JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M504347200 on July 18, 2005

J. Biol. Chem., Vol. 280, Issue 36, 31587-31594, September 9, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/36/31587    most recent
M504347200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bachhuber, T.
Right arrow Articles by Kunzelmann, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bachhuber, T.
Right arrow Articles by Kunzelmann, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Cl– Interference with the Epithelial Na+ Channel ENaC*

Tanja Bachhuber, Jens König, Thilo Voelcker, Bettina Mürle, Rainer Schreiber, and Karl Kunzelmann{ddagger}

From the Institut für Physiologie, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany

Received for publication, April 20, 2005 , and in revised form, July 7, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A and ATP-regulated Cl– channel that also controls the activity of other membrane transport proteins, such as the epithelial Na+ channel ENaC. Previous studies demonstrated that cytosolic domains of ENaC are critical for down-regulation of ENaC by CFTR, whereas others suggested a role of cytosolic Cl– ions. We therefore examined in detail the anion dependence of ENaC and the role of its cytosolic domains for the inhibition by CFTR and the Cl– channel CLC-0. Coexpression of rat ENaC with human CFTR or the human Cl– channel CLC-0 caused inhibition of amiloride-sensitive Na+ currents after cAMP-dependent stimulation and in the presence of a 100 mM bath Cl– concentration. After activation of CFTR by 3-isobutyl-1-methylxanthine and forskolin or expression of CLC-0, the intracellular Cl– concentration was increased in Xenopus oocytes in the presence of a high bath Cl– concentration, which inhibited ENaC without changing surface expression of {alpha}{beta}{gamma}ENaC. In contrast, a 5 mM bath Cl– concentration reduced the cytosolic Cl– concentration and enhanced ENaC activity. ENaC was also inhibited by injection of Cl– into oocytes and in inside/out macropatches by exposure to high cytosolic Cl– concentrations. The effect of Cl– was mimicked by Br–, , and I–. Inhibition by Cl– was reduced in trimeric channels with a truncated COOH terminus of {beta}ENaC and {gamma}ENaC, and it was no longer detected in dimeric {alpha}{Delta}C{beta} ENaC channels. Deletion of the NH2 terminus of {alpha}-, {beta}-, or {gamma}ENaC, mutations in the NH2-terminal phosphatidylinositol bisphosphate-binding domain of {beta}ENaC and {gamma}EnaC, and activation of phospholipase C, all reduced ENaC activity but allowed for Cl–-dependent inhibition of the remaining ENaC current. The results confirm a role of the carboxyl terminus of {beta}ENaC for Cl–-dependent inhibition of the Na+ channel, which, however, may only be part of a complex regulation of ENaC by CFTR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cystic fibrosis transmembrane conductance regulator (CFTR)1 is a cAMP-regulated Cl– channel and a regulator of other channels. A large number of membrane proteins and cellular processes are reported to be controlled by CFTR (1, 2). Most work has been devoted to the inhibitory effect of CFTR on the epithelial Na+ channel ENaC. Missing inhibition of ENaC by defective CFTR in cystic fibrosis (CF) is regarded as an important reason for the enhanced epithelial Na+ conductance detected in the airways of CF patients (3–5). A regulatory relationship has been shown for CFTR and ENaC in human and murine airway and intestinal epithelia (5, 6), and enhanced Na+ absorption has been demonstrated in airways and colonic epithelia carrying the CF defect (3). Subsequent studies showed that the epithelial Na+ channel is inhibited in recombinant and native cells during activation of CFTR (5–13).

Studies were performed to uncover the mechanism of the reciprocal interaction between CFTR and ENaC. Two-hybrid analysis and co-immunoprecipitation suggested a direct molecular interaction between CFTR and {alpha}ENaC, and the first nucleotide binding domain of CFTR was found to be crucial for inhibition of ENaC (14–16). Another study found a role of the carboxyl terminus of {beta}ENaC for down-regulation of ENaC by activation of CFTR in Xenopus oocytes (15). When CFTR and ENaC were reconstituted in a cell-free planar lipid bilayer, carboxyl-terminal domains of {beta}- and {gamma}ENaC were found to modulate the kinetics of the channel, whereas the NH2 terminus {beta}- and {gamma}ENaC were in charge of CFTR-dependent regulation of heterodimeric {alpha}{beta}- and {alpha}{gamma}ENaC but not in heterotrimeric {alpha}{beta}{gamma}ENaC (17). We and others found that truncations or Liddles disease mutations in the COOH terminus of {alpha}-, {beta}-, and {gamma}ENaC do not abolish CFTR-dependent inhibition of ENaC in trimeric channels (18, 19). However, the COOH-terminal 20 amino acid residues of {alpha}ENaC conferred species specificity of ENaC inhibition by CFTR in another study (20). Most studies agreed that CFTR affects ENaC channel kinetics and open probability rather than surface expression of ENaC (15, 17, 19). For mutant CFTR, which is unable to inhibit ENaC, genistein was shown to restore the functional interaction between CFTR and ENaC (8, 21, 22). Interestingly, the reverse, namely an up-regulation of CFTR currents by ENaC coexpression, has also been observed (12, 13).

