Epithelial Na channel mutants causing Liddle

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作者：Muriel Auberson, Nicole Hoffmann-Pochon, A. Vandewalle, Stephan Kellenberger,  Laurent Schild作者单位：1 Institut de Pharmacologie et Toxicologie,Université de Lausanne, CH-1005 Lausanne, Switzerland; and Institut National de la Santé et de laRecherche Médicale, Unité 47 Faculté de MédecineXavier Bichat, 75870 Paris Cedex France
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2 o7 l! [+ T  c# K          【摘要】, G8 X! Q2 }3 X( ]: c' m8 j6 I# c' E
      Liddle's syndrome is a monogenic form of hypertension caused by mutationsin the PY motif of the COOH terminus of - and -epithelialNa   channel (ENaC) subunits. These mutations lead to retention ofactive channels at the cell surface. Because of the critical role of this PYmotif in the stability of ENaCs at the cell surface, we have investigated itscontribution to the ENaC response to aldosterone and vasopressin. Mutants ofthe PY motif in - and -ENaC subunits ( -Y618A, -P616L, -R564stop, and -K570stop) were stably expressed by retroviralgene transfer in a renal cortical collecting duct cell line(mpkCCD cl4 ), and transepithelial Na   transport wasassessed by measurements of the benzamil-sensitive short-circuit current( I sc ). Cells that express ENaC mutants of the PY motifshowed a five- to sixfold higher basal I sc compared withcontrol cells and responded to stimulation by aldosterone(10 - 6 M) or vasopressin (10 - 9 M)with a further increase in I sc. The rates of the initial increases in I sc after aldosterone or vasopressinstimulation were comparable in cells transduced with wild-type and mutant ENaCs, but reversal of the effects of aldosterone and vasopressin was slowerin cells that expressed the ENaC mutants. The conserved sensitivity of ENaCmutants to stimulation by aldosterone and vasopressin together with theprolonged activity at the cell surface likely contribute to the increasedNa   absorption in the distal nephron of patients with Liddle'ssyndrome. : d! v6 ?8 `; b1 I% q
          【关键词】 collecting duct PY motif hypertension2 q9 U" B! t6 E5 L1 K2 R
                  REGULATION OF NA   AND WATER homeostasis by the distalnephron of the kidney is critical for the maintenance of normal arterial bloodpressure. The recent identification of mutations in genes that encode theepithelial Na   channel (ENaC), the mineralocorticoid receptor (MR),and the 11 -hydroxysteroid dehydrogenase that causes monogenic forms ofhypertension strongly supports this notion( 13, 20, 36 ).2 g/ x$ w7 ^% T" |
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The ENaCs in the apical membrane of the kidney connecting tubule andcortical collecting duct allow vectorial Na   absorption from thetubule lumen ( 45 ). ENaCactivity is under the control of aldosterone and vasopressin in response tostimuli such as volume contraction, salt depletion, or hyperkalemia.! S+ F2 _- X: f  I4 O, E% b( g# [
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Liddle's syndrome is a Mendelian form of low plasma renin hypertension dueto inappropriate Na   absorption in the distal nephron ( 19 ). Genetic linkage studiesin families of Liddle's syndrome patients permitted the identification ofpathogenic mutations in the - and -subunits of ENaCs( 15, 16, 36, 43 ). These mutations, bydeleting or modifying a conserved PY motif (PPPxY sequence) in the cytoplasmicCOOH terminus of -and -ENaCs, cause an increase in ENaC activityand channel stability at the surface of target cells( 31, 39 ). Proline-rich motifs aregenerally involved in protein-protein interactions( 42 ). In the case of -and -ENaC subunits, the PY motif can bind to the WW domain of theubiquitin protein ligase Nedd4-2( 18 ). In the Xenopus oocyte expression system, this binding interaction leads to ENaCubiquitination, which tags the channel for endocytosis and degradation.Consistent with these observations, mutations in the PY motif result indeficient ubiquitination of ENaC and lead to retention at the cell surface( 41 ). A second model proposesthat the PY motif is part of an endocytic motif that is recognized by theclathrin-adaptor protein (AP)-2 complex; this model does not preclude theinvolvement of Nedd4-2 in regulation of channel activity at the cell surface( 35 ). Although the primarymechanism of internalization and degradation of ENaC in the cell has not yetbeen clearly established, mutagenesis experiments have demonstrated that thePY motif in - and -ENaC subunits represents a criticaldeterminant of channel activity at the cell surface.0 U! T. z8 r% y! u
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The stimulation of Na   absorption in the distal nephron by aldosterone is mainly due to an increase in the number of active ENaCs at thecell surface ( 21, 26 ). This upregulation of ENaC by aldosterone involves transcriptional and posttranscriptional events thatare initiated on the binding of aldosterone to its cytosolic receptor andsubsequent translocation of the complex to the nucleus( 45 ). Because the PY motif inthe COOH terminus of - and -ENaC subunits seems to control ENaCdensity at the cell surface, this regulatory domain represents a potential target site for the ENaC response to aldosterone stimulation ( 29 ). In support of thishypothesis, recent experiments on the Xenopus oocyte have shown thatthe aldosterone-induced serum and glucocorticoid-regulated kinase (SGK) canmodulate the interaction between Nedd4-2 and the PY motif of ENaC( 8 ). Vasopressin alsostimulates Na   absorption in cultured epithelia by increasing thedensity of active ENaCs at the cell surface ( 24 ). The cAMP-mediated effectof vasopressin might also involve the PY motif of ENaCs as the target site forthe modulation of channel stability at the cell surface( 37 ). According to theserecent data, ENaC mutants that cause Liddle's syndrome would be lessresponsive or even unresponsive to stimulation by aldosterone and/orvasopressin.9 N- B" ~5 E9 Q4 w
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To address the question of the contribution of the PY motif of the -and -ENaC subunits in the ENaC response to aldosterone or vasopressinstimulation, we have stably expressed mutants of the - and -ENaCsubunits in the mpkCCD cl4 cortical collecting duct cells, which isa cell line that responds to aldosterone and vasopressin. We show that thesecells, which stably express - and -ENaC mutants of the PY motif,exhibit a higher benzamil-sensitive Na   current that is consistentwith the ENaC gain-of-function phenotype in Liddle's syndrome. The ENaCchannels with mutations in the PY motif retain the ability to mediate anincreased Na   absorption in response to aldosterone or vasopressinstimulation. These findings are consistent with early clinical observations made by Liddle, who found that patients with pseudoaldosteronism (Liddle'ssyndrome) responded to a challenge with aldosterone by a decrease in renalNa   excretion( 19 ).
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# J7 r2 Q9 f8 L2 E# N7 X1 BMATERIALS AND METHODS
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Vector construction. The Epstein-Barr virus-based retroviral vector (LZRS) was kindly provided by G. Nolan and was used for transfection ofthe Phoenix retrovirus producer cells( 27 ). This vector takes upsemistable residence as episomes within the virus producer cell line. The rat - and -subunits of ENaC cDNAs were subcloned into the LZRSvector. The -cDNA was modified by the addition of the -G525Cmutation, which causes low affinity for amiloride block with or withoutdifferent Liddle's mutations (Y618A, P616L, or R564stop; Ref. 32 ). In addition, the K570stop mutation was introduced in -ENaCs( 15 ).9 j0 T8 A. d8 g1 ?% D

