solid lines) was obtained according to equation described (see MATERIALS AND METHODS), where Isc measured is the sum of two components, the rate of appearance of newly active ENaC {Delta}Isc/{Delta}t (in µA/min), and the rate of ENaC disappearance (kremove, in min-1) at the cell surface. Fit parameters were {Delta}Isc/{Delta}t = 0.12 µA/min before and 0.6 µA/min after aldosterone stimulation in control and {beta}-G525C + Y618A mutants that express mpkCCDcl4 cells; kremove remained unchanged by aldosterone and was 0.012 min-1 in control cells and 0.0017 min-1 to account for the higher basal Isc in cells that express {beta}-G525C + Y618A mutant. B: fit of the Isc increase measured under the same conditions in response to aldosterone stimulation in cells that express both {beta}- and {gamma}-COOH-terminal truncated mutant (top trace, {blacksquare}) and control {beta}-G525C (bottom trace, {bullet}) ENaCs. Fit parameters were {Delta}Isc/{Delta}t increase from 0.06 to 0.25 µA/min in response to aldosterone in both cells; kremove = 0.011 min-1 for the control cells and 0.0009 min-1 for cells that express the mutant; kremove did not change in response to aldosterone. Gray lines and points represent the average of four Isc measures taken every 20 s in n = 4 experiments. Error bars show SE.

 

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 {beta}-Y618A mutant, whereas in control cells, Isc returned to baseline values. This stronger and prolonged response to aldosterone by the Liddle's {beta}-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.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5. Isc in mpkCCDcl4 cells transduced with wild-type ({bullet}) and {beta}-Y618A ({blacksquare}) ENaCs during the late aldosterone response. Cells were grown to confluent monolayers and were maintained 16 h before Isc measurements were obtained in a low-apical Na+ (10 mM) medium with amiloride (10 µM). Aldosterone (10-6 M) was added at time 0. At times 0 and 4 and 16 h, instantaneous Isc was measured in a 100 mM apical Na+ medium without amiloride. Each point represents a mean of 4-8 Isc measurements.

 

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 {beta}-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 {beta}-G525C subunit (Fig. 6). In cells that express the {beta}-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 {beta}-G525C + Y618A mutant ENaCs respond to stimulation by vasopressin.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6. Effects of vasopressin on mpkCCDcl4 cells that express {beta}-G525C or {beta}-G525C + Y618A ENaCs. Benzamil (500 µM)-sensitive Isc was measured under basal conditions and 100 min after stimulation by vasopressin (10-9 M). Hatched portions of the bars represent the fraction of Isc due to the endogenous channels that were sensitive to amiloride (10 µM). Bars represent means ± SE of 8-10 different filters. *P < 0.05 vs. untreated cells.

 

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 {beta}-G525C and {beta}-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 {beta}-G525C + Y618A mutant, or the double-{beta}- and {gamma}-ENaC COOH-terminal truncated mutants. All three cell lines responded to vasopressin and exhibited a similar increase in the magnitude of Isc ({Delta}Isc). In the mpkCCDcl4 cells that were transduced with GFP or that express control {beta}-G525C and {beta}-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 {beta}- and {gamma}-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 {beta}-G525C ENaCs, the {beta}-Y618A ENaC mutation does not seem to greatly affect the ENaC response to vasopressin. Therefore, the PY motif in the COOH terminus of {beta}- and {gamma}-ENaCs does not seem to be required for ENaC stimulation by vasopressin. However, in cells that express the double-{beta}- and -{gamma} 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 {beta}- or {gamma}-ENaCs other than the PY motif.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 7. Effects of vasopressin or forskolin on Isc in cells that express Liddle's mutant ENaCs. A: Isc was measured in the presence of amiloride (10 µM) after addition of vasopressin (10-9 M; arrow) to cells transduced with {beta}-G525C (black line, left axis) or {beta}-G525C + Y618A (gray line, right axis) ENaCs. At the end of the experiment, benzamil (500 µM) was added to the apical side of the cells. B: similar Isc recording in confluent mpkCCDcl4 cells transduced with {beta}-G525C (black line, left axis)or {beta}-G525C + Y618A ENaC (gray line, right axis) after addition of forskolin (10 µM; arrow). C: time course of Isc recovery from vasopressin stimulation. Arrow denotes the time when hormone was removed. Half-lives for Isc decreases were 35.42 ± 8.9 min for the control cells ({bullet}) and 245.3 ± 45.3 min for the {beta}-G525C + Y618A ({blacktriangleup}) mutant. Each point represents the mean of n = 4-6 experiments; error bars show SE.