Possible contributions by additional proteins and factors to the CFTR regulation of ENaC has been examined in subsequent studies. CFTR carries a so-called PDZ-binding domain, a common site for protein interaction at the COOH terminus, which binds regulatory proteins such as NHERF (23). This PDZ domain is essential for proper expression of CFTR in the apical plasma membrane of polarized cells (24, 25). Whereas in Xenopus oocytes this domain was neither important for expression of CFTR nor for inhibition of ENaC by CFTR, in polarized epithelial cells NHERF affects expression of both CFTR and ENaC (26, 27). Other mechanisms were excluded, such as release of ATP by CFTR, followed by purinergic inhibition of ENaC (28). Also changes in cell volume, which may occur during activation of Cl– conductances, are unlikely to explain the negative effects of CFTR on ENaC (29). However, independent reports demonstrated a role of Cl– ions (15, 30). It has known for some time that Na+ and Cl– conductances are controlled by the cytosolic Cl– concentration in intralobular duct cells of the mouse mandibular gland (31). Subsequent studies demonstrated that the control of ENaC currents by intracellular anions is mediated by G proteins in salivary duct cells, but not in Xenopus oocytes (32, 33). Different mechanisms have been shown for inhibition of ENaC by cytosolic Na+ and Cl– (34). Our initial work suggested that influx of Cl– into the oocyte rather than the efflux is inhibiting ENaC (9). Although a role of Cl– for the inhibition of ENaC by CFTR was not found in all studies (19, 35), subsequent work in both epithelial tissues and Xenopus oocytes confirmed that intracellular Cl– ions decrease expression of ENaC protein (36) and acutely inhibit ENaC currents (15, 27, 30, 37). Recent studies with polarized epithelial cells expressing ENaC along with CFTR and Ca2+-activated Cl– channels, demonstrate an increase in intracellular Cl– by stimulation of CFTR or Ca2+-dependent Cl– channels (38). This increase in intracellular Cl– is paralleled by inhibition of amiloride-sensitive Na+ conductance. Amiloride-sensitive Na+ currents are also inhibited in patch clamp experiments, when high Cl– concentrations are present on the cytosolic side (27, 38). These results strongly suggest a role of Cl– ions for inhibition of ENaC by CFTR or other Cl– channels. These channels cause an increase in the intracellular Cl– concentration when a large parallel Na+ conductance is present (39). We therefore tried in the present study to identify a Cl–-sensitive side in ENaC. We found that amino and carboxyl termini of {alpha}-, {beta}-, and {gamma}ENaC control ENaC activity and show a role of the COOH terminus of {beta}ENaC for inhibition by Cl–.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Ussing Chamber Experiments—Mouse M1 collecting duct cells were grown to confluence on permeable supports and were mounted into a modified Ussing chamber with a circular aperture of 0.95 mm2. M1 cells are derived from mouse cortical collecting duct and have properties of principal cells (kindly provided by C. Korbmacher, Physiologisches Institut, Universität Erlangen, Germany). Luminal and basolateral sides of the epithelium were perfused continuously at a rate of 10 ml/min (chamber volume 2 ml). The bath solution had the following composition (mmol/liter): NaCl, 145; KH2PO4, 0.4; K2HPO4, 1.6; D-glucose, 5; MgCl2, 1; HEPES, 5; calcium gluconate, 1.3. pH was adjusted to 7.4. Bath solutions were heated to 37 °C using a water jacket. Experiments were carried out under open circuit conditions. Values for transepithelial voltages (Vte) were referred to the serosal side of the epithelium. Transepithelial resistance (Rte) was determined by applying short (1 s) current pulses ({Delta}I = 0.5 µA) and the resistance of the empty chamber was subtracted. Rte was calculated according to Ohms law (Rte = {Delta}Vte/{Delta}I). The equivalent short circuit current (Isc) was calculated (Isc = Vte/Rte) and the amiloride-sensitive Isc (Isc-Amil) is used to express the amount of equivalent short circuit current that is inhibited by 10 µmol/liter amiloride. Tissue preparations were only accepted if the transepithelial resistance exceeded that of an empty chamber at least by a factor of 3. The transepithelial resistance of filter grown M1 cells was 387 ± 39 ohm cm2 (n = 20).

cRNAs for ENaC Subunits, CFTR, and CLC-0 and ENaC Mutants—cDNAs encoding rat {alpha}{beta}{gamma}ENaC (kindly provided by Prof. Dr. B. Rossier, Pharmacological Institute of Lausanne, Switzerland) and Cl– channels CFTR or CLC-0 were linearized in pBluescript or pTLN (40) with NotI or MluI, and in vitro transcribed using T7, T3, or SP6 promotor and polymerase (Promega). For some experiments cDNAs of FLAG-tagged {alpha}{beta}{gamma}ENaC subunits were used (kindly provided by Prof. B. Rossier, University of Lausanne, Switzerland) (41). Isolation and microinjection of oocytes have been described in a previous report (8). In brief, after isolation from adult Xenopus laevis female frogs (Xenopus express, South Africa and Kähler, Germany), oocytes were dispersed and defolliculated by a 45-min treatment with collagenase (type A, Boehringer, Germany). Subsequently, oocytes were rinsed and kept at 18 °C in NMDG/ND96 buffer (in mmol/liter): NMDG3, 96; HCl, 80; KCl, 2; CaCl2, 1.8; MgCl2, 1; HEPES, 5; sodium pyruvate, 2.5; pH 7.55), supplemented with theophylline (0.5 mmol/liter) and gentamicin (5 mg/liter). Mutations in the NH2-terminal phosphatidylinositol bisphosphate (PIP2)-binding domains of {beta}- and {gamma}ENaC were generated by replacing 2 ({beta}2N, {gamma}2N) and 4 ({beta}4N, {gamma}4N) positively charged amino acids by non-polar (N) amino acids as described in Ref. 42. {alpha}{Delta}N, {beta}{Delta}N, and {gamma}{Delta}N indicate that the NH2 terminus of {alpha}-, {beta}-, and {gamma}-subunits was shortened by removing the first 94, 50, and 94 initial amino acids, respectively, and in some cases replaced by other N-terminal subunits (43). All other mutations and truncations ({alpha}P671A, {alpha}{Delta}C = {alpha}H647X; {beta}{Delta}C = {beta}R561X and {beta}V554X; {gamma}{Delta}C = {gamma}S608X and {gamma}R565X) have been generated by PCR techniques and correct sequences were verified by sequencing.

Double Electrode Voltage Clamp—Oocytes were injected with cRNA (1–10 ng) after dissolving in 47 nl of double-distilled water (Nanoliter Injector WPI, Germany). Water-injected oocytes served as controls. 2–4 days after injection, oocytes were impaled with two electrodes (Clark instruments) that had a resistances of <1 megohm when filled with 2.7 mol/liter KCl. Using two bath electrodes and a virtual-ground head-stage, the voltage drop across Rserial was effectively zero. Membrane currents were measured by voltage clamping of the oocytes (Warner oocyte clamp amplifier OC725C) in intervals from –90 to +30 mV, in steps of 10 mV, each 1 s. Conductances were calculated according to Ohms law, and amiloride-sensitive conductances (GAmil) were used in the present report to express the amount of whole cell conductance that is inhibited by 10 µmol/liter amiloride. During the whole experiment, the bath was continuously perfused at a rate of 5–10 ml/min. All experiments were conducted at room temperature (22 °C).

Chemiluminescence Measurements—Oocytes were incubated for 60 min at 4 °C in ND96 media (in mM: 96 NaCl, 2 KCl, 1.8 CaCl2·2 H2O, 1 MgCl2·6 H2O, 5 HEPES, 2.5 sodium pyruvate, adjusted to pH 7.5 with NaOH) with 1% bovine serum albumin (BSA) to block nonspecific binding of antibodies. Afterward oocytes were incubated for 60 min at 4 °C with 1 µg/ml mouse monoclonal anti-FLAG M2 antibody (clone M2, Sigma) in 1% BSA/ND96, washed eight times at 4 °C with 1% BSA/ND96, and incubated with sheep anti-mouse IgG peroxidase-linked whole antibody (Amersham Bioscience) diluted 1:20000 in 1% BSA/ND96 for 40 min at 4 °C. Then oocytes were washed for 60 min at 4 °C in 1% BSA/ND96 and in ND96 (60 min, 4 °C) and placed separately in 50 µl of ECL Plus Western blotting Detection Reagents (Amersham Biosciences). After an incubation period of 5 min at room temperature, chemiluminescence was measured in a BioOrbit 1250 Luminometer (Turku, Finland). The integration period was 1 s and the results were obtained in millivolts (mV).