! l" n6 t% k+ E( s$ |# e* PTransfection of retrovirus producer cells. Virus producer cells (Phoenix cells) were grown in DMEM that contained 10% FCS, 100 U/mlpenicillin, and 100 µg/ml streptomycin. When transfected with the LZRSvector, Phoenix cells were capable of producing recombinant retroviruses after48 h. Transfections of the retrovirus producer Phoenix cells with the LZRSvectors 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 particlesproduced by the packaging cells were harvested from the supernatant 4-5 daysafter transfection and were filtered through a 0.45-µm filter.
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Cell infection. We used mpkCCD cl4 cells, which are aclone of principal cells that have been derived from microdissected corticalcollecting ducts of a transgenic mouse( 3 ). Cells were routinely grownon plastic tissue-culture flasks in a modified Ham's F-12 medium (LifeTechnologies) 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, pH7.4, 100 U/ml penicillin, and 100 µg/ml streptomycin. The recombinantviruses harvested from the Phoenix producer cells were used immediately fortransduction of the mpkCCD cl4 cells. After 24 h, the mediumcontaining the viruses was replaced with fresh modified Ham's F-12 medium.Delivery of more than one ENaC-subunit gene in the mpkCCD cl4 cellswas obtained by performing sequential infections. Expression of the exogenousENaC subunit proteins remained stable in the 12passages after infection. Cells were studied between passages 32 and 42.' w8 k7 q. j- s* F9 G' ?! C7 {, |

/ c7 m) Q! E) J; a0 m: p! gElectrophysiological studies. Electrophysiological studies were performed on confluent cell monolayers grown on collagen-coated filters(Transwell 0.4-µm pore, 4.7 cm 2 or Snap-well 0.4-µm pore, 1cm 2; Corning Costar, Cambridge, MA). Cells were maintained for 5days in the modified Ham's F-12 medium described above and then transferred ina medium of identical composition but deprived of EGF, transferrin, and FSC.Before measurements, filters were maintained overnight in Ham's F-12 mediumsupplemented with 11 mM D -glucose, 100 U/ml penicillin, and 100µg/ml streptomycin. Transepithelial short-circuit currents( I sc ) were recorded on confluent mpkCCD cl4 cells grown on filters and mounted in Ussing chambers. The epithelium wasmaintained under a current-clamp condition instead of a voltage-clampcondition to avoid high transepithelial Na   flux, which couldsaturate the transport capacities of the cells. The I sc (µA/cm 2 ) and the transepithelial resistance (k ) werecalculated from ±10-µA pulses of 20-ms duration elicited by acomputer-controlled voltage-clamp apparatus (Physiological Instruments, SanDiego, CA). The amiloride-sensitive I sc defines the I sc sensitive to 10 µM amiloride carried byNa   ions through endogenously expressed ENaC channels. Theamiloride-resistant I sc defines the I sc sensitive to 500 µM benzamil but resistant to 10µM amiloride and represents I sc carried byNa   ions through ENaC with the -G525C mutation.Electrophysiological recordings were performed in symmetrical solutions thatcontained (in mM) 120 NaCl, 5 KCl, 25 NaHCO 3, 1 sodium pyruvate,0.9 sodium phosphate, 10 glucose, and 1 MgCl 2 or in Ham's F-12 medium supplemented with 11 mM D -glucose, 100 U/ml penicillin, and100 µg/ml streptomycin, both bubbled with 95% O 2 -5% CO 2.9 V) B! `6 c! m( C
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The patch-clamp technique in the outside-out configuration was used tomeasure ENaC activity from confluent mpkCCD cl4 cells grown ontransparent filters. The extracellular (bath) solution contained (in mM) 135lithium or sodium methanesulfonate, 2 CaCl 2, 1 MgCl 2, 5BaCl 2, 10 HEPES, and 2 glucose, pH 7.4. The pipette solutioncontained (in mM) 103 potassium aspartate, 7 KCl, 20 CsOH, 20tetraethylammonium chloride, 5 EGTA, and 10 HEPES, pH 7.4 (with KOH).
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' u' L4 B  |0 f; Q2 G+ iImmunoprecipitation studies. Immunoprecipitation studies were doneusing polyclonal-specific anti- antibodies( 9 ). Cells were grown onfilters for 5 days in modified Ham's F-12 and 5 days in the same medium butdeprived of EGF, transferrin, and FCS. Filters were rinsed three times in amethionine-free medium and pulsed at 37°C for 30 min with 200 µl of methionine-free medium that contained 1 mCi/ml [ 35 S]methionine added to the basal side of inverted filters. Cells were washed two times withice-cold 1 x PBS, scraped on ice in 250 µl of lysis buffer (50 mMHEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl 2, 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. Theamount of incorporated [ 35 S]methionine was determined bytrichloroacetic acid (10%) precipitation and similar counts per minute were submitted to immunoprecipitation. The samples were incubated with 10 µl ofthe specific antibody and 25 µl of protein A-Sepharose beads overnight at4°C. The beads were centrifuged and washed five times with lysis buffer.The immunoprecipitated proteins were recovered in Laemmli sample buffer. Thesamples were boiled at 95°C for 5 min and then loaded on 5-10% SDS-polyacrylamide gradient gel. After electrophoresis, gels were fixed andtreated with sodium salicylate and exposed on X-Omat S films (Eastman Kodak)at -70°C for 24 h to 3 days.
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Data analyses. The changes in I sc induced byaldosterone were fitted to a simple model in which changes in I sc result from both the addition of newly active ENaCs atthe cell surface and the rate of ENaC removal ( k remove )from the cell surface. We assume that new channels are added at a constantrate that corresponds to a constant addition of current( I sc,new ).9 n) C7 e4 Y3 S3 e$ U( ]$ D3 E* r

7 I0 b0 C8 Z0 M! m) z! T1 T) wAt any given time, I sc is given by the equation
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# q7 g5 D, Y/ A# ~, x+ o1 Wwhere I sc( t ) is I sc at time t; I sc,new is the rate of I sc increase due to insertion of newly active ENaCs(expressed in µA/min); and k remove is the rate constantof I sc decrease due to removal of ENaCs from the cellsurface and is determined from the half-life of I sc decline after suppression of aldosterone stimulation (expressed inmin - 1; see Table1 ). The I sc half-life at the cell surface isgiven as t 1/2 = ln2 /k remove.  N6 P3 G7 x4 l/ E) f
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Table 1. Effects of aldosterone on I sc in transducedmpkCCD c/4 cells9 Y. O. J" @& C% T7 k1 o