 

View this table:
[in this window]
[in a new window]
 
Table 2. Effects of vasopressin on Isc in transduced mpkCCDcl4 cells

 

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 {beta}-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 {beta}-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 {beta}- or {gamma}-ENaCs might contribute the vasopressin response.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our study demonstrates that ENaC mutant subunits can be expressed and analyzed functionally in a differentiated mammalian collecting duct cell line that responds to aldosterone and vasopressin. We have generated mpkCCDcl4 cells that express {beta}-and {gamma}-ENaC subunits with mutations in the conserved PY motif of the cytoplasmic COOH terminus. Consistent with hyperactive ENaC mutants that cause Liddle's syndrome, these cells are characterized by a higher baseline Isc due to a longer half-life of ENaC activity at the cell surface. Despite their higher basal Isc, the cells transduced with ENaC mutants that cause Liddle's syndrome still responded to aldosterone with an increase in Na+ transport that was similar in magnitude during the first hours of stimulation. Furthermore, the kinetics of activation of the Na+ transport by aldosterone were comparable in cells transduced with control or Liddle's mutant, which indicates that Liddle's mutant ENaCs retain their sensitivity to the hormone. Cells that express ENaC mutants differ from control cells by a prolonged response to aldosterone due to retention of active ENaCs at the cell surface. Similar observations were done on Na+ transport stimulated by vasopressin.

Transduced mpkCCDcl4 cells that express amiloride-resistant ENaC mutants. The exogenous rat {beta}-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 {beta}-G525C ENaC subunit was therefore constitutively expressed and not transcriptionally regulated by aldosterone in the mpkCCDcl4 cells as in the endogenous {beta}-ENaCs. The cells transduced with the exogenous {beta}-G525C ENaCs showed a higher expression of {beta}-ENaC subunits at the protein level without a corresponding change in the basal level of Isc. The reason is that {beta}-ENaC subunits alone do not form homomeric functional channels at the cell surface; only the heteromeric {alpha},{beta},{gamma}-ENaC is functional (5). Thus it is likely that in mpkCCDcl4 cells transduced with and overexpressing the {beta}-G525C subunits, {alpha}- and {gamma}-ENaC subunits become limiting for expression of ENaC activity at the cell surface. Accordingly, overexpression of {beta}-ENaCs only will not increase the level of baseline Isc.

Despite the fact that the exogenous {beta}-G525C ENaC subunit is constitutively expressed in the mpkCCDcl4 cells, the channels made of the transduced {beta}-ENaC subunit respond to aldosterone. In target epithelia, the stimulation of Na+ transport by aldosterone requires de novo protein synthesis of {alpha}-ENaCs as well as other aldosterone-induced proteins that regulate ENaC activity (23, 28). In the intact distal nephron, aldosterone induces the expression of {alpha}-ENaC, whereas {beta}- and {gamma}-ENaCs are constitutively expressed (11). Thus in the mpkCCDcl4 cells transduced with the exogenous {beta}-G525C subunit, the newly synthesized {alpha}-ENaC subunits in response to aldosterone stimulation likely assemble with the available {beta}- and {gamma}-ENaC subunits; in the case of cells that overexpress the {beta}-G525C ENaC subunit, channel assembly will occur preferentially with the exogenous {beta}-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 {beta}-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 {beta}-G525C ENaCs have incorporated an exogenous mutant {beta}-ENaC subunit and responded to aldosterone.

mpkCCDcl4 cells that express {beta}-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 {beta}-and {gamma}-ENaC subunits.

