Cystic Fibrosis Transmembrane Conductance Regulator Inhibits
Epithelial Na+ Channels Carrying Liddle's Syndrome
Mutations*
Anna
Hopf,
Rainer
Schreiber,
Marcus
Mall,
Rainer
Greger, and
Karl
Kunzelmann
From the Physiologisches Institut, Albert-Ludwigs-Universität
Freiburg, Hermann-Herder-Straße 7, 79104 Freiburg, Germany
 |
ABSTRACT |
Epithelial Na+ channels (ENaC)
are inhibited by the cystic fibrosis transmembrane conductance
regulator (CFTR) upon activation by protein kinase A. It is, however,
still unclear how CFTR regulates the activity of ENaC. In the present
study we examined whether CFTR interacts with ENaC by interfering with
the Nedd4- and ubiquitin-mediated endocytosis of ENaC. Various
C-terminal mutations were introduced into the three
-,
-, and
-subunits of the rat epithelial Na+ channel, thereby
eliminating PY motifs, which are important binding domains for the
ubiquitin ligase Nedd4. When expressed in Xenopus oocytes,
most of the ENaC stop (
-H647X,
-P565X,
-S608X) or point
(
-P671A,
-Y618A,
-P(624-626)A) mutations induced enhanced Na+ currents when compared with wild type
,
,
-rENaC. However, ENaC currents formed by either of the
mutant
-,
-, or
-subunits were inhibited during activation of
CFTR by forskolin (10 µmol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l). Antibodies to dynamin or ubiquitin enhanced
,
,
-rENaC
whole cell Na+ conductance but did not interfere with
inhibition of ENaC by CFTR. Another mutant,
-T592M,T593A-ENaC, also showed enhanced Na+
currents, which were down-regulated by CFTR. Moreover, activation of
ENaC by extracellular proteases and xCAP1 does not disturb CFTR-dependent inhibition of ENaC. We conclude that
regulation of ENaC by CFTR is distal to other regulatory limbs and does
not involve Nedd4-dependent ubiquitination.
 |
INTRODUCTION |
Recent studies have indicated that epithelial Na+
channels (ENaC)1 are
inhibited during activation of Cl
secretion in human
airways and the colonic epithelium
(1).2 These tissues coexpress
both CFTR and
,
,
-rENaC and demonstrate that CFTR, when
activated by cAMP, inhibits epithelial Na+ channels. In
fact, recent studies in cells expressing both recombinant CFTR and ENaC
indicate CFTR-dependent inhibition of ENaC (3, 4).
Currently, several models for regulation may be proposed: (i) we have
shown recently that CFTR and
,
,
-rENaC may interact directly
(5). Two independent groups detected inhibition of ENaC single channel
currents by CFTR in isolated membranes (6, 7), while other studies
demonstrate inhibition of ENaC by increase of cytosolic
Cl
activities (8, 9). The results of the latter study are supported by the notion that Cl
movement through the
activated CFTR Cl
channel is essential for the inhibition
of ENaC (10). Very recently, a C-terminal CFTR domain was detected that
binds to PDZ domains of proteins that anchor CFTR to the cytoskeleton
(11, 12). This could provide a potential mechanism through which CFTR
can affect the activity of other membrane proteins. Along this line,
the cytoskeleton has been suggested for a long time to participate in
the regulation of the epithelial Na+ channel (13).
Nedd4-dependent ubiquitination turned out to be an
important regulatory pathway for ENaC that is also of clinical
relevance in Liddle's disease (14-16). Regulation by Nedd4 includes
binding of Nedd4 to a PY motif in the C terminus of all three ENaC
subunits via WW domain interaction with subsequent ubiquitination- and dynamin-mediated endocytosis of ENaC (17-19). In the present paper we
addressed the question to what extent the PY motifs in the three rENaC
subunits contribute to the CFTR-dependent regulation of
rENaC and whether elimination of respective motifs interferes with the
ability of CFTR to down-regulate rENaC. This seems to be of particular
importance since Liddle's disease patients do not suffer from
pulmonary symptoms that would be caused by an enhanced Na+
conductance in the airways along with hyperabsorption of the airway
surface fluid. In addition, preliminary results demonstrate inhibition
of
,
,
573X-ENaC by CFTR in Madin-Darby canine kidney cells
(20). In this study we coexpressed various ENaC mutants that show a
gain of function. We examined the inhibitory effects of CFTR on these
ENaC mutants. In addition, we analyzed whether another, recently
identified regulatory pathway for ENaC, the epithelial serine protease
CAP1 (21), interferes with CFTR-dependent inhibition of
ENaC.