# r* |, @: j# Q( m; xKnowing the k remove value, it was possible to determine I sc,new from the equilibrium baseline value of I sc considering that at the steady state, the number ofnew ENaCs added is equal to the number of ENaCs removed
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Data are expressed as means ± SE. Statistical differences betweengroups were calculated using Student's t -test.3 W0 M0 l1 I0 f. g2 F: u
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Stable expression of ENaC mutants in mpkCCD cl4 cells. We have used mpkCCD cl4 cells, an immortalized mousecortical collecting duct cell line, for the expression of the rat -ENaCsubunit carrying mutations in the PY motif of the COOH terminus. These cellsexpress an endogenous ENaC composed of the three -, -, and -subunits and exhibit amiloride-sensitive Na   currents thatrespond to stimulation by aldosterone and vasopressin ( 3, 44 ). As a control, we haveretrovirally transduced these cells with the rat -ENaC subunit carryingthe -G525C mutation in the amiloride-binding domain that confers to thefunctional heteromeric,, -ENaC channel a resistance toamiloride ( 32 ). This mutationwas introduced in the -ENaC sequence to enable us to functionallydistinguish the channels that contained the transduced -G525C ENaCsubunit from the endogenous ENaC expressed by the mpkCCD cl4 cells.. S; E6 E# I4 ^9 y4 v
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The missense mutation Y618H in the PY motif -ENaC is associated withLiddle's syndrome ( 43 ). Wehave introduced the -Y618A mutation in the -G525C mutantbackground, and cells were transduced with this -G525C   Y618A ENaCconstruct ( 31 ). ThempkCCD cl4 cells that express -G525C   Y618A ENaCs werecompared 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. 1 A show that inthe absence of aldosterone, anti- -ENaC antibodies could not clearlyrecognize the endogenous -ENaC subunit in mpkCCD cl4 transduced with GFP due to the low level of -ENaC expression. In cellstransduced with the exogenous -G525C subunit, a clear band is detectedat 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 ofsubunit protein compared to endogenous -ENaCs. Expression of theexogenous -G525C ENaC protein carrying the mutation -Y618A wasslightly lower than the expression of the control -G525C ENaCsubunit.
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Fig. 1. Expression of exogenous -G525C endothelial Na   channel(ENaC) subunits in transduced mpkCCD cl4 cells. A :confluent cells transduced with -G525C ENaCs, -G525C   Y618AENaCs, or with green fluorescent protein (GFP) as control were grown onfilters and deprived of steroid hormones the day before the experiments. -ENaC subunit was immunoprecipitated with anti- -ENaC antibody, andpreimmune serum (PIS) was used as control. B : sensitivity ofshort-circuit current ( I sc ) to amiloride was measured inthe presence (open symbols) or absence (closed symbols) of aldosterone inconfluent mpkCCD cl4 cells transduced with GFP (, ), -G525C ENaCs (, ), or -G525C   Y618A ENaCs(, ). Fit of data ( ) to Langmuir isotherms yielded anamiloride IC 50 of 0.2 µM; data ( ) and ( ) were fittedassuming a biphasic dose-response relationship and yielded an IC 50 of 0.2 µM for the high-affinity component of amiloride block and 250 µMfor the low-affinity component. Each point represents 5-7 different filters. C : single-channel recording in mpkCCD cl4 cells transducedwith -G525C   Y618A ENaCs. Patches were obtained from the apicalmembrane in the outside-out configuration. Recording was performed at avoltage ( V pipet ) of -100 mV with Li   ions inthe extracellular bath as charge carrier. Channel openings correspond todownward deflections. Dark line above the trace indicates the period whenbenzamil (500 µM) was added to the external side of the channel.( P& D% E. s, K

$ R) W2 T5 z) m, ^1 k0 C' Y$ jThe -ENaC subunits cannot form functional homomeric channels at thecell surface ( 12 ). To conferamiloride resistance to functional ENaCs, the -G525C ENaC subunits haveto coassemble with the endogenous - and -ENaC subunits. OnlyENaC channels that contain an exogenous -G525C ENaC subunit remainactive in the presence of inhibitory concentrations of amiloride (10 µM). Figure 1 B shows thedose-dependent amiloride inhibition curve of the I sc intransduced mpkCCD cl4 cells. The cells transduced with GFP exhibitan amiloride-sensitive I sc with an inhibitory constant(IC 50 ) of 0.2 µM, whereas the cells that express the exogenous -G525C or -G525C   Y618A ENaC subunits show a biphasic inhibitioncurve with a large amiloride-resistant component of I sc.In these latter cells, only 15% of the I sc was sensitiveto 10 µM amiloride, a concentration that maximally inhibited I sc carried by endogenous ENaCs in cells transduced withGFP ( Fig. 1 A ). Thelarge amiloride-resistant component of I sc (IC 50 = 250 µM) is carried by channels that have incorporatedthe -G525C ENaC subunit; this amiloride-resistant I sc component is completely inhibited by 500 µMbenzamil, which is a more potent analog of amiloride. In both cell lines thatexpress the -G525C or the -G525C   Y618A ENaC subunits, thefraction of the amiloride-sensitive I sc represents of the overall benzamil-blockable I sc, which indicatesthat 85% of the channels expressed have incorporated the exogenous -ENaC subunit. The titration curves of the I sc carried by the -G525C or the -G525C   Y618A ENaCs were similar inthe presence or absence of aldosterone, which indicates that the fraction ofactive endogenous ENaCs relative to ENaCs with an exogenous -subunitdoes not vary after aldosterone stimulation. These data also show that in thetransduced mpkCCD cl4 cells, the ratio of functional expression ofENaCs containing -G525C or -G525C   Y618A subunits relative to theendogenous ENaCs is identical.& \/ F; X  ]/ F0 ~: Z9 Z