Fluorescence Measurements—Oocytes were incubated for 60 min at 4 °C in ND96 media (in mM: 96 NaCl, 2 KCl, 1.8 CaCl2·2H2O, 1 MgCl2·6 H2O, 5 HEPES, 2.5 sodium pyruvate, adjusted to pH 7.5 with NaOH) with 1% BSA to block nonspecific binding of antibodies. Afterwards oocytes were incubated for 60 min at 4 °C with 1 µg/ml mouse monoclonal anti-FLAG M2 antibody in 1% BSA/ND96, washed eight times at 4 °C with 1% BSA/ND96, and incubated with goat anti-mouse IgG1 fluorescein isothiocyanate-conjugated (Santa Cruz Biotechnology) in 1% BSA/ND96 for 60 min at room temperature and in the dark. Oocytes were washed for 60 min at room temperature and in the dark in 1% BSA/ND96 and then under the same conditions for 60 min in ND96. Images were obtained using a Zeiss Axiovert 35-inverted microscope with a x3.2 objective and digitized with a Nikon D100 camera.

Macropatch—Xenopus oocytes expressing ENaC were defolliculated and were placed on the stage of an inverted microscope (IM35, Zeiss, Oberkochen, Germany). The bath was continuously perfused with Ringer solution at a rate of about 10 ml/min. Patch clamp experiments were performed in cell-excised inside/out and outside/out configurations. The patch pipettes had an input resistance of 2–4 ohm when filled with a solution containing (mmol/liter) 96 NaCl, 2 KCl, 1.8 CaCl2·2H2O, 1 MgCl2·6H2O, 5 HEPES, 2.5 sodium pyruvate, 1 diphenylcarboxylate at pH 7.5. Currents (voltage clamp) and voltages (current clamp) were recorded using a patch clamp amplifier (EPC 9, HECA, Lambrecht, Germany), and data were stored continuously on a computer hard disc. In regular intervals, membrane voltages (Vc) were clamped in steps of 10 mV from –50 to +50 mV. G was calculated from the measured I and Vc values according to Ohms law.

Materials and Statistical Analysis—All compounds were of highest available grade of purity. Nystatin, amiloride, NMDG, PIP2, phosphatidylinositol-specific phospholipase C from Bacillus cereus and ATP were from Sigma. U73122 [GenBank] was from Calbiochem. The anti-PIP2 antibody was from Echelon. Student's t test p values <0.05 were accepted to indicate statistical significance. n indicates the number of experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Whole cell currents produced by the epithelial Na+ channel ENaC were partially inhibited by activation of CFTR through stimulation with IBMX (1 mmol/liter) and forskolin (2 µmol/liter). Inhibition was reversible when extracellular (bath) Cl– concentration was reduced from 100 to 5 mM. This is shown as a continuous whole cell current recording of an oocyte coexpressing CFTR and ENaC (Fig. 1A) and is summarized in Fig. 1B. In oocytes expressing ENaC only, the amiloride-sensitive conductance (GAmil) of 59.2 ± 7.9 microsiemens was not changed when the extracellular Cl– concentration was reduced to 5 mmol/liter (GAmil = 59.9 ± 8.2 µS; n = 9). All experiments were performed using a bath clamp, which minimizes voltage drops along the series resistances. However, we included two sets of control experiments. (i) We permeabilized ENaC expressing oocytes with 10 µM nystatin, which induced a nonselective conductance without affecting amiloride-sensitive membrane currents (Fig. 1D). GAmil was 19.3 ± 2.7 and 23.3 ± 3 µS (n = 7) in the absence and presence of nystatin, respectively. (ii) ENaC was coexpressed with KVLQT1 K+ channels, and amiloride-sensitive currents were assessed in the absence or presence of the K+ channel blocker Ba2+ (5 mM), which did not affect Na+ transport (Fig. 1E): GAmil was 7.9 ± 1.3 and 7.5 ± 1.1 µS (n = 9) in the absence and presence of Ba2+, respectively. Thus, additional currents do not compromise the measurement of ENaC currents in our setup. Cl– currents generated by CFTR but not non-selective currents or K+ currents inhibited ENaC.



View larger version (47K):
[in this window]
[in a new window]
  		FIG. 1.
CFTR inhibits ENaC in Xenopus oocytes. A, original recording of the whole cell current in a Xenopus oocyte coexpressing rat {alpha}{beta}{gamma}ENaC together with human CFTR. Whole cell currents were measured during continuous voltage clamp from –90 to +30 mV in steps of 10 mV. CFTR Cl– currents were activated by IBMX/forskolin (1 mM/2 µM) and were measured in the presence of a high (100 mM) or low (5 mM) bath Cl– concentration. The effects of amiloride (A, 10 µM) on whole cell currents were largely inhibited after activation of CFTR and in the presence of high bath Cl– concentration. B, summary of the effects of IBMX/forskolin (I/F) on amiloride-sensitive conductance (GAmil) in coexpressing oocytes. C, typical current/voltage relationships of coexpressing oocytes before and after stimulation with IBMX/forskolin and the effect of amiloride. D, original recording of the whole cell current in an ENaC expressing oocyte and the effect of amiloride before and after inducing a nonselective conductance with nystatin (10 µM). E, original recording of the whole cell current in a Xenopus oocyte coexpressing rat {alpha}{beta}{gamma}ENaC together with human KVLQT1 K+ channels. Asterisk (*) indicates significant difference (paired t test). n, number of experiments.