 |
MATERIALS AND METHODS |
Liddle and
T592M,T593A Mutations--
Mutations of the rat
epithelial Na+ channel subunits were generated by
polymerase chain reaction. For the C-terminal stop mutations
-H647X,
-P565X,
-S608X the following sense (s) and antisense (as)
oligonucleotides (5'-3') were used:
-H647X, CGACCCACGCGTCGCG (s); AGGACAGAAACGGGACG (as);
-P565X, CCCACGCGTCCGACC (s),
GCCGCCTCCTGCGCA (as);
-S608X, TCGACCCACGCGTCC (s),
AGGTAAAAGTGGGCAGGTC (as). C-terminal point mutations
-P671A,
-Y618A,
-P (624-626)A were generated using
GACAGCCCCTCCAGCTGCCTATGCTACT (s), AGTAGCATAGCCAGCTGGAGGG-GCTGTC (as)
for
-P671A); GCACTCCACCTCCCAATGCGCACTCCCTGAGGCTG (s),
CAGCCTCAGGGAGTGGCGATTGGGAGGTGGAGTGC (as) for
-Y618A;
GGTGCCTGGCACAGCGGCCGCCAGATACAATA (s), TATTGTATCTGGCGGCCGCTGTGCCTGGTATT (as) for
-P (624-626)A).
T592M,T593A was created
using the oligonucleotides TCTTCCAGCCTGACATGGCTAGCTGCAGGCCCAAT (s),
ATTGGGCCTGCAGCTAGCCATGTCAGGCTGGAA (as). ENaC mutations were
checked for correct sequences by restriction digest and by cycle
sequencing (PRISM, Perkin-Elmer). cDNAs encoding
,
,
-rENaC
and the serine protease xCAP1 were kindly provided by Prof. Dr. B. Rossier, Pharmacological Institute of Lausanne, Switzerland.
cRNAs for CFTR, wt, and Mutant Epithelial Na+ Channel
(rENaC) Subunits and xCAP1--
cDNAs encoding wild type human
CFTR, the three (
,
,
) subunits of the rat
amiloride-inhibitable Na+ channel ENaC, and the
Xenopus protease xCAP1 were linearized using either
NotI or KpnI, and cRNA was in vitro
transcribed using T7, T3, or SP6 polymerases and a 5'-cap (mCAP
mRNA capping kit, Stratagene).
Preparation of Oocytes and Microinjection of cRNA--
Isolation
and microinjection of oocytes have been described in a previous report
(10). In brief, after isolation from adult Xenopus laevis
female frogs oocytes were dispersed and defolliculated by a 0.5-h
treatment with collagenase (type A, Boehringer, Mannheim, Germany).
Subsequently oocytes were rinsed and kept in ND96 buffer (in mmol/l):
96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES,
2.5 sodium pyruvate, pH 7.55, supplemented with theophylline (0.5 mmol/l) and gentamycin (5 mg/l) at 18 °C. Oocytes of identical batches were injected with cRNA of either wt or mutant
,
,
-rENaC (each subunit 10 ng), xCAP1, and CFTR (each 20 ng),
respectively, after dissolving cRNAs in about 50 nl of double-distilled
water (PV830 pneumatic pico pump, WPI, Berlin, Germany). Oocytes
injected with 50 nl of double-distilled water served as controls. For
some experimental protocols oocytes were injected with 50 ng of either sense or antisense oligonucleotides of the serine protease
xCAP1 (21).
Electrophysiological Analysis of Xenopus Oocytes--
2-4 days
after injection oocytes were impaled with two electrodes (Clark
instruments), which had resistances of 1 M
when filled with 2.7 mol/l KCl. A flowing (2.7 mol/l) KCl electrode served as bath
reference. Membrane currents were measured by voltage clamping of the
oocytes (OOC-1 amplifier, WPI) in intervals from
90 to +30 mV in
steps of 10 mV. Current data were filtered at 400 Hz (OOC-1 amplifier).