/ \7 n9 F& ?6 j, y1 K+ i/ jThe single-channel characteristics of the -G525C   Y618A ENaC mutantwere verified by the patch-clamp technique in transduced cells grown on afilter. As shown in Fig.1 C, the ENaC activity was detected in outside-out patchesfrom the apical membrane, and channel activity was determined by the productof the number of channels ( N ) times the open probability( P o ). The channel activity in this patch ( N x P o = 0.453) was not affected by benzamil at aconcentration of 0.5 µM ( N x P o = 0.437, data not shown) that normally completely inhibits endogenous wild-type ENaC.ENaC activity was largely inhibited ( N x P o = 0.009) by high doses (500 µM) of benzamil, which is consistent with thechannel resistance to amiloride ( Fig. 1 C ); in addition, the single-channel conductance( G ) in the presence of Na   or Li   ions wasslightly lower ( G Na = 2.1 and G Li =4.2 pS) than the wild-type ENaCs expressed in Xenopus oocytes, whichis another functional signature of the, -G525C -ENaCchannel ( 32 ). Taken together,these experiments demonstrate that exogenous -G525C mutant subunitsexpressed in mpkCCD cl4 cells can assemble with endogenous ENaCsubunits to form functional channels at the cell surface with the expectedfunctional characteristics.
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Transduced -ENaC mutants respond to aldosterone. Totest whether the functional ENaC that contains transduced -ENaC subunits responds to aldosterone, mpkCCD cl4 cells transduced with GFP, -G525C, or -G525C   Y618A ENaC mutants were grown on filters. After formation of a confluent monolayer, they were challenged with10 - 6 M aldosterone for 4-5 h. This high concentration ofaldosterone was used to induce a maximal effect on Na   transport byoccupation of both types of MR receptors by the ligand. Figure 2 shows that in cellstransduced with GFP as a control, the benzamil (500 µM)-inhibitable I sc was entirely sensitive to a low concentration ofamiloride (10 µM), which is expected for cells that exclusively expressendogenous ENaC. This amiloride-sensitive current increased by about threefold after 4 h of incubation with aldosterone. Overexpression of the -G525CENaC subunit did not significantly change the level of the basal I sc; the I sc was mainly resistant to10 µM amiloride but sensitive to 500 µM benzamil, which is consistent with a majority of channels made of the amiloride-resistant ENaC that containsthe exogenous -G525C subunit. Aldosterone stimulated bothamiloride-sensitive and amiloride-resistant fractions of I sc by about sixfold, thereby indicating that ENaCchannels that contain either endogenous -ENaC or exogenous -G525CENaC subunits respond to aldosterone. Cells that express the Liddle -G525C   Y618A ENaC mutant subunit exhibited a higher basal I sc that was almost completely resistant to 10 µM amiloride, which is consistent with the previously reported hyperactivity ofthe -Y618A ENaC mutant at the cell surface of Xenopus oocytes.In these cells, both amiloride-sensitive (10 µM) and amiloride-resistantfractions of I sc increased on aldosterone stimulation,thereby indicating that the -G525C   Y618A mutant retains its ability torespond to aldosterone.% g% Q) ~9 [9 R& {0 g8 ~+ {: J# A
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Fig. 2. ENaC mutants transduced in mpkCCD cl4 cells respond toaldosterone. Benzamil (500 µM)-sensitive I sc ( I sc benz ) was measured in the absence and 5 h afteraddition of aldosterone (10 - 6 M) in mpkCCD cl4 cells transduced with GFP, -G525C ENaCs, and -G525C   Y618A ENaCs.Hatched portions of bars represent the fraction of I scbenz due to the endogenous channels that was sensitive to amiloride (10µM). Transepithelial voltages (in mV) in the absence and presence ofaldosterone were, respectively, -18.6 ± 7 and -55.2 ± 5.4 forGFP, -18.7 ± 6.4 and -88.2 ± 13.5 for -G525C, and -51.9± 8.5 and -86 ± 15 for -G525C   Y618A. Transepithelialresistance (in k ) did not significantly change with aldosterone andranged from 1.3 ± 0.1 to 3.4 ± 0.8 in the different experiments.Bars represent the means ± SE of 9 different filters; * P4 r- Z5 y% v8 ?# ~' l; P+ w+ P. D
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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 ofthese cell lines, we choose mpkCCD cl4 cells with a low level ofbaseline I sc to avoid possible saturation of thetranscellular Na   transport capacity in cells that express ENaCwith activating mutations. In Table1, we compared I sc measured in the presence of10 µM amiloride in cells transduced with these Liddle's ENaC mutants, with parental cells transduced with their respective control -G525C ENaCconstruct. In these experiments, the amiloride-sensitive component of I sc that reflects the activity of endogenous ENaCs represented 15-25% of the 500 µM benzamil-inhibitable current carried bythe ensemble of the functional ENaCs. The results obtained with thempkCCD cl4 cells that express ENaCs with missense mutations ordeletions in the PY motif of -ENaCs all show a higher baseline I sc than the respective control mpkCCD cl4 cells. The aldosterone-induced absolute increase in I sc ( I sc ) after 5 h (early response to aldosterone) was similar in magnitude in cells transduced with ENaC Liddle's mutants and incontrol cells, which indicates identical responses to the hormone. Similarresults were obtained with mpkCCD cl4 cells that express both - and -ENaC truncation mutants in the COOH terminus. According tofunctional and biochemical studies, these mutations in the PY motif of -and -ENaCs are sufficient to nearly abolish the interaction betweenENaCs and WW domains of rNedd4-1( 30, 31, 40 ).% r, g' m; b3 f3 f- Q9 ^. E8 G$ X

% e  w5 P; D# h' b* N6 }- a3 XA large body of evidence indicates that aldosterone stimulation of thetransepithelial Na   transport in tight epithelia results from theappearance of newly active ENaCs at the cell surface ( 45 ). We have measured thekinetics of appearance of newly active ENaC mutants at the cell surface. Anexperiment is shown in Fig.3 A with the recording of the time course of I sc changes after the addition of aldosterone to cellsthat express -G525C and -G525C  Y618A ENaCs. The recording startsafter the addition of aldosterone, and I sc measurements inthe presence of 10 4-h time period. Typically, the cells that express the -Y618A mutant have ahigher baseline I sc value than the control cells. After a40-min period of latency, the I sc 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 linetransduced with exogenous -ENaC mutants, the rate of I sc increase( I sc / t, in µA/min) wascomparable to that in its respective control cells transduced with -G525C ENaCs. These results indicate that aldosterone has similareffects during the early phase of the response on the magnitude of theENaC-dependent Na   transport in cells that express ENaC controlsand in cells transduced with -and -ENaC mutant subunits lackingthe PY motif.. x4 }' y+ ?% X1 P9 ]" e  T1 j