 
We examined the Cl– dependence of the CFTR effect on ENaC in more detail and found that in oocytes, in which CFTR had been activated by IBMX/forskolin, a gradual increase in the bath Cl– concentration from 2 to 100 mM, gradually inhibited ENaC currents, in parallel to the increase of CFTR Cl– currents (Fig. 2, A and C). Moreover, we coexpressed the Cl– channel CLC-0 together with ENaC and found a comparable decrease of ENaC currents with increasing bath Cl– concentrations (Fig. 2, B and D). Thus, Cl– ions play an essential role in both CFTR, and CLC-0 induced inhibition of ENaC. When ENaC was inhibited by amiloride, oocyte membranes are dominated by a Cl– conductance. Thus it is possible to measure the Cl– diffusion potential and to calculate for the apparent intracellular Cl– concentration. We found that in the presence of activated CFTR or when CLC-0 was coexpressed, increase in bath Cl– from 5 to 100 mM resulted in a change of the apparent intracellular Cl– concentration from about 10 to roughly 60 mM (Fig. 2, E and F). In parallel, ~50% of the initial amiloride-sensitive Na+ current was inhibited, indicating that an increase in cytosolic Cl– inhibits ENaC. We observed a recovery of amiloride-sensitive conductances over the time course of 30 min, when bath Cl– was reduced from 100 to 5 mM in ENaC/CLC-0 coexpressing oocytes. Finally, injection of 23 nl of 50 mM NaCl also inhibited ENaC, whereas injection of water or 5 mM NaCl had no effect (data not shown). In summary, increase in intracellular Cl– inhibits ENaC, which may be in charge of the inhibitory effects of CFTR or CLC-0 on ENaC.

This was further confirmed in experiments with macropatches from ENaC expressing oocytes, after verifying expression by double electrode voltage clamp. Patch pipettes were filled with 1 mM diphenylcarboxylate, to minimize endogenous Cl– currents. A current noise was recorded from excised inside/out macropatches, which was largely increased when the cytosolic Cl– concentration was reduced from 100 to 5 mM (Fig. 3, A and B). 95 mM Na+ were replaced by NMDG+ in the presence of low or high bath Cl–. We found that the NMDG+ that inhibited Na+ conductance in macropatches was 86.2 ± 6.2 microsiemens in the presence of 5 mM cytosolic Cl–, which was significantly larger when compared with that observed at 100 mM Cl– (27.3 ± 2.1 microsiemens). We then exposed the cytosolic side of the macropatches to different Cl– concentrations, ranging from 5 to 100 mM. As shown in Fig. 3B, a gradual increase in cytosolic Cl– inhibited the conductance of excised inside out membrane patches. Finally, we confirmed expression of ENaC in excised outside/out patches and found inhibition of membrane conductance by amiloride (10 µM) from 228 ± 70 to 140 ± 60 microsiemens (n = 5). Thus ENaC may be directly inhibited by Cl– ions or attached Cl–-sensitive proteins control ENaC activity.



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 2.
Cl–-dependent inhibition of ENaC by CFTR and CLC-0. Original recording of the whole cell current in Xenopus oocytes coexpressing {alpha}{beta}{gamma}ENaC together with CFTR (A) or CLC-0 (B). Whole cell currents were measured during continuous voltage clamp from –90 to +30 mV in steps of 10 mV. CFTR Cl– currents were activated by IBMX/forskolin (I/F) (1 mM/2 µM). Inhibition of whole cell currents by amiloride (A, 10 µM) were examined in the presence of bath Cl– concentrations ranging from 2 to 100 mM. Summary of the CFTR (C) and CLC-0 (D) induced whole cell conductances and amiloride-sensitive whole cell conductances at different bath Cl– concentrations. Relationship between amiloride-sensitive whole cell conductance and calculated intracellular Cl– concentration in CFTR (E) and CLC-0 (F) expressing oocytes. Asterisk (*) indicates significant difference (paired t test). n, number of experiments.

 
We examined if the change in cytosolic Cl– affects membrane expression of ENaC. To that end we expressed FLAG-tagged ENaC subunits together with CFTR or CLC-0 and exposed the oocytes to 5 or 100 mM bath Cl– concentration. For these experiments CFTR had been pre-activated by IBMX and forskolin. FLAG-tagged ENaC subunits were detected by staining with primary anti-FLAG and secondary fluorescein isothiocyanate-conjugated antibodies. There was no obvious change in fluorescence during exposure to high or low extracellular Cl– (data not shown). We quantified surface expression of individual ENaC subunits by chemiluminescence and found no change in membrane expression in ENaC/CFTR (Fig. 4A) or ENaC/CLC-0 (Fig. 4B) coexpressing oocytes, during change of the bath Cl– concentration from 5 to 100 mM Cl–. Chemiluminescence was also measured for {beta}ENaC and {gamma}ENaC mutants, in which 2 ({beta}2N, {gamma}2N) or 4 ({beta}4N, {gamma}4N) positively charged amino acids in the NH2 terminus have been replaced by neutral amino acids. These amino acids are located in PIP2 binding sides of {beta}ENaC and {gamma}ENaC and control ENaC activity (42, 44, 45). However, membrane expression of the mutant ENaCs was not affected by changes in the bath Cl– concentration (Fig. 4, A and B). To further validate the chemiluminescence methods used here, we expressed FLAG-tagged ENaC subunits, which were truncated either at the COOH terminus of either {beta}ENaC (R561X, {beta}{Delta}C) or {gamma}ENaC (Ser-608, {gamma}{Delta}C). These truncations have been shown to lead to enhanced membrane expression of ENaC. In fact, expression of {beta}{Delta}C and {gamma}{Delta}C was enhanced 1.70 ± 0.2 (n = 15) and 1.73 ± 0.3 (n = 15)-fold, respectively, when compared with WT {beta}-ENaC and WT {gamma}-ENaC. Moreover, when {beta}{Delta}C and {gamma}{Delta}C were coexpressed together with CLC-0 or CFTR, membrane expression was not affected by a change of bath Cl– from 5 to 100 mM (n = 15 for all series, data not shown).

The role of Cl– for inhibition of ENaC was further confirmed by blocking CLC-0 channels with 500 µM Zn2+ (46). Zn2+ inhibited about 50% of the CLC-0 conductance in 100 mM extracellular Cl– and enhanced amiloride-sensitive conductance (18.3 ± 4.3 versus 26.3 ± 4.5 µS). Zn2+ applied in a low bath Cl– concentration abolished CLC-0 conductance completely and further enhanced ENaC conductance (29.1 ± 4.4 µS, n = 9). Zn2+ itself had no effects on ENaC. We examined if the effect of Cl– on ENaC can be mimicked by other anions and therefore replaced bath Cl– by equimolar concentrations of NO–3, I–, or Br– (Fig. 5A). CFTR conductance and inhibition of ENaC were detected in the presence of the different anions, but were both reduced in the presence of extracellular I– (Fig. 5, B and C). ENaC is inhibited by an increase in intracellular Na+ (31). This was reported as feedback inhibition and is abolished by ENaC mutations occurring in Liddles disease (18). Because both intracellular Cl– and Na+ may change in parallel, we examined if Cl–-dependent inhibition of ENaC is also observed with ENaC carrying Liddles disease mutations or truncations in the COOH terminus of all three subunits (Fig. 5, D and E). The ENaC mutants were coexpressed either with CFTR (Fig. 5D) or with CLC-0 (Fig. 5E). The summaries show that the mutant channels were also inhibited by Cl– in a concentration-dependent manner, although inhibition appeared somewhat reduced when compared with wtENaC. Thus, Cl– dependence can be clearly separated from Na+-dependent inhibition.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 3.
Cl–-dependent inhibition of ENaC in macropatches. A, current noise in an inside out macropatch of a ENaC expressing oocyte at different clamp voltages and in the presence of high (100 mM) or low (5 mM) cytosolic Cl– concentration. B, Cl– dependence of the conductance of inside/out macropatches of ENaC expressing oocytes. C, current/voltage relationships of the current noise in inside/out macropatches of ENaC expressing oocytes in the presence of low or high cytosolic Cl– concentration. n, number of experiments.

