From the Hanson Centre for Cancer Research, Institute
of Medical and Veterinary Science, Frome Road, Adelaide, South
Australia 5000, Australia and the § Department of
Physiology, University of Sydney, Sydney, New South Wales 2006, Australia
Received for publication, December 21, 2000, and in revised form, January 4, 2001
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ABSTRACT |
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The amiloride-sensitive epithelial sodium channel
(ENaC) plays a critical role in fluid and electrolyte homeostasis and
consists of The amiloride-sensitive epithelial sodium channel
(ENaC)1 plays a critical role
in fluid and electrolyte homeostasis and is widely expressed in
absorptive epithelia such as the renal collecting duct, the colon, the
lung, and sweat and salivary ducts (1-4). It consists of The PY motifs in the carboxyl termini of ENaC subunits are believed to
be necessary for interaction with the WW domains of Nedd4 (20), a
widely expressed ubiquitin-protein ligase (20-24). Nedd4 has been
proposed to down-regulate Na+ channel activity in response
to increases in intracellular Na+ (17, 25, 26) by
ubiquitination of the channel (16, 25, 27) leading to its
endocytosis and degradation (16, 27, 28). The detailed mechanisms by
which Nedd4 interacts with Na+ channels remain, however,
unclear. Nedd4 consists of a ubiquitin-protein ligase domain, multiple
WW domains, and a Ca2+ and lipid binding domain (20-22).
The ubiquitin-protein ligase domain of Nedd4 is required for
ubiquitination of ENaC (16, 24, 27, 28). The Ca2+ and lipid
binding domain has been shown to mediate
Ca2+-dependent redistribution of Nedd4 from the
cytoplasm to the cell membrane (29) and has recently been demonstrated
to associate in a Ca2+-dependent manner with
annexin XIIIb, a protein concentrated in apical rafts (30).
In addition to Nedd4, there are now a number of proteins known to
share a similar modular structure to Nedd4. Some of the Nedd4
family members have been implicated in ubiquitin-mediated regulation of
several cellular proteins (31). The Nedd4-like protein that is most
homologous to human Nedd4 is human KIAA0439, encoded by an unpublished
cDNA sequence in the GenBankTM data base
(accession no. AB007899). These observations prompted us to
investigate whether KIAA0439 could also regulate ENaC function. Our
hypothesis was further supported by our observation that a Nedd4-like
Xenopus protein, claimed to be the homolog of mammalian Nedd4 (32), had been shown to regulate ENaC activity in
Xenopus oocytes (28). A close examination of the homology
and sequence alignment of this protein with the Nedd4-like proteins in
the data base suggested that the Xenopus protein is more
closely related to KIAA0439 than to Nedd4 (see below). Given that
KIAA0439 and Nedd4 are highly related but distinct proteins in mammals,
the results obtained by Abriel et al. (28) suggest that
mammalian KIAA0439 could also play a key role in the regulation of
ENaC. In this communication we present evidence that mammalian KIAA0439 and Nedd4 may have overlapping or redundant roles in the regulation of
ENaC function.
Sequence Retrieval and Homology Analysis--
Nedd4 family
members in the GenBankTM data base were identified by
protein and nucleotide BLAST and TBLASTN programs using the NCBI
server. Multiple sequence alignments and the construction of
phylogenetic trees were carried out using the Bionavigator software
packages Protpars and Protml at the Australian National Genome
Information Services server. The near full-length sequence of mouse
KIAA0439 cDNA was compiled from a number of EST sequence entries in
the GenBankTM data base.
Northern Blot Analysis--
A 1.2-kb fragment of mouse
KIAA0439 cDNA was generated by reverse
transcriptase-PCR2
using RNA from mouse salivary glands. This fragment covers the mouse
KIAA0439 coding region for the four WW domains. The 1.2-kb cDNA was
used as a probe for RNA blot analysis using a mouse multiple tissue
Northern (MTN) blot from CLONTECH.
Expression Plasmids and Production of GST Fusion
Proteins--
The expression construct used to generate protein
containing all three WW domains of mouse Nedd4 fused to glutathione
S-transferase (GST) has been described previously (25, 26).