$ ^; D( [4 G6 e& X3 \1 A0 MFig. 3. Effects of aldosterone and recovery from stimulation inmpkCCD cl4 cells that express -G525C or -G525C   Y618AENaC subunits. A : representative traces of I sc recordings performed on cells transfected with -G525C ENaCs (solid blackline, left axis ) and -G525C   Y618A ENaCs (solid gray line, right axis ) after the addition of aldosterone(10 - 6 M) at time = 0. I sc was recorded in the presence of amiloride (10 µM). Fraction of the I sc sensitive to amiloride (10 µM) corresponded to 10and 15% of the benzamil-inhibitable I sc for -G525Cand -G525C   Y618A ENaCs, respectively (not shown). Time when benzamil(500 µM) was added is indicated (arrow). I sc in cellstransfected with -G525C and -G525C   Y618A ENaCs in the absence ofaldosterone are indicated by dotted black and gray lines, respectively. B : time course of I sc changes after aldosteroneremoval (circles) or after the addition (arrow, time = 0 ) of0.5 mg/ml cycloheximide (triangles) to mpkCCD cl4 cells transducedwith -G525C (closed symbols) or -G525C   Y618A ENaCs (opensymbols) constructs. I sc was recorded in the presence ofamiloride (10 µM). Values are means ± SE from 4-9 separateexperiments.
$ w. a3 S& @/ j! |4 ?, y
( C& S6 F. P$ S1 |) ]8 n% qWe also measured the recovery of the I sc fromaldosterone stimulation in the different mpkCCD cl4 cell lines thatwe generated. As shown in Fig.3 B, 40 min after aldosterone removal, I sc started to decline faster in control cells transducedwith -G525C ENaCs than in cells that expressed the -G525C   Y618Amutant. Similar results were obtained with addition of the protein synthesis inhibitor cycloheximide (0.5 mg/ml) while aldosterone was maintained in thebath. The half-time for I sc decline in controlmpkCCD cl4 cells after aldosterone removal ranged between 50 and 60min (see Table 1 ) and wascomparable to the I sc decline measured after cycloheximidetreatment (56.4 ± 8.7 min, n = 4). These values are consistentwith the half-life of ENaCs at the cell surface that was recently reported inENaC-transfected Madin-Darby canine kidney cells( 17 ). The rate of I sc decline in mpkCCD cl4 cells that expressedthe different ENaC Liddle's mutants was significantly slower (see Table 1 ). This observation isconsistent with a longer half-life of activity for ENaC Liddle's mutants atthe cell surface compared with control ENaCs.1 |& ~% d0 V) Z7 [( }& r. f
9 G4 h% }+ P% J( u
Compared with mpkCCD cl4 cells that express wild-type ENaCs, those that express ENaCs with mutations in the PY motif exhibit two mainfeatures. First, these cells have a longer half-life of channel activity atthe cell surface that can account for a larger baseline I sc. Second, they respond to aldosterone with a similarincrease in Na   transport during the first hours of stimulation.These observations are illustrated in Fig.4 with the mean I sc changes before and afteraldosterone administration in mpkCCD cl4 cells that express the ENaCLiddle's mutants -G525C   Y618A and the - and -truncatedENaC subunits together with their respective controls. The question remainswhether a higher stimulation of Na   transport by aldosterone shouldbe expected in cells that express ENaCs with a longer half-life of activity atthe cell surface. We addressed this question using a simple mathematicalmodel, assuming that aldosterone stimulates Na   transport simply byincreasing the rate of appearance of newly active ENaCs at the cell surface(see MATERIALS AND METHODS ). Such a model for aldosteronestimulation can be justified by evidence that shows that aldosterone indeedincreases the number of active ENaCs at the cell surface( 21, 26 ). In our model, thesteady-state I sc basically results from an equilibrium between the rates of insertion and retrieval of active ENaCs at the cellsurface. We have used this model to predict the I sc response to aldosterone in cells that express control and Liddle's mutantsthat differ in their respective activity half-lives. The fit of our data tosuch a model reveals that the differences in the baseline I sc values between cells that express control and Liddle'smutant ENaCs can account for an ENaC half-life at the cell surface of 60 minfor the wild-type ENaC, 410 min for the -G525C   Y618A mutant, and 650min for the -and -COOH-terminal truncated ENaC mutants. Our modelalso predicts that the rate of Na   transport( I sc ) increase is basically the same during the first 2-3h of aldosterone stimulation in cells transduced with ENaC control and ENaCLiddle's mutants and that we should not expect large differences in the earlyaldosterone response.
5 E6 j- y3 i0 r4 \& O/ }& j
% q( t" d+ p- \) T( I; D8 B% S0 zFig. 4. I sc recordings during the early aldosterone response inmpkCCD cl4 cells that express control and Liddle's mutant ENaCs. A : mean I sc values in cells that express -G525C   Y618A ENaCs ( top trace,, n = 6) orcontrol -G525C ENaC cells ( bottom trace,, n =4) were determined in the presence of amiloride (10 µM) and afterstimulation by aldosterone (10 - 6 M) at time = 0. Fit of data (thin solid lines) was obtained according to equationdescribed (see MATERIALS AND METHODS ), where I sc measured is the sum of two components, the rate ofappearance of newly active ENaC I sc / t (in µA/min), and the rateof ENaC disappearance ( k remove, inmin - 1 ) at the cell surface. Fit parameters were I sc / t = 0.12 µA/min before and0.6 µA/min after aldosterone stimulation in control and -G525C  Y618A mutants that express mpkCCD cl4 cells; k remove remained unchanged by aldosterone and was 0.012min - 1 in control cells and 0.0017min - 1 to account for the higher basal I sc in cells that express -G525C   Y618A mutant. B : fit of the I sc increase measured under thesame conditions in response to aldosterone stimulation in cells that expressboth - and -COOH-terminal truncated mutant ( top trace, ) and control -G525C ( bottom trace, ) ENaCs. Fitparameters were I sc / t increase from0.06 to 0.25 µA/min in response to aldosterone in both cells; k remove = 0.011 min - 1 for thecontrol cells and 0.0009 min - 1 for cells that expressthe mutant; k remove did not change in response toaldosterone. Gray lines and points represent the average of four I sc measures taken every 20 s in n = 4experiments. Error bars show SE.) c3 i1 u2 S0 w+ N

  o% s) b( F! ~  IDuring the late phase of the aldosterone response, the ENaC Liddle'smutants are likely to continue to accumulate with time at the plasma membranedue to their longer half-life at the cell surface, while the I sc in cells transduced with control ENaCs will havealready reached a steady-state level due to the equilibrium between the rateof channel insertion and removal from the cell surface. Thus we expected tosee, after several hours of stimulation, a larger aldosterone-induced I sc in mpkCCD cl4 cells that express ENaCLiddle's mutants. In these experiments investigating the long-term effects ofaldosterone, mpkCCD cl4 cells that express control and Liddle'smutant ENaCs were maintained during the time of aldosterone stimulation (16 h)in an apical low-Na   medium that contained amiloride to inhibitapical Na   entry. The I sc was then measured 4and 16 h after addition of aldosterone in the presence of a normal apicalNa   medium without amiloride. The experiments in Fig. 5 show that in both mpkCCD cl4 cells that express wild-type ENaC and ENaCs carrying theY618A mutation, aldosterone did not induce a sustained I sc stimulation for a 16-h time period. Rather, I sc reached apeak 4 h after the addition of aldosterone and then slowly decreased, therebyshowing no clear late response of aldosterone in both mpkCCD cl4 cell lines. Under conditions of low baseline Na   transport usingamiloride and low-apical Na   medium during incubation, theinstantaneous measurement of I sc revealed that after 4 h,the aldosterone-induced I sc was already higher and after16 h, it was still measurable in mpkCCD cl4 cells that express the -Y618A mutant, whereas in control cells, I sc returned to baseline values. This stronger and prolonged response to aldosterone by the Liddle's -Y618A mutant ENaCs compared with controlENaCs further supports a longer half-life and the retention of the active ENaCmutants at the cell surface. Thus we can conclude that mpkCCD cl4 cells that express ENaCs with mutations in the PY motif respond to aldosteroneduring the early stimulatory phase with changes in Na   transportthat 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.
5 [* N/ p- i1 E1 s: e( b4 u
4 h- k6 o' I- ~0 F, m' p7 ~' hFig. 5. I sc in mpkCCD cl4 cells transduced withwild-type ( ) and -Y618A ( ) ENaCs during the late aldosteroneresponse. Cells were grown to confluent monolayers and were maintained 16 hbefore I sc measurements were obtained in a low-apicalNa   (10 mM) medium with amiloride (10 µM). Aldosterone(10 - 6 M) was added at time 0. At times0 and 4 and 16 h, instantaneous I sc was measured in a100 mM apical Na   medium without amiloride. Each point represents amean of 4-8 I sc measurements.
6 O  P: |6 N# P3 ^9 I$ s3 H1 H' s+ b3 ~
Effects of vasopressin on Na   absorption in cellsthat express ENaC mutants. Vasopressin and cAMP both stimulateNa   transport in the cortical collecting duct and in culturedmpkCCD cl4 cells ( 3, 6 ). In mpkCCD cl4 cells transfected with GFP, the 500 µM benzamilinhibitable I sc was completely blocked by 10 µM amiloride in theabsence or after stimulation by 10 - 9 M vasopressin. InmpkCCD cl4 cells that express -G525C ENaCs, the largest fraction of the benzamil-inhibitable I sc was insensitiveto 10 µM amiloride. The addition of 10 - 9 Mvasopressin for 2 h stimulated I sc mediated by bothendogenous amiloride-sensitive ENaCs and amiloride-resistant ENaCs thatcontained the -G525C subunit ( Fig.6 ). In cells that express the -G525C   Y618A mutant, thebasal I sc was higher than in control cells, as expectedfor a Liddle's mutation, and in the presence of vasopressin, bothamiloride-sensitive and -resistant components of I sc increased. These experiments indicate that the -G525C   Y618A mutantENaCs respond to stimulation by vasopressin.' _& Y7 C* Y6 l6 V2 i4 p, ?7 }2 b* E2 v