Between intervals, oocytes were voltage-clamped to their spontaneous
membrane voltage for 20 s. Data were collected continuously on a
computer hard disc and analyzed by using the programs chart and scope
(McLab, AD-Instruments, Macintosh). Conductances were calculated for
the voltage clamp range of
90 to +30 mV according to Ohm's law.
During the whole experiment the bath was continuously perfused at a
rate of 5-10 ml/min.
Materials and t Test--
All used compounds were of highest
available grade of purity. 3-Isobutyl-1-methylxanthine (IBMX),
forskolin, trypsin, and amiloride were all from Sigma (Deisenhofen,
Germany). The dynamin and ubiquitin antibodies were purchased from
Calbiochem (Bad Soden, Germany) and Dianova (Hamburg, Germany). Sense
and antisense oligonucleotides of the protease xCAP1 were
synthesized by the facility of the local University and were stabilized
by phosphorothioate modification. The oligonucleotides had the
following (5'-3') sequence: ATGGAGCCTCTTCCACTTCTC (sense) and
GAGAAGTGGAAGTGGCTCCAT (antisense). Statistical analysis was performed
according to Student's t test. p values <0.05 were accepted to indicate statistical significance. All experiments were
conducted at room temperature (22 °C).
 |
RESULTS |
Effects of Deletion of C-terminal PY Motifs in
,
,
-rENaC--
PY motifs in either
-,
-, or
-subunits
of rENaC were deleted by either C-terminal point (
-P671A,
-Y618A,
-P(624-626)A) or C-terminal stop mutations (
-H647X,
-P565X,
-S608X). A representative record of the whole cell current from an
oocyte coexpressing
-P565X with wild type
- and
-subunits is
shown in the lower trace of Fig.
1. This current is enhanced when compared
with that induced by
,
,
-rENaC (Fig. 1, upper
trace). Amiloride-sensitive whole cell conductances
(GENaC-mut) were calculated for the various mutations and
were compared with that of
,
,
-rENaC
(GENaC-mut/GENaC-wt). As demonstrated in Fig.
2 GENaC was significantly
enhanced for
-P565X,
-S608X,
-P671A, and
-Y618A, while
-H647X and
-P(624-626)A did not produce enhanced conductances.
These results confirm those of previous studies (17, 22) and indicate
the importance of the C-terminal PY motif for the regulation of
ENaC.

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Fig. 1.
Representative examples of whole cell
currents and inhibition by amiloride (10 µmol/l) observed in oocytes expressing
, , -rENaC
(upper trace) or
, -P565X, -rENaC
(lower trace). Oocytes were voltage-clamped in
steps of 10 mV (for 1 s) from 90 mV to +30 mV.
|
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Fig. 2.
Effects of C-terminal deletions and
elimination of PY motifs in -,
-, and -subunits of ENaC
on amiloride-sensitive Na+ currents. cRNAs encoding
either of the -, -, or -mutants were coinjected with the
complementary wild type subunits. The corresponding bars indicate whole
cell conductances normalized for wild type ENaC values
(GENaC-mut/GENaC-wt). Results are means ± S.E. (number of experiments). * indicates significant difference from
Gwt.
|
|
ENaC Carrying Liddle Mutations Are Inhibited by
CFTR--
Individual ENaC mutants were coexpressed together with CFTR,
and we examined whether these ENaC Liddle mutants are still
down-regulated by CFTR. As demonstrated in Fig.
3A, the whole cell conductance that is produced by
,
,
-rENaC and is inhibited by 10 µmol/l amiloride (A) is attenuated after stimulation of CFTR by
IBMX (1 mmol/l) and forskolin (10 µmol/l). The summary (Fig.
3C) indicates significant inhibition of
,
,
-rENaC by
CFTR, while no effects of IBMX and forskolin could be detected in the
absence of CFTR. Similar to
,
,
-rENaC, also
,
,
-S608X-rENaC whole cell conductances were down-regulated
upon stimulation of CFTR with IBMX and forskolin (Fig. 3B).