The PY motifs of {beta}- or {gamma}-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 {beta}- and {gamma}-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 {beta}-G525C or {beta}-G525C + Y618A ENaC subunits. Recovery of Isc from aldosterone stimulation showed that the active {beta}-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 {alpha}-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 {alpha}-ENaCs (30, 31). This relatively small effect of {alpha}-ENaC mutations correlates with the reduced ability of the PY motif of {alpha}-ENaCs to interact with WW domains Nedd4-1, whereas mutations in the PY motif of {beta}- or {gamma}-ENaCs impair most of the ability to bind this Nedd4 protein (40).

Aldosterone response of Liddle's mutant ENaCs. In the cells that express {beta}- and {gamma}-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 {beta}-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 {beta}-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 {beta}- and {gamma}-ENaCs is required for the ability of the channel to respond to aldosterone. Therefore, the phosphorylation of the COOH terminus of {beta}-and {gamma}-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 {alpha},{beta},{gamma}-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 {beta}-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 {beta}-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 {beta}-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 {beta}- and {gamma}-ENaC mutant show a slightly attenuated response to vasopressin, which suggests the possibility that sites in the COOH termini of {beta}- and {gamma}-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 {beta}- and {gamma}-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.


    ACKNOWLEDGMENTS
 
The authors thank J.-D. Horisberger and B. R. Rossier for helpful discussion and critical review of the manuscript.

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.


    FOOTNOTES
 

Address for reprint requests and other correspondence: L. Schild, Institut de Pharmacologie et Toxicologie, rue du Bugnon 27, CH-1005 Lausanne (E-mail: Laurent.Schild{at}ipharm.unil.ch).