. |9 z" w" W/ o1 K5 @  lFig. 6. Effects of vasopressin on mpkCCD cl4 cells that express -G525C or -G525C   Y618A ENaCs. Benzamil (500 µM)-sensitive I sc was measured under basal conditions and 100 min afterstimulation by vasopressin (10 - 9 M). Hatched portions ofthe bars represent the fraction of I sc due to theendogenous channels that were sensitive to amiloride (10 µM). Barsrepresent means ± SE of 8-10 different filters. * P
* e0 o9 a( R. A% w. a7 r; d
8 [3 j! _  E# \9 @2 a1 P( lRecent evidence shows that vasopressin promotes the appearance of newlyactive channels at the cell surface. We asked whether the rate of appearanceof newly active channels is similar in mpkCCD cl4 cells that expresscontrol -G525C and -G525C   Y618A mutant ENaCs. As shown in Fig. 7 A, in both celllines, vasopressin induced a rapid increase in I sc duringthe first 10 min followed by a slower rate of I sc increasethat lasted 1 h. Then, I sc started to decline despite thepresence of vasopressin in the bathing solution. The vasopressin-inducedincrease in I sc measured in the presence of 10 µMamiloride to block endogenous ENaCs was inhibited by the addition of 500 µM benzamil at the apical surface. Similarly, forskolin, a nonspecific agonistfor adenylyl cyclase, was also able to stimulate I sc through the amiloride-resistant ENaCs with and without the Y618A Liddle'smutation ( Fig. 7 B ).The absolute magnitude and the rate of changes of I sc werecomparable in mpkCCD cl4 cells that expressed control and mutantENaCs. Table 2 summarizescharacteristics of the vasopressin response in cells that express controlENaCs, the -G525C   Y618A mutant, or the double- - and -ENaCCOOH-terminal truncated mutants. All three cell lines responded to vasopressin and exhibited a similar increase in the magnitude of I sc ( I sc ). In the mpkCCD cl4 cells that weretransduced with GFP or that express control -G525C and -G525C  Y618A mutant ENaCs, the rate of I sc increase wascomparable. A slightly but significantly lower rate of I sc increase was measured in cells transduced with both - and -ENaCCOOH-terminal truncated mutants. Considering the variations in the rate ofvasopressin-induced increase in I sc in both control cellstransduced with GFP or -G525C ENaCs, the -Y618A ENaC mutation doesnot seem to greatly affect the ENaC response to vasopressin. Therefore, the PYmotif in the COOH terminus of - and -ENaCs does not seem to berequired for ENaC stimulation by vasopressin. However, in cells that expressthe double- - and - ENaC COOH-terminal truncated mutants, theslightly lower rate of I sc increase could be related to the deletion of sequences in the COOH terminus of - or -ENaCs other than the PY motif." m! S0 A2 y- K* [' u2 ]& W& s
0 J9 U& A* _( f; m& L0 J0 _7 z8 e8 o* L
Fig. 7. Effects of vasopressin or forskolin on I sc in cellsthat express Liddle's mutant ENaCs. A : I sc wasmeasured in the presence of amiloride (10 µM) after addition of vasopressin(10 - 9 M; arrow) to cells transduced with -G525C(black line, left axis ) or -G525C   Y618A (gray line, rightaxis ) ENaCs. At the end of the experiment, benzamil (500 µM) was addedto the apical side of the cells. B : similar I sc recording in confluent mpkCCD cl4 cells transduced with -G525C(black line, left axis )or -G525C   Y618A ENaC (gray line, right axis ) after addition of forskolin (10 µM; arrow). C : time course of I sc recovery from vasopressinstimulation. Arrow denotes the time when hormone was removed. Half-lives for I sc decreases were 35.42 ± 8.9 min for the controlcells ( ) and 245.3 ± 45.3 min for the -G525C   Y618A( ) mutant. Each point represents the mean of n = 4-6experiments; error bars show SE.
/ f  g0 L! L3 _4 R* B& m5 n" {! V) S3 P: J7 n3 P% W& X
Table 2. Effects of vasopressin on I sc in transducedmpkCCD cl4 cells9 Z  A, F: j& t+ f

8 V7 [5 o9 M, C# rThe vasopressin effect of Na   transport was rapidly reversible on removal of vasopressin from the bath: I sc decreasedwith 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.7 C ). This suggests that the recovery from vasopressinstimulation likely involves retrieval of ENaCs from the cell surface andrequires functional PY motifs. In summary, these data indicate that mutationof the Tyr within the PY motif of -ENaCs does not affect the magnitude of the response to vasopressin of the cortical collecting duct cells thatexpress the ENaCs; we cannot exclude that sites other than the PY motif in theCOOH terminus of - or -ENaCs might contribute the vasopressinresponse.: x: S$ y( T  y" ~$ |0 I

0 \: h: N: c1 g0 z) ^, B2 jDISCUSSION" X0 z& Y, f- Q1 e) `1 i( ~  t
$ E7 T5 G. e: x+ j6 W8 K
Our study demonstrates that ENaC mutant subunits can be expressed andanalyzed functionally in a differentiated mammalian collecting duct cell linethat responds to aldosterone and vasopressin. We have generatedmpkCCD cl4 cells that express -and -ENaC subunits withmutations in the conserved PY motif of the cytoplasmic COOH terminus.Consistent with hyperactive ENaC mutants that cause Liddle's syndrome, thesecells are characterized by a higher baseline I sc due to alonger half-life of ENaC activity at the cell surface. Despite their higherbasal I sc, the cells transduced with ENaC mutants thatcause Liddle's syndrome still responded to aldosterone with an increase inNa   transport that was similar in magnitude during the first hoursof stimulation. Furthermore, the kinetics of activation of the Na   transport by aldosterone were comparable in cells transduced with control orLiddle's mutant, which indicates that Liddle's mutant ENaCs retain theirsensitivity to the hormone. Cells that express ENaC mutants differ fromcontrol cells by a prolonged response to aldosterone due to retention ofactive ENaCs at the cell surface. Similar observations were done onNa   transport stimulated by vasopressin.' A  R8 E3 a5 _' ~# j# u: S