The results from the various ENaC mutants coexpressed with CFTR are
summarized in Fig. 4. It is shown that
GENaC caused by each of the C-terminal stop (A)
or point (B) mutants is significantly attenuated upon
stimulation of CFTR by IBMX and forskolin (black bars). We,
therefore, conclude that PY motifs do not participate in
CFTR-dependent regulation of ENaC.

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Fig. 3.
Inhibition of
, , -rENaC
(A) and
, , S608X-rENaC
(B) by CFTR. Whole cell conductances were blocked
reversibly by amiloride (A, 10 µmol/l). The effects of
amiloride on both wt , , -rENaC and , , S608X-rENaC were
attenuated after activation of a CFTR Cl conductance by
IBMX (1 mmol/l) and forskolin (IBMX/Fors, 10 µmol/l).
C, summary of the amiloride-sensitive Na+
conductance (GENaC) in Xenopus oocytes
expressing , , -rENaC or coexpressing , , -rENaC together
with CFTR. Results are means ± S.E. (number of experiments). *
indicates significant effects of amiloride. # indicates significant
down-regulation of ENaC by CFTR.
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Fig. 4.
CFTR-dependent inhibition of ENaC
carrying C-terminal deletions (A, B)
or point mutations (A, C) in
either -, -, or
-subunits of rENaC. A, continuous
recording of the whole cell conductances produced by either point or
stop mutations of ENaC and effect of amiloride before and after
activation of CFTR. Summary of GENaC produced by ENaC
carrying either stop (B) or point (C) mutations
before (white bars) and after (black bars)
stimulation with IBMX and forskolin (IBMX/Fors). Results are
means ± S.E. (number of experiments). * indicates significant
effects of amiloride. # indicates significant inhibition of ENaC by
CFTR.
|
|
Antibodies for Ubiquitin and Dynamin Enhanced ENaC Conductance and
Do Not Interfere with the Inhibition by CFTR--
In order to further
examine the role of the Nedd4/ubiquitin cascade in the regulation of
ENaC by CFTR, we made use of two different antibodies, which bind to
either ubiquitin and dynamin. When coinjected with ENaC, both of these
antibodies enhanced GENaC when compared with
amiloride-sensitive whole cell conductances measured in oocytes from
the same batch and injected solely with
,
,
-rENaC (Fig.
5A). In another series of
experiments, we coinjected
,
,
-rENaC together with CFTR and
ubiquitin or dynamin antibodies. The summary of these experiments
indicates that even after interfering with both intracellular ubiquitin
and dynamin, CFTR is able to inhibit ENaC (Fig. 5B).

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Fig. 5.
A, effects of dynamin and ubiquitin
antibodies, respectively, on GENaC. Oocytes injected with
ENaC and equal amounts of water (H2O) served as controls. # indicates significantly enhanced GENaC in coinjected
oocytes compared with oocytes injected with ENaC only. B,
summary of GENaC in Xenopus oocytes coinjected
with CFTR and dynamin or ubiquitin antibodies. GENaC is
shown before (white bars) and after (black bars)
activation of CFTR by IBMX (1 mmol/l) and forskolin
(IBMX/Fors, 10 µmol/l). Results are means ± S.E.
(number of experiments). * indicates significant effects of amiloride.
# indicates significant inhibition of ENaC by CFTR.
|
|
CFTR-dependent Inhibition of
T592M,T593A-ENaC--
According to previous reports, a mutation in
the
-subunit of the human ENaC (
-T594M) causes a loss
of protein kinase C inhibition and leads to salt-sensitive hypertension
in the African-American population (23). We examined whether the
respective mutations in the rat ENaC
-subunit interferes with
CFTR-dependent inhibition.
T592M,T593A-rENaC, when coexpressed with equal amounts
of
- and
-subunits, led to an amiloride sensitive Na+
conductance that was significantly higher than in oocytes from the same
batch injected with wt
,
,
-ENaC (Fig.
6B). Stimulation with IBMX and
forskolin had no effect on
,
T592M,T593A,
-rENaC Na+ conductance (13.7 ± 1.2 versus
13.5 ± 1.9 microsiemens; n = 4). When coexpressed
with CFTR,
,
T592M,T593A,
-rENaC conductance was
significantly attenuated by stimulation with IBMX and forskolin (Fig.
6, A and C). Thus, gain of function mutations of
ENaC do not limit inhibition of ENaC by CFTR.