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. 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.[Abstract/Free Full Text]
  2. Alvarez de la Rosa DA, Li H, and Canessa CM. Effects of aldosterone on biosynthesis, traffic, and functional expression of epithelial sodium channels in A6 cells. J Gen Physiol 119: 427-442, 2002.[Abstract/Free Full Text]
  3. 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.[Abstract/Free Full Text]
  4. Botero-Velez M, Curtis J, and Warnock DG. Liddle's syndrome revisited—a disorder of sodium reabsorption in the distal tubule. N Engl J Med 330: 178-181, 1994.[Free Full Text]
  5. Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, and Rossier BC. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367: 463-467, 1994.[ISI][Medline]
  6. Chen L, Williams SK, and Schafer JA. Differences in synergistic actions of vasopressin and deoxycorticosterone in rat and rabbit CCD. Am J Physiol Renal Fluid Electrolyte Physiol 259: F147-F156, 1990.[Abstract/Free Full Text]
  7. 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.[Abstract/Free Full Text]
  8. Debonneville C, Flores SY, Kamynina E, Plant PJ, Tauxe C, Thomas MA, Munster C, Chraibi A, Pratt JH, Horisberger JD, Pearce D, Loffing J, and Staub O. Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na+ channel cell surface expression. EMBO J 20: 7052-7059, 2001.[Abstract/Free Full Text]
  9. Duc C, Farman N, Canessa CM, Bonvalet JP, and Rossier BC. Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J Cell Biol 127: 1907-1921, 1994.[Abstract]
  10. Ecelbarger CA, Kim GH, Terris J, Masilamani S, Mitchell C, Reyes I, Verbalis JG, and Knepper MA. Vasopressin-mediated regulation of epithelial sodium channel abundance in rat kidney. Am J Physiol Renal Physiol 279: F46-F53, 2000.[Abstract/Free Full Text]
  11. Escoubet B, Coureau C, Bonvalet JP, and Farman N. Noncoordinate regulation of epithelial Na channel and Na pump subunit mRNAs in kidney and colon by aldosterone. Am J Physiol Cell Physiol 272: C1482-C1491, 1997.[Abstract/Free Full Text]
  12. Firsov D, Schild L, Gautschi I, Mérillat AM, Schneeberger E, and Rossier BC. Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach. Proc Natl Acad Sci USA 93: 15370-15375, 1996.[Abstract/Free Full Text]
  13. Geller DS, Farhi A, Pinkerton N, Fradley M, Moritz M, Spitzer A, Meinke G, Tsai FTF, Sigler PB, and Lifton RP. Activating mineralocorticoid receptor mutation in hypertension exacerbated by pregnancy. Science 289: 119-123, 2000.[Abstract/Free Full Text]
  14. Grunder S, Firsov D, Chang SS, Jaeger NF, Gautschi I, Schild L, Lifton RP, and Rossier BC. A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel. EMBO J 16: 899-907, 1997.[Abstract/Free Full Text]
  15. Hansson JH, Nelson-Williams C, Suzuki H, Schild L, Shimkets R, Lu Y, Canessa C, Iwasaki T, Rossier B, and Lifton RP. Hypertension caused by a truncated epithelial sodium channel gamma subunit: genetic heterogeneity of Liddle syndrome. Nat Genet 11: 76-82, 1995.[ISI][Medline]
  16. Hansson JH, Schild L, Lu Y, Wilson TA, Gautschi I, Shimkets R, Nelson-Williams C, Rossier BC, and Lifton RP. A de novo missense mutation of the beta subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity. Proc Natl Acad Sci USA 92: 11495-11499, 1995.[Abstract]
  17. Hanwell D, Ishikawa T, Saleki R, and Rotin D. Trafficking and cell surface stability of the epithelial Na+ channel expressed in epithelial Madin-Darby canine kidney cells. J Biol Chem 277: 9772-9779, 2002.[Abstract/Free Full Text]
  18. Kamynina E, Debonneville C, Bens M, Vandewalle A, and Staub O. A novel mouse Nedd4 protein suppresses the activity of the epithelial Na+ channel. FASEB J 15: 204-214, 2001.[Abstract/Free Full Text]
  19. Liddle GW, Bledsoe T, and Coppage WS. A familial renal disorder simulating primary aldosteronism but with negligible aldosterone secretion. Trans Assoc Am Physicians 76: 199-213, 1963.[ISI]
  20. Lifton RP. Molecular genetics of human blood pressure variation. Science 272: 676-680, 1996.[Abstract]
  21. Loffing J, Zecevic M, Feraille E, Kaissling B, Asher C, Rossier BC, Firestone GL, Pearce D, and Verrey F. Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK. Am J Physiol Renal Physiol 280: F675-F682, 2001.[Abstract/Free Full Text]
  22. Marunaka Y and Eaton DC. Effects of vasopressin and cAMP on single amiloride-blockable Na channels. Am J Physiol Cell Physiol 260: C1071-C1084, 1991.[Abstract/Free Full Text]
  23. 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 alpha subunit in A6 renal cells. J Am Soc Nephrol 8: 1813-1822, 1997.[Abstract]