 



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 4.
Expression of FLAG-tagged ENaC-subunits. Detection of surface expression of ENaC subunits by chemiluminescence in ENaC/CFTR (A) or ENaC/CLC-0 (B) coexpressing oocytes. FLAG-tagged ENaC subunit were detected by staining with a primary anti-FLAG and secondary IgG peroxidase-linked antibody. Chemiluminescence was integrated over 1 s and is expressed as surface expression relative to that of {alpha}-ENaC at 100 mM Cl–. Chemiluminescence was assessed in a bath solution containing 100 (black bars) or 5 mM (white bars) Cl–. {beta}2N, {gamma}2N and {beta}4N, {gamma}4N indicate that 2 or 4 positively charged amino acids have been removed from the NH2 terminus of either {beta}ENaC or {gamma}ENaC. The number of experiments was for each series between 5 and 15.

 



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 5.
Anion-dependent inhibition of ENaC. A, original recording of the whole cell current in a oocyte coexpressing ENaC and CFTR and the effect of amiloride in the presence of different anions in the bath. Summary of the CFTR conductance (B) and inhibition of amiloride-sensitive Na+ conductance (C) in the presence of different anions in the bath. D, summary of the CFTR conductance and inhibition of little mutants of the amiloride-sensitive Na+ channel in the presence of different bath Cl– concentrations. E, summary of the CLC-0 conductance and inhibition of little mutants of the amiloride-sensitive Na+ channel in the presence of different bath Cl– concentrations. Asterisk (*) indicates significant difference (paired t test). n, number of experiments. Fors, forskolin.

 
ENaC is inhibited by CFTR and by stimulation of puringeric receptors with ATP or UTP, which leads to hydrolysis of PIP2 via activation of phospholipase C{beta} (42). We previously found a Cl– dependence also for purinergic inhibition of ENaC (47) and therefore asked whether both mechanisms are additive. To that end we measured Na+ absorption in polarized epithelial cells of the collecting duct, which express CFTR, ENaC, and Ca2+-activated Cl– channels along with purinergic receptors. ENaC was shown to be controlled by CFTR and purinergic receptors in these cells (10, 42, 48). In Ussing chamber experiments we observed attenuation of amiloride-sensitive transport after activation of CFTR by IBMX and forskolin, indicating inhibition of ENaC by CFTR (Fig. 6A). After preinhibition of ENaC by purinergic stimulation, the amiloride-sensitive short circuit current was further reduced by 1.6 ± 0.3 µA/cm2 when CFTR was activated. This, however, was significantly less than inhibition of ENaC by CFTR without purinergic pre-stimulation (3.9 ± 0.4 µA/cm2, n = 6). Moreover when phospholipase C{beta} was inhibited by U73122 [GenBank] (10 µM), inhibition of amiloride-sensitive transport by CFTR was significantly enhanced (7.8 ± 0.9 µA/cm2, n = 8). This data suggest that both inhibitory mechanisms are additive. The NH2 terminus of {beta}- and {gamma}ENaC play a central role for the purinergic regulation of ENaC and may also participate in CFTR and Cl– dependent control of ENaC. We examined this issue in ENaC and CLC-0 expressing Xenopus oocytes and performed maneuvers, which have been shown to affect PIP2 binding and thus ENaC activity (42). Injection of PIP2 (30 µM, 30 nl) slightly but significantly enhanced amiloride-sensitive whole cell currents. This additional current was inhibited by an increase in intracellular Cl– (Fig. 6B). Control injection of oocytes with water had no effects. In contrast, injection of a PIP2 antibody (1:100, 30 nl) slightly reduced GAmil and the remaining current was inhibited by Cl– to the same level as before injection (Fig. 6C). Moreover, both injection of phospholipase C (2 units/mg, 30 nl) or incubation of the oocytes in pH 6.0 for 1 h largely reduced GAmil and the amount of GAmil blocked by Cl– (Fig. 6, D and E). These results suggest that PIP2 binding of {beta}- and probably {gamma}ENaC determines the amount of ENaC current inhibited by Cl– ions.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 6.
Inhibition of ENaC by ATP and Cl–. Continuous recording of the transepithelial voltage (Vte) of M1 collecting duct cells in a perfused micro-Ussing chamber, and effects of amiloride (A; 10 µmol/l) under control conditions and after activation of CFTR by IBMX and forskolin. B–E, change in the intracellular Cl– concentration (Cl–i) by an increase in the bath Cl– concentration from 5 to 100 mM in CLC-0/ENaC coexpressing oocytes and change in the amiloride-sensitive Na+ conductance. Effects of injection of PIP2 (23 nl, 25 µM, n = 16) (B), injection of PIP2 antibody (23 nl, 1:100, n = 6) (C), injection of PLC (23 nl, 2 units/ml, n = 9) (D), or incubation for 30 min at pH 6.0; n = 26 (E). Dashed lines indicate inhibition of ENaC by an increase in Cl–i in control oocytes (n = 10-26). Asterisk (*) indicates significant difference (paired t test). Number sign (#) indicates significant difference in Na+ conductance and slope for Cl–-dependent inhibition (unpaired t test).