) e6 m" C% l  n1 O, C  K/ e. nTransduced mpkCCD cl4 cells that expressamiloride-resistant ENaC mutants. The exogenous rat -G525C mutantENaC subunit that confers to the functional ENaC a resistance to amiloride wasretrovirally transduced in the mpkCCD cl4 cells under the control ofa viral promoter. This exogenous -G525C ENaC subunit was thereforeconstitutively expressed and not transcriptionally regulated by aldosterone inthe mpkCCD cl4 cells as in the endogenous -ENaCs. The cellstransduced with the exogenous -G525C ENaCs showed a higher expression of -ENaC subunits at the protein level without a corresponding change inthe basal level of I sc. The reason is that -ENaCsubunits alone do not form homomeric functional channels at the cell surface;only the heteromeric,, -ENaC is functional( 5 ). Thus it is likely that inmpkCCD cl4 cells transduced with and overexpressing the -G525Csubunits, - and -ENaC subunits become limiting for expression ofENaC activity at the cell surface. Accordingly, overexpression of -ENaCsonly will not increase the level of baseline I sc.1 P& _. ~' n" n# }$ [5 J4 n% c

- ~+ B) W6 G" ^. \: X' sDespite the fact that the exogenous -G525C ENaC subunit is constitutively expressed in the mpkCCD cl4 cells, the channels madeof the transduced -ENaC subunit respond to aldosterone. In targetepithelia, the stimulation of Na   transport by aldosterone requiresde novo protein synthesis of -ENaCs as well as other aldosterone-induced proteins that regulate ENaC activity( 23, 28 ). In the intact distalnephron, aldosterone induces the expression of -ENaC, whereas -and -ENaCs are constitutively expressed( 11 ). Thus in thempkCCD cl4 cells transduced with the exogenous -G525C subunit,the newly synthesized -ENaC subunits in response to aldosteronestimulation likely assemble with the available - and -ENaCsubunits; in the case of cells that overexpress the -G525C ENaC subunit,channel assembly will occur preferentially with the exogenous -G525Csubunit because of its higher level of protein expression (see Fig. 1 A ). 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 I sc sensitivity to amiloride indicate 85% of the ENaCs at the surface of cells transfected with -G525CENaCs have incorporated an exogenous mutant -ENaC subunit and respondedto aldosterone.
% h, c9 b: H$ l0 a( e/ S7 Y& Y5 c! y! E
mpkCCD cl4 cells that express -ENaC mutants causing Liddle's syndrome. Gain-of-functionmutations that cause Liddle's syndrome have been identified in the conservedPY motif in the COOH-terminal end of -and -ENaC subunits.. H2 D8 j, M/ O6 M
5 E7 s% u" h# _  m
The PY motifs of - or -ENaCs are involved in ENaCinternalization and/or its degradation as shown by mutations of this motif that increase ENaC activity at the cell surface( 29 ). In mpkCCD cl4 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 I sc compared with control cells. This higher baseline I sc was observed for comparable levels of expression of -G525C or -G525C   Y618A ENaC subunits. Recovery of I sc from aldosterone stimulation showed that the active -G525C   Y618A ENaC mutant is more stable at the cell surface than thewild-type ENaC with a longer half-life of activity. Taken together, theseobservations are consistent with experimental evidence obtained from Xenopus oocytes and suggest the possibility that the increasedactivity at the cell surface of ENaC mutants that cause Liddle's syndromemight be due to a deficient binding interaction with its partner such asNedd4-2. Mutations in the PY motif of -ENaCs have only little effectson ENaC function in Xenopus oocytes, and to date no mutations thatcause the syndrome have been found in the PY motif of -ENaCs( 30, 31 ). This relatively smalleffect of -ENaC mutations correlates with the reduced ability of the PYmotif of -ENaCs to interact with WW domains Nedd4-1, whereas mutationsin the PY motif of - or -ENaCs impair most of the ability to bindthis Nedd4 protein ( 40 ).
8 u1 X  a5 \1 P+ H6 Y5 R/ o+ H: S
# l4 L0 F1 S; S; y2 ]+ uAldosterone response of Liddle's mutant ENaCs. In the cells thatexpress - and -ENaCs with mutations in the PY motif, aldosterone induced a significant increase in I sc that after 4-5 h was comparable in absolute magnitude to the response of the cells that expresscontrol ENaCs. The kinetics of activation of the amiloride-sensitive I sc during the early phase of aldosterone stimulation werecomparable in cells transduced with control or Liddle's mutant ENaCs. We canconclude that the early response of the mpkCCD cl4 cells toaldosterone is maintained and not affected by mutations in the PY motif ofENaCs. These results are expected if aldosterone acts on cortical collectingduct cells primarily by promoting the appearance of newly active ENaCs at thecell surface. This is supported by recent immunochemical experiments that haveevidenced a rapid translocation of ENaC subunits from a cytoplasmic pool tothe apical membrane 2-4 h after aldosterone stimulation( 21, 45 ). The rapid insertion ofnewly active ENaCs at the cell surface together with an inhibition of theirinternalization could theoretically be a very efficient way for aldosterone toincrease Na   transport. In our experiments comparing thealdosterone response of cells that express wild-type ENaCs and mutants thatdiffer 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 ENaCstability at the cell surface does not play a major role in the aldosteroneresponse. Recent biochemical evidence indicates that aldosterone does notchange the half-life of ENaCs in the cell and at the cell surface( 2, 23 ).! v; Y# l% ~% g/ R