  24. Morris RG and Schafer JA. cAMP increases density of ENaC subunits in the apical membrane of MDCK cells in direct proportion to amiloride-sensitive Na+ transport. J Gen Physiol 120: 71-85, 2002.[Abstract/Free Full Text]
  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.[Abstract/Free Full Text]
  26. 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]
  27. Pear WS, Nolan GP, Scott ML, and Baltimore D. Production of high titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci USA 90: 8392-8396, 1993.[Abstract/Free Full Text]
  28. Robert-Nicoud M, Flahaut M, Elalouf JM, Nicod M, Salinas M, Bens M, Doucet A, Wincker P, Artiguenave F, Horisberger JD, Vandewalle A, Rossier BC, and Firsov D. Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin. Proc Natl Acad Sci USA 98: 2712-2716, 2001.[Abstract/Free Full Text]
  29. Rotin D, Kanelis V, and Schild L. Trafficking and cell surface stability of ENaC. Am J Physiol Renal Physiol 281: F391-F399, 2001.[Abstract/Free Full Text]
  30. Schild L, Canessa CM, Shimkets RA, Gautschi I, Lifton RP, and Rossier BC. A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system. Proc Natl Acad Sci USA 92: 5699-5703, 1995.[Abstract]
  31. Schild L, Lu Y, Gautschi I, Schneeberger E, Lifton RP, and Rossier BC. Identification of a PY motif in the epithelial Na channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome. EMBO J 15: 2381-2387, 1996.[Abstract]
  32. Schild L, Schneeberger E, Gautschi I, and Firsov D. Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation. J Gen Physiol 109: 15-26, 1997.[Abstract/Free Full Text]
  33. Shi HK, Asher C, Chigaev A, Yung Y, Reuveny E, Seger R, and Garty H. Interactions of beta and gamma ENaC with Nedd4 can be facilitated by an ERK-mediated phosphorylation. J Biol Chem 277: 13539-13547, 2002.[Abstract/Free Full Text]
  34. Shimkets RA, Lifton R, and Canessa CM. In vivo phosphorylation of the epithelial sodium channel. Proc Natl Acad Sci USA 95: 3301-3305, 1998.[Abstract/Free Full Text]
  35. Shimkets RA, Lifton RP, and Canessa CM. The activity of the epithelial sodium channel is regulated by clathrin-mediated endocytosis. J Biol Chem 272: 25537-25541, 1997.[Abstract/Free Full Text]
  36. Shimkets RA, Warnock DG, Bositis CM, Nelson-Williams C, Hansson JH, Schambelan M, Gill JR Jr, Ulick S, Milora RV, Findling JW, Canessa CM, Rossier BC, and Lifton RP. Liddle's syndrome: heritable human hypertension caused by mutations in the beta subunit of the epithelial sodium channel. Cell 79: 407-414, 1994.[ISI][Medline]
  37. 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.[Abstract/Free Full Text]
  38. Snyder PM, Olson DR, and Thomas BC. Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na+ channel. J Biol Chem 277: 5-8, 2002.[Abstract/Free Full Text]
  39. Snyder PM, Price MP, McDonald FJ, Adams CM, Volk KA, Zeiher BG, Stokes JB, and Welsh MJ. Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na+ channel. Cell 83: 969-978, 1995.[ISI][Medline]
  40. Staub O, Dho S, Henry PC, Correa J, Ishikawa T, McGlade J, and Rotin D. WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome. EMBO J 15: 2371-2380, 1996.[Abstract]
  41. Staub O, Gautschi I, Ishikawa T, Breitschopf K, Ciechanover A, Schild L, and Rotin D. Regulation of stability and function of the epithelial Na channel (ENaC) by ubiquitination. EMBO J 16: 6325-6336, 1997.[Abstract/Free Full Text]
  42. Sudol M, Chen HI, Bougeret C, Einbond A, and Bork P. Characterization of a novel protein binding module—the WW domain. FEBS Lett 369: 67-71, 1995.[ISI][Medline]
  43. Tamura H, Schild L, Enomoto N, Matsui N, Marumo F, Rossier BC, and Sasaki S. Liddle disease caused by a missense mutation of beta subunit of the epithelial sodium channel gene. J Clin Invest 97: 1780-1784, 1996.[Abstract/Free Full Text]
  44. Vandewalle A, Bens M, and Van Huyen JPD. Immortalized kidney epithelial cells as tools for hormonally regulated ion transport studies. Curr Opin Nephrol Hypertens 8: 581-587, 1999.[ISI][Medline]
  45. Verrey F, Hummler E, Schild L, and Rossier BC. Control of Na+ transport by aldosterone. In: The Kidney, edited by Seldin DW and Giebisch G. Philadelphia, PA: Lippincott Williams & Wilkins, 2001, p. 1441-1472.
  46. Wang J, Barbry P, Maiyar AC, Rozansky DJ, Bhargava A, Leong M, Firestone GL, and Pearce D. SGK integrates insulin and mineralocorticoid regulation of epithelial sodium transport. Am J Physiol Renal Physiol 280: F303-F313, 2001.[Abstract/Free Full Text]