 
We further tried to identify the ENaC subunits and cytosolic domains that participate in Cl– dependent regulation. To that end, {alpha}{beta}{gamma}-subunits were truncated at amino ({Delta}N) or carboxyl ({Delta}C) termini and coexpressed in various combinations. Moreover, PIP2 binding domain mutants of {beta}- and {gamma}ENaC were generated by eliminating 2 or 4 positively charged amino acids ({beta}2N, {beta}4N, {gamma}2N, and {gamma}4N) (42). ENaC subunits were coexpressed together with CLC-0 or CFTR, activated with IBMX and forskolin. The whole cell currents generated by CLC-0 or CFTR in the presence of 100 mM extracellular Cl– were between 5 and 10 µA and were reduced by replacing extracellular Cl– by gluconate (data not shown). Because virtually identical results were obtained for coexpression of ENaC with ClC-0 and CFTR and for {alpha}WT, {alpha}{Delta}C, {alpha}P671A, the data are not listed separately. Fig. 7A shows the ratio of GAmil generated by mutant ENaC to GAmil produced by WT ENaC at low (5 mM) bath Cl– (black bars). The white bars indicate the ratio of the inhibition of mutant ENaC to inhibition of WT ENaC by an increase in bath Cl– to 100 mM (white bars). It is shown that individual truncations ({Delta}C) of the carboxyl terminus of {alpha}-, {beta}-, and {gamma}ENaC in trimeric channels lead to enhanced Na+ currents compared with WT ENaC, but attenuated down-regulation by Cl– only for {beta}{Delta}C. Interestingly, the double mutant {beta}{Delta}C{gamma}{Delta}C showed reduced ENaC currents and down-regulation by Cl–. In contrast, truncation of the amino terminus reduced ({beta}{Delta}N, {gamma}{Delta}N) or even abolished ({alpha}{Delta}N) ENaC currents. Individual mutations in the PIP2 binding domain ({beta}2N, {beta}4N, {gamma}2N, and {gamma}4N) inhibited ENaC currents, particularly in {beta}ENaC, but did not affect Cl– inhibition. The double mutant {beta}4N{gamma}4N did not produce significant whole cell currents. As further shown, expression of various other combinations of double mutants showed compromised inhibition of ENaC by Cl–, when the carboxyl terminus of {beta}ENaC was truncated.

Expression of {alpha}ENaC alone generated a small but significant Na+ current, which was not down-regulated by Cl– (Fig. 7B). Additional mutations in the carboxyl terminus of {alpha}ENaC did not change Cl– sensitivity (data not shown). ENaC currents were reduced in dimeric channels formed by {alpha}ENaC together with {beta}ENaC or {gamma}ENaC, but were all inhibited by Cl–, except of the dimeric channel containing {beta}{Delta}C (Fig. 7B). Taken together, the results suggest that (i) {alpha}ENaC does not participate in the control of ENaC by intracellular Cl–, (ii) the COOH terminus of {beta}ENaC confers Cl– sensitivity and has a central role for the regulation of ENaC by CFTR, and (iii) both NH2 termini of {beta}ENaC and {gamma}ENaC containing the PIP2 binding motif determine the activity of ENaC, and the amount of ENaC controlled by Cl–.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present results clearly demonstrate inhibition of amiloride-sensitive Na+ currents by activation of CFTR or CLC-0 Cl– channels. In contrast, ENaC currents were not affected by a parallel K+ conductance or by a nonselective conductance induced by nystatin. Inhibition of ENaC by CFTR has been questioned in two studies and it was suggested that the inhibitory effect of CFTR on ENaC could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, such as a large series resistance and/or insufficient feedback voltage gain (49, 50). However, several facts argue against the possibility of a series resistance error as an explanation for the present and previous results: (i) in contrast to Refs. 49 and 50, CFTR and ENaC currents were of a much smaller amplitude in the previous reports (8, 9, 12, 14–16, 18, 26, 30, 35, 47). (ii) Inhibition of ENaC by CFTR was observed at whole cell currents as low as 1.5 µA (8, 9, 28, 29), which is roughly 10-fold below the currents reported in Refs. 49 and 50. (iii) Experiments were performed with large dimension, low resistance KCl electrodes, avoiding a significant artificial voltage drop along series resistances (8, 9, 14, 16). (iv) Experiments performed with different oocyte voltage clamp amplifiers and two bath electrodes with compensation circuit confirmed previous results (22, 26, 30, 47). (v) Including the present report, a roughly 50% inhibition of ENaC currents by activation of CFTR was found in most of the previous studies (8, 9, 15, 18–20, 27, 30). (vi) Coexpression of large K+ conductances together with ENaC should exert the same artifact as activation of CFTR Cl– currents according to Ref. 50. This, however, was not observed in the present and a previous report (19). (vii) Partial permeabilization of oocytes with amphotericin B, which induced a large leak conductance did not compromise detection of GAmil (30). (viii) Partial permeabilization of oocytes with nystatin in the present study, or coexpression of another parallel K+ conductance did not compromise the measurement of GAmil. Taken together, it is rather unlikely that the reported inhibition of ENaC by CFTR is a result from compromised measurements of whole cell currents in Xenopus oocytes.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 7.
Summary of double voltage clamp experiments with oocytes coexpressing WT ENaC or various ENaC mutants in different combinations together with CFTR of CLC-0. CFTR was preactivated with IBMX (1 mM) and forskolin (2 µM). Black bars indicate the ratio of the conductance generated by mutant ENaC to that produced by WT ENaC at low (5 mM) bath Cl– concentration. White bars indicate the ratio for the inhibition of mutant ENaC to that of WT ENaC by an increase of bath Cl– from 5 to 100 mM. A, all three {alpha}{beta}{gamma}ENaC subunits were coexpressed to/from trimeric channels. B, monomeric channels were formed by {alpha}ENaC only, or dimeric channels were formed by {alpha}{beta}ENaC or {alpha}{gamma}ENaC. Asterisk (*) indicates significant effects of amiloride (black bars) and significant inhibition of ENaC during an increase of Cl– from 5 to 100 mM (white bars; paired t test). Number sign (#) indicates significant difference in the amplitude of amiloride-sensitive currents generated by mutant ENaC when compared with WT ENaC (black bars) and significantly attenuated inhibition of mutant ENaC by Cl– (white bars, unpaired t test). The number for each series of experiments was n = 6–36.

 
It was discussed that in epithelial tissues such as the reabsorptive sweat duct, where both CFTR and ENaC reside in the same apical membrane, the absence of CFTR necessarily reduces absorption of Na+ because of coupled movement of both Cl– and Na+ (49–51). Whereas this may accurately describe the situation for a tight epithelium such as the sweat duct, where all electrolyte transport occurs transcellularly, the situation is quite different for the airways, which is a leaky epithelium unable to maintain larger osmotic gradients (52, 53). Additional Na+ conductance, as present in the airways of cystic fibrosis patients, will necessarily lead to an increase in Na+ and Cl– absorption, with consecutive loss of airway surface liquid and reduced mucociliary clearance. This has been convincingly demonstrated in a novel transgenic mouse model with airway-specific overexpression of ENaC. In contrast to CFTR-transgenic animals, these mice reproduce a lung disease with all typical features of a CF lung, including an increased salt absorption and reduced airway surface liquid (54). Thus, enhanced electrolyte absorption because of increased Na+ conductance in the airways takes place even in the absence of an additional Cl– selective shunt.