5 O9 w. J) {4 S% w9 k; @However, the question remains as to whether the longer half-life of theLiddle's mutant ENaCs could have functional consequences on transepithelialNa   transport during the late aldosterone response. One couldexpect that by promoting the insertion at the apical membrane of ENaC mutantswith longer half-lives, aldosterone should ultimately induce a higher I sc in cells that express Liddle's mutant compared withcontrol ENaCs. We have measured amiloride-sensitive I sc upto 16 h of aldosterone stimulation in mpkCCD cl4 cells and observedthat despite the continuous presence of the hormone, the I sc declines after 4-5 h. Nevertheless, after 16 h ofstimulation, a significant higher aldosterone-induced I sc was measured in mpkCCD cl4 cells that express the Liddle's mutant,whereas in the controls cells I sc returned to the baselinevalues. This observation is consistent with retention of active ENaC mutantsat the cell surface. It is interesting to note that the inhibition of ENaCtransport activity during early aldosterone stimulation seems to enhance the I sc response in mpkCCD cl4 cells that expressthe ENaC -Y618A mutant (see Figs. 2 and 5 for comparison). Thisobservation further supports the ability of the Liddle's mutant ENaCs to respond to aldosterone and suggests that the high transcellular Na   transport in mpkCCD cl4 cells that express the mutants ENaCs mayinduce feedback-inhibitory effects on the aldosterone response.5 g! p  N6 s, f
$ {; B- G/ B/ R" g6 R3 P7 z
The aldosterone-signaling pathway initiated by the binding of the ligand tothe mineralocorticoid receptor and ending with the activation of ENaCs at thecell surface remains to be deciphered. In the aldosterone-sensitive distalnephron, the SGK is an early aldosterone-induced protein, and itsintracellular accumulation seems to precede the apical translocation of ENaCs( 21 ). In addition,coexpression of ENaCs and SGK in Xenopus oocytes increases cellsurface expression of active ENaCs. Together, these experiments stronglysuggest that SGK mediates at least in part the aldosterone-inducedtranslocation 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 aninhibition of the binding interaction between Nedd4-2 and ENaCs and aretention of ENaCs at the cell surface( 8, 38 ). The physiologicalrelevance of this signaling pathway involving SGK and Nedd4-2 phosphorylationhas not yet been established in aldosterone-responding epithelia. Our results obtained from cortical collecting duct cells that express mutants ENaCs showthat the PY motif is not essential for the early response to aldosteronestimulation.3 @5 h; T* M: M: E: H7 n/ h" K" o

  s9 d9 P1 W: X  `( \+ ?& VInsulin and aldosterone have synergetic actions on Na   transport. The signaling pathway for both hormones requiresphosphatidylinositol 3-kinase( 46 ). Recently it was shown inA6 kidney cells that phosphatidylinositol 3-kinase is necessary for thealdosterone effect on Na   transport and on the phosphorylation ofSGK. Considering that insulin also increases SGK phosphorylation, theseobservations suggest that SGK represents a critical point of convergencebetween the aldosterone- and the insulin-signaling pathways. We found thatcells that express the -Y618A mutant respond to insulin in a way similarto the cells that express control ENaCs (data not shown). This furtherindicates that the target sequence crucial for the SGK effect on ENaCregulation is likely not the PY motif in the COOH-terminal end of ENaCs.1 t( P3 I, U3 X) L- l& h$ G3 ?' s

: K* a  k) P4 X! n1 w7 v- {7 OOur data indicate that neither the PY motif nor the COOH terminus of - and -ENaCs is required for the ability of the channel torespond to aldosterone. Therefore, the phosphorylation of the COOH terminus of -and -ENaC cells and its modulation observed in Madin-Darbycanine kidney cells do not seem to be directly related to the early responseof the channel to aldosterone ( 34 ). The contribution ofother potential phosphorylation sites, in particular in the NH 2 terminus of the,, -ENaC subunits in the channel responseto aldosterone, remains to be investigated.# Z. ]- y) ]( C4 [+ H5 G
* ]/ r6 H; @& |4 t% x
Vasopressin response. The increased Na   absorption inresponse to vasopressin is due to a stimulation of ENaCs at the cell surface( 10 ). The vasopressin effectis mediated by the binding of vasopressin to the V 2 receptor, whichleads to an increase in intracellular cAMP content. Patch-clamp experiments inA6 cells showed that vasopressin and cAMP increase the number of active ENaCsper 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 inNa   current( 24 ).& r5 N  h+ U. A
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Our results demonstrate that cells that express the Y618A ENaC mutantretain the ability to respond to vasopressin with a comparable increase in theabsolute magnitude of I sc. Furthermore, the rate of theinitial increase in I sc was comparable in mpkCCD cl4 cells transduced with control ENaCs and with the -G525C   Y618A Liddle's mutant, which suggests similar rates ofactivation for ENaCs at the cell surface. Thus cortical collecting duct cellsthat express either wild-type or -Y618A mutant ENaCs have similarresponses to vasopressin stimulation. This response is dependent on cAMP,since forskolin induces similar stimulation of I sc in bothcells that express wild-type and mutant ENaC. Recent experiments in ratthyroid cells transfected with ENaCs showed that the -Y618A mutationdisrupts 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 todifferences in the cellular environment of ENaCs in thyroid cells that do notphysiologically respond to vasopressin or express ENaCs. ThempkCCD cl4 cells that express the double COOH-terminal truncated - and -ENaC mutant show a slightly attenuated response tovasopressin, which suggests the possibility that sites in the COOH termini of - and -ENaC other than the PY motif could contribute to thestimulation of ENaCs by vasopressin. It has recently been reported that theCOOH termini of - and -ENaCs can be phosphorylated in vitro inthe vicinity of the PY motif, but the physiological role of thesephosphorylation sites remains to be established( 33, 34 ).1 U$ `# c$ N7 e2 |6 @

  `5 ?% J, {1 ~! A! f- lPhysiological relevance. We have studied the aldosterone and vasopressin response in transduced mpkCCD c14 cells that overexpress ENaC mutants causing Liddle's syndrome. It is possible that the ENaC biologyin these engineered epithelial cells as in any other cell expression systemdoes not necessarily reflect the precise biology of ENaCs in the nativetissue. Thus how can we consider our results in the context of the earlyclinical observation made by Liddle in his patients with pseudoaldosteronism ( 19 )? Typically, patients withpseudoaldosteronism have a low plasma level of aldosterone and low plasmarenin activity ( 4 ). Interestingly, Liddle observed in one of his patients maintained on a 30-meqNa   /day diet that treatment with exogenous aldosterone decreasedthe urinary Na   excretion to 19 ). Together with ourresults, this observation strongly suggests that Liddle's patients respondedefficiently to aldosterone stimulation because of the conserved responsivenessof the ENaC mutant to the hormone.+ Q- u: o* a* q+ e/ C0 o

) e7 K" r& f  g. k) O7 qDoes this conserved response together with a prolonged activity at the cellsurface of the ENaC mutants to aldosterone have a pathophysiological relevancein patients with Liddle's syndrome, considering that their plasma levels ofaldosterone are chronically low? Hypertension in patients with Liddle'ssyndrome usually starts at an early age. The sequence of pathophysiological events in these patients that starts from an abnormally high distalNa   absorption and leads ultimately to the development ofhypertension remains unclear. It is conceivable that before the development ofvolume expansion and hypokalemia, the plasma aldosterone levels in thesepatients remain in the normal range. At this preclinical stage, it is possiblethat a prolonged response of the distal nephron to aldosterone and/orvasopressin, 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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+ E+ c& R5 Q: p( E4 e1 `0 A5 dACKNOWLEDGMENTS( c3 U2 {1 p4 P7 s

+ D1 `0 ~% M1 Y- j! G; ~The authors thank J.-D. Horisberger and B. R. Rossier for helpful discussion and critical review of the manuscript.
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) b$ H( I3 ?( s$ q* W9 t" M3 lThis work was supported by Grant 31-59,217.99 from the Swiss NationalScience Foundation to L. Schild. S. Kellenberger was supported by a NationalInstitutes of Health Specialized Center of Research grant for hypertension,and M. Auberson was supported by Human Frontier Science Program GrantRG00261/2000.3 K9 L  ]6 A; A: v$ w. N
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