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Fig. 6.
CFTR-dependent inhibition of the
ENaC mutant T593A/T594M. A,
continuous recording of the whole cell conductance and effect of
amiloride before and after activation of CFTR. B, summary of
GENaC produced by wild type , , -ENaC or
, -T593A/594M, -ENaC. C, summary of
GENaC produced by , -T593A/594M, -ENaC
before (white bar) and after (black bar)
stimulation with IBMX and forskolin (IBMX/Fors). Results are
means ± S.E. (number of experiments). * indicates significant
effects of amiloride. § indicates significantly enhanced ENaC
conductance when compared with wt ENaC. # indicates significant
inhibition of ENaC by CFTR.
|
|
ENaC Conductance Activated by Extracellular Trypsin Is Inhibited by
CFTR--
It was shown recently that extracellular proteases activate
ENaC probably by interfering with the extracellular loop of ENaC (21).
We also found augmentation of GENaC by trypsin in a
dose-dependent manner (Fig.
7B). In an oocyte coexpressing
both ENaC and CFTR the amiloride-sensitive Na+ conductance
was enhanced after exposure to 2 µg/ml trypsin (Fig. 7A).
Upon stimulation by IBMX (1 mmol/l) and forskolin (10 µmol/l) and
activation of a CFTR whole cell conductance, the inhibitory effect of
amiloride was largely attenuated. The summary shown in Fig.
7C indicates a significant increase of GENaC by
trypsin and inhibition of GENaC by CFTR. The results
demonstrate the ability of CFTR to inhibit ENaC in the presence of
extracellular protease activity and suggest that the portion of
GENaC that is activated by trypsin is also subjected to
down-regulation by CFTR.

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Fig. 7.
A, activation of , , -rENaC by
trypsin (2 µg/ml) and inhibition of , , -rENaC by CFTR. Whole
cell conductances were blocked reversibly by amiloride (A,
10 µmol/l). The effect of amiloride was augmented after application
of trypsin and was attenuated after activation of a CFTR
Cl conductance by IBMX (1 mmol/l) and forskolin
(IBMX/Fors, 10 µmol/l) in the continuous presence of
trypsin. B, concentration-response curve for the effects of
trypsin on GENaC. C, summary of
GENaC coexpressed with CFTR. Effect of incubation with
trypsin and activation of CFTR by IBMX and forskolin
(IBMX/F) is shown. Results are means ± S.E. (number of
experiments). * indicates significant effects of amiloride. ° indicates significant difference from control. # indicates significant
inhibition of ENaC by CFTR.
|
|
Inhibtion of ENaC by CFTR in the Presence or Absence of the
Epithelial Protease xCAP1--
In order to investigate the impact of
the serine protease xCAP1, we blocked expression of xCAP1 by injection
of xCAP1 antisense oligonucleotides. The summary of the results is
shown in Fig. 8, A and
B, and demonstrate attenuation of GENaC in
oocytes coinjected with CFTR/ENaC/xCAP1 antisense compared with oocytes
coinjected with CFTR/ENaC/xCAP1 sense. However, when CFTR was activated
by IBMX and forskolin, GENaC was inhibited in both sense-
and antisense-injected oocytes. In a second approach we overexpressed
xCAP1 together with ENaC and xCAP1 together with ENaC and CFTR. The
summary of the results from these experiments is shown in Fig.
8C. The data demonstrate that GENaC was
approximately doubled when ENaC was coexpressed with xCAP1. However, a
smaller GENaC value was detected when CFTR was coexpressed
with both rENaC and xCAP1. It is shown that in the presence of xCAP1,
CFTR was still able to down-regulate ENaC. We conclude that larger ENaC
currents that are detected in the presence of protease activity are
inhibited by CFTR.

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Fig. 8.
Effects of xCAP1 sense (A)
and xCAP1 antisense (B) oligonucleotides on the
expression of GENaC inhibition by CFTR. # indicates
significantly attenuated GENaC in antisense compared with
sense-injected oocytes. ° indicates significant inhibition of ENaC by
CFTR. Black bars show GENaC when CFTR was
activated by IBMX and forskolin (IBMX/F). C,
GENaC in oocytes injected with , , -rENaC only or
coinjected with , , -rENaC and xCAP1. ° indicates
significantly enhanced GENaC in coinjected ooyctes.