As outlined above, an inhibitory effect of CFTR on ENaC has been detected under very different experimental conditions and accordingly enhanced Na+ absorption has been detected in CF tissues, including human airways and intestine (3–6, 55). In contrast, CFTR knock-out mice do not demonstrate a CF lung disease (56). In the mouse, expression of CFTR takes place in the nasal epithelium, but is essentially irrelevant in trachea and more distal airways (57). Mouse airways are obviously dominated by a Ca2+-activated Cl– conductance and thus lack of CFTR does not affect ion transport in the airways, except the nasal epithelium. Consequently, enhanced Na+ absorption is found in CFTR knock-out mice only in the nasal epithelium, but not in the remaining airways (58, 59). A thorough analysis of the CFTR expression in human airways using a new high affinity CFTR antibody revealed that CFTR protein is expressed in the apical membrane of ciliated cells within the superficial epithelium and in gland ducts, but hardly detectable in the gland acinus and alveolar epithelium. This suggests an essential role of CFTR for the regulation of Na+ absorption (60).

The present results demonstrate an increase in the intracellular Cl– concentration in ENaC expressing oocytes, when a parallel Cl– conductance and a high bath Cl– concentration is present. This is not surprising, because a large Cl– shunt allows for electroneutral uptake of NaCl into the oocyte. The results show that Cl– or other anions have an inhibitory effect on ENaC, when accumulating on the cytosolic side. It should be pointed out that this inhibitory effect of Cl– ions on epithelial Na+ channels confirms results from earlier studies on mandibular duct cells, which shows an inhibitory effect of Cl– on epithelial Na+ conductance (31–33). Our results are also in excellent agreement with a previous report showing that deletion of the cytoplasmic NH2 terminus of {alpha}-, {beta}-, and {gamma}ENaC dramatically reduces GAmil (61). The present results confirm that the carboxyl terminus of {beta}ENaC is crucial for down-regulation of ENaC by CFTR (15). We conclude that the inhibitory effect of CFTR on ENaC is at least in part Cl– mediated. Evidence is presented that cytosolic domains of ENaC, in a complex and not yet fully understood, participate in this regulation, not excluding the possible role of partner proteins. Moreover, it is possible that additional partner proteins may form a complex together with ENaC and CFTR, possibly in raft-like lipid platforms (62, 63). Although participation of some anion-sensitive proteins has been excluded recently (33, 47), others have not yet been examined. In this regard, the recently discussed metabolic coupling of ENaC and CFTR via AMP-activated protein kinases would provide a good candidate (64, 65).


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grant Ku756/7-1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 49-0-941-943-4302; Fax: 49-0-941-943-4315; E-mail: uqkkunze{at}mailbox.uq.edu.au.

1 The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; IBMX, 3-isobutyl-1-methylxanthine; ENaC, epithelial Na+ channel; CF, cystic fibrosis; BSA, bovine serum albumin; WT, wild-type; PIP2, phosphatidylinositol bisphosphate; NMDG, N-methyl-D-glucamine. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kunzelmann, K. (1999) Rev. Physiol. Biochem. Pharmacol. 137, 1–70[Medline] [Order article via Infotrieve]
  2. Schwiebert, E. M., Benos, D. J., Egan, M. E., Stutts, M. J., and Guggino, W. B. (1999) Physiol. Rev. 79, S145–S166[Medline] [Order article via Infotrieve]
  3. Boucher, R. C., Cotton, C. U., Gatzy, J. T., Knowles, M. R., and Yankaskas, J. R. (1988) J. Physiol. (Lond.) 405, 77–103[Abstract/Free Full Text]
  4. Kunzelmann, K., Kathöfer, S., and Greger, R. (1995) Pflügers Arch. 431, 1–9[CrossRef][Medline] [Order article via Infotrieve]
  5. Mall, M., Bleich, M., Greger, R., Schreiber, R., and Kunzelmann, K. (1998) J. Clin. Investig. 102, 15–21[Medline] [Order article via Infotrieve]
  6. Mall, M., Bleich, M., Kühr, J., Brandis, M., Greger, R., and Kunzelmann, K. (1999) Am. J. Physiol. 277, G709–G716[Medline] [Order article via Infotrieve]
  7. Stutts, M. J., Canessa, C. M., Olsen, J. C., Hamrick, M., Cohn, J. A., Rossier, B. C., and Boucher, R. C. (1995) Science 269, 847–850[Abstract/Free Full Text]
  8. Mall, M., Hipper, A., Greger, R., and Kunzelmann, K. (1996) FEBS Lett. 381, 47–52[CrossRef][Medline] [Order article via Infotrieve]
  9. Briel, M., Greger, R., and Kunzelmann, K. (1998) J. Physiol. (Lond.) 508, 825–836[Abstract/Free Full Text]
  10. Letz, B., and Korbmacher, C. (1997) Am. J. Physiol. 272, C657–C666[Medline] [Order article via Infotrieve]
  11. Ismailov, I. I., Awayda, M. S., Jovov, B., Berdiev, B. K., Fuller, C. M., Dedman, J. R., Kaetzel, M. A., and Benos, D. J. (1996) J. Biol. Chem. 271, 4725–4732[Abstract/Free Full Text]
  12. Jiang, Q., Li, J., Dubroff, R., Ahn, Y. J., Foskett, J. K., Engelhardt, J., and Kleyman, T. R. (2000) J. Biol. Chem. 275, 13266–13274[Abstract/Free Full Text]
  13. Konstas, A. A., Koch, J. P., Tucker, S. J., and Korbmacher, C. (2002) J. Biol. Chem. 277, 25377–25384[Abstract/Free Full Text]
  14. Kunzelmann, K., Kiser, G., Schreiber, R., and Riordan, J. R. (1997) FEBS Lett. 400, 341–344[CrossRef][Medline] [Order article via Infotrieve]
  15. Ji, H. L., Chalfant, M. L., Jovov, B., Lockhart, J. P., Parker, S. B., Fuller, C. M., Stanton, B. A., and Benos, D. J. (2000) J. Biol. Chem. 275, 27947–27951[Abstract/Free Full Text]
  16. Schreiber, R., Hopf, A., Mall, M., Greger, R., and Kunzelmann, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 5310–5315[Abstract/Free Full Text]
  17. Berdiev, B. K., Shlyonsky, V. G., Karlson, K. H., Stanton, B. A., and Ismailov, I. I. (2000) Biophys. J. 78, 1881–1894[Abstract/Free Full Text]
  18. Hopf, A., Schreiber, R., Greger, R., and Kunzelmann, K. (1999) J. Biol. Chem. 274, 13894–13899[Abstract/Free Full Text]
  19. Konstas, A. A., Koch, J. P., and Korbmacher, C. (2003) Pflugers Arch. 445, 513–521[Medline] [Order article via Infotrieve]
  20. Yan, W., Samaha, F. F., Ramkumar, M., Kleyman, T. R., and Rubenstein, R. C. (2004) J. Biol. Chem. 279, 23183–23192[Abstract/Free Full Text]
  21. Suaud, L., Carattino, M., Kleyman, T. R., and Rubenstein, R. C. (2002) J. Biol. Chem. 277, 50341–50347[Abstract/Free Full Text]
  22. Suaud, L., Li, J., Jiang, Q., Rubenstein, R. C., and Kleyman, T. R. (2001) J. Biol. Chem. 277, 8928–8933[Medline] [Order article via Infotrieve]
  23. Wang, S., Raab, R. W., Schatz, P. J., Guggino, W. B., and Li, M. (1998) FEBS Lett. 427, 103–108[CrossRef][Medline] [Order article via Infotrieve]
  24. Moyer, B. D., Denton, J., Karlson, K. H., Reynolds, D., Wang, S., Mickle, J. E., Milewski, M., Cutting, G. R., Guggino, W. B., Li, M., and Stanton, B. A. (1999) J. Clin. Investig. 104, 1353–1361[Medline] [Order article via Infotrieve]
  25. Moyer, B. D., Duhaime, M., Shaw, C., Denton, J., Reynolds, D., Karlson, K. H., Pfeiffer, J., Wang, S., Mickle, J. E., Milewski, M., Cutting, G. R., Guggino, W. B., Li, M., and Stanton, B. A. (2000) J. Biol. Chem. 275, 27069–27074[Abstract/Free Full Text]
  26. Boucherot, A., Schreiber, R., and Kunzelmann, K. (2001) Biochem. Biophys. Acta 1515, 64–71[Medline] [Order article via Infotrieve]
  27. Schreiber, R., Boucherot, A., Mürle, B., Sun, J., and Kunzelmann, K. (2004) J. Membr. Biol. 199, 89–98
  28. König, J., Schreiber, R., Mall, M., and Kunzelmann, K. (2002) Biochim. Biophys. Acta 1565, 17–28[Medline] [Order article via Infotrieve]
  29. Schreiber, R., König, J., Sun, J., Markovich, D., and Kunzelmann, K. (2003) J. Membr. Biol. 192, 101–110[CrossRef][Medline] [Order article via Infotrieve]
  30. König, J., Schreiber, R., Voelcker, T., Mall, M., and Kunzelmann, K. (2001) EMBO Rep. 2, 1–5[Medline] [Order article via Infotrieve]
  31. Dinudom, A., Young, J. A., and Cook, D. I. (1993) J. Membr. Biol. 135, 289–295[Medline] [Order article via Infotrieve]
  32. Dinudom, A., Komwatana, P., Young, J. A., and Cook, D. I. (1995) J. Physiol. (Lond.) 487, 549–555[Medline] [Order article via Infotrieve]
  33. Hübner, M., Schreiber, R., Boucherot, A., Sanchez-Perez, A., Poronnik, P., Cook, D. I., and Kunzelmann, K. (1999) FEBS Lett. 459, 443–447[CrossRef][Medline] [Order article via Infotrieve]
  34. Komwatana, P., Dinudom, A., Young, J. A., and Cook, D. I. (1998) J. Membr. Biol. 162, 225–232[CrossRef][Medline] [Order article via Infotrieve]
  35. Chabot, H., Vives, M. F., Dagenais, A., Grygorczyk, C., Berthiaume, Y., and Grygorczyk, R. (1999) J. Membr. Biol. 169, 175–188[CrossRef][Medline] [Order article via Infotrieve]
  36. Niisato, N., Eaton, D. C., and Marunaka, Y. (2004) Am. J. Physiol. Renal Physiol. 287, F932–F939[Abstract/Free Full Text]
  37. Xie, Y., and Schafer, J. A. (2004) Am. J. Physiol. 287, F722–F731
  38. Adam, G., Ousingsawat, J., Schreiber, R., and Kunzelmann, K. (2005) Pflügers Arch. 449, 470–478[CrossRef][Medline] [Order article via Infotrieve]
  39. Kunzelmann, K. (2003) Pflügers Arch. 445, 505–512
  40. Kieferle, S., Fong, P., Bens, M., Vandewalle, A., and Jentsch, T. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6943–6947[Abstract/Free Full Text]
  41. Firsov, D., Schild, L., Gautschi, I., Merillat, A. M., Schneeberger, E., and Rossier, B. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 15370–15375[Abstract/Free Full Text]
  42. Kunzelmann, K., Bachhuber, T., Regeer, R. R., Markovich, D., Sun, J., and Schreiber, R. (2004) FASEB J. 18, 142–163
  43. Kunzelmann, K. (1997) Kidney Blood Press. Res. 20, 151–153[Medline] [Order article via Infotrieve]
  44. Yue, G., Malik, B., and Eaton, D. C. (2002) J. Biol. Chem. 277, 11965–11969[Abstract/Free Full Text]
  45. Ma, H. P., Saxena, S., and Warnock, D. G. (2002) J. Biol. Chem. 277, 7641–7644[Abstract/Free Full Text]
  46. Chen, T. Y. (1998) J. Gen. Physiol. 112, 715–726[Abstract/Free Full Text]
  47. Kunzelmann, K., and Boucherot, A. (2001) Kidney Int. 60, 455–461[CrossRef][Medline] [Order article via Infotrieve]
  48. Cuffe, J. E., Bielfeld-Ackermann, A., Thomas, J., Leipziger, J., and Korbmacher, C. (2000) J. Physiol. (Lond.) 524, 77–90[Abstract/Free Full Text]
  49. Nagel, G., Szellas, T., Riordan, J. R., Frierich, T., and Hartung, K. (2001) EMBO Rep. 2, 249–254[CrossRef][Medline] [Order article via Infotrieve]
  50. Nagel, G., Barbry, P., Chabot, H., Brochiero, E., Hartung, K., and Grygorczyk, R. (2005) J. Physiol. 564, 671–682[Abstract/Free Full Text]
  51. Reddy, M. M., and Quinton, P. M. (1999) Nature 402, 301–304[CrossRef][Medline] [Order article via Infotrieve]
  52. Boucher, R. C. (1999) J. Physiol. 516, 631–638[Abstract/Free Full Text]
  53. Matsui, H., Davis, C. W., Tarran, R., and Boucher, R. C. (2000) J. Clin. Investig. 105, 1419–1427[Medline] [Order article via Infotrieve]
  54. Mall, M., Grubb, B. R., Harkema, J. R., O'Neal, W. K., and Boucher, R. C. (2004) Nat. Med. 10, 452–454[CrossRef][Medline]