GENaC was lower in oocytes coexpressing , , -rENaC,
xCAP1, and CFTR and was significantly (#) inhibited after activation of
CFTR by IBMX and forskolin (IBMX/Fors). Results are
means ± S.E. (number of experiments). * indicates significant
effects of amiloride.
|
|
 |
DISCUSSION |
The amplitude of the epithelial Na+ current is
determined by the number of ENaC channels present in the cell membrane
and by the activity of membrane resident channels (18, 19, 22). Correspondingly, dual effects of Liddle's mutation on
Po and the number of Na+ channels
expressed at the cell surface have been described (19). However,
endocytosis of ENaC by a Nedd4/ubiquitin/dynamin-dependent pathway is probably the predominant mechanism for regulation of epithelial Na+ absorption (14). Thus, a loss of Nedd4
recognition site causes enhanced Na+ absorption in kidney
collecting ducts and a salt-sensitive hypertension (15-17). The well
known Na+ feedback (24) detected in mouse mandibular duct
cells occurs via activation of G0 proteins through increase
of intracellular Na+ and probably also via
Nedd4-dependent retrieval of Na+ channels from
the cell membrane (25). However, in Xenopus oocytes inhibition of ENaC channels by Na+ occurs without changing
the number of ENaC channels (2, 19).
Because Nedd4 and ubiquitination play such a central role in ENaC
regulation, we examined whether down-regulation of ENaC by CFTR occurs
via this regulatory axis. According to the present results, however,
this seems rather unlikely, since ENaC channels containing either
-,
-, or
-subunits with defective PY motifs were inhibited during
activation of CFTR. Moreover, the data indicate strong inhibition by
CFTR for most of the ENaC mutants, which may suggest that the
additional portion of Na+ conductance that is due to
limited retrieval from the cell membrane and enhanced ENaC activity is
also inhibited by CFTR. Similar holds true for
T592M,T593A-ENaC and ENaC currents activated by proteases. We may therefore assume that CFTR-dependent
inhibition of ENaC is superior to the regulation by the
ubiquitin-dependent pathway and extracellular proteases,
including xCAP1. This also applies to other gain of function mutations
like
T592M,T593A-rENaC, which was not regulated directly
by cAMP in our hands (23). The results of the present report also
explain why Liddle's disease patients, unlike CF patients, do not show
enhanced Na+ conductance in their airways and therefore do
not suffer from pulmonary symptoms (20).
Nedd4/ubiquitin-dependent regulation of ENaC does not
provide a mechanism through which ENaC is regulated by CFTR. The
relatively fast onset and reversibility of the inhibitory effects of
CFTR on ENaC (10) are in good agreement with such a result. The
question regarding the mechanisms underlying the
CFTR-dependent regulation of ENaC can therefore not
completely be answered at the moment. We know from previous reports
that interaction of CFTR with ENaC takes place even in isolated
membranes and after reconstitution into planar lipid bilayers (6, 7).
The interaction does not require full-length CFTR but only cytosolic
domains of CFTR (5) and largely depends on CFTR's ability to conduct
Cl
ions (10). Additional studies are needed to show
whether the recently identified PDZ domain interaction enables
functional coupling of CFTR and ENaC.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the expert
technical assistance of H. Schauer.
 |
FOOTNOTES |
*
This work was supported by Deutsche Forschungsgemeinschaft
Grant Ku756/2-3, Zentrum klinische Forschung 1 (ZKF1), and Fritz Thyssen Stiftung Grant 1996/1044.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by a Heisenberg fellowship. To whom correspondence
should be addressed. Tel.: 49-761-203-5153; Fax: 49-761-203-5191; E-mail: kkunzel{at}sibm2.ruf.uni-freiburg.de.
2
M. Mall, M. Bleich, J. Kühr, M. Brandis,
R. Greger, and K. Kunzelmann, submitted for publication.
 |
ABBREVIATIONS |
The abbreviations used are:
ENaC, epithelial
Na+ channel(s);
CFTR, cystic fibrosis transmembrane
conductance regulator;
wt, wild type;
l, liter;
IBMX, 3-isobutyl-1-methylxanthine.
 |
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