Other WW domain-GST constructs were generated by amplifying WW domain
regions from cDNAs for the various Nedd4 family members by PCR
using appropriate primers2 followed by cloning into
pGEX-2TK or pGEX-4T3 (Amersham Pharmacia Biotech). The regions encoding
the carboxyl termini of human and mouse SDS-PAGE and Far-Western Analysis--
32P-labeled
protein probes were produced by directly labeling the appropriate GST
fusion protein using protein kinase A (New England Biolabs).
Glutathione beads containing bound fusion protein were incubated with
protein kinase A and [ Whole-cell Patch Clamping--
Isolated granular duct cells were
prepared by collagenase digestion of mouse mandibular glands from male
mice as described previously (34). The standard bath solution (pH 7.4)
contained 145 mM NaCl, 5.5 mM KCl, 1 mM CaCl2, 1.2 mM MgCl2,
1.2 mM NaH2PO4, 7.5 mM HEPES, and 10 mM glucose. The standard (zero
Na+) pipette solution (pH 7.2) contained 150 mM
N-methyl-D-glucamine (NMDG)-glutamate, 1 mM MgCl2, 10 mM HEPES, 5 mM EGTA, and 10 mM glucose. In the 70 mM Na+ pipette solution, Na+ was
adjusted by substituting Na+-glutamate for NMDG-glutamate.
Standard whole-cell patch clamp techniques were used (25, 34). After
establishing the whole-cell configuration, the bath solution was
replaced with a solution containing 145 mM
Na+-glutamate, 5 mM NaCl, 1 mM
MgCl2, 10 mM HEPES, 1 mM EGTA, and 10 mM glucose (pH 7.4). The amiloride-sensitive current was
measured as described previously (25, 34). The chord conductance was measured between KIAA0439 and Nedd4 Are Distinct Proteins in Mammals--
There are
eight predicted human members of the Nedd4 family of proteins in the
current GenBankTM data base. A sequence comparison using
multiple alignment programs indicates that the human Nedd4-like
proteins can be clustered into four subgroups. The first of these
subgroups contains Nedd4 and KIAA0439, which are highly related to each
other (~78% identity) (Fig.
1A). In this phylogenetic
scheme, WWP1, WWP2, and AIP-4 (human homolog of mouse Itch protein)
form the second subgroup, and Smurf1 and Smurf2 form the third
subgroup of Nedd4-like proteins (Fig. 1A). KIAA0322 is the
only protein in the fourth subgroup as it is distantly related to the
other three subgroups (Fig. 1A). The first two subgroups are
more related to each other than to the last two subgroups. We then
compared the available sequences of Nedd4 and KIAA0439 orthologs from
various species to ensure that these two proteins evolved as distinct
proteins in mammals. As shown in the phylogenetic tree in Fig.
1B, the Xenopus Nedd4-like protein, published
previously as the Xenopus homolog of Nedd4 (32), is more
highly related to the mouse and human KIAA0439 proteins than to the
mouse, rat, and human Nedd4 proteins. The current EST data base also
contains partial cDNA sequences for the porcine and bovine Nedd4
and KIAA0439 proteins (data not shown). Additionally, there are partial
chicken ESTs in the GenBankTM (accession nos. AJ397021,
BE012727, and AJ399234) that encode two distinct but closely related
Nedd4-like proteins that appear to represent avian Nedd4 and KIAA0439
proteins. A BLAST search of the recently completed Drosophila
melanogaster genomic sequence suggests that there are three
Nedd4-like proteins in the fly. One of these (which we call
Drosophila Nedd4) appears to be a common ancestor for both
the Nedd4 (~73% identity) and KIAA0439 (~67% identity) proteins.
Because the gene cloned by Rebhun and Pratt (32) is more closely
related to KIAA0439 than to Nedd4, it is possible that there is another
protein in Xenopus that is the true ortholog of Nedd4.
However, so far we have not been able to clone such a protein and there
are currently no Xenopus ESTs that correspond to Nedd4.
KIAA0439 mRNA Is Expressed in Multiple Tissues--
The Nedd4
transcript and protein are widely expressed during embryonic
development and in adult tissues (22, 35-37). To test the possibility
that KIAA0439 may have a more tissue-specific expression, we analyzed
KIAA0439 mRNA expression by Northern blotting. As shown in Fig.
2, an ~4.0-kb KIAA0439 transcript was
present in all tissues examined. The expression was particularly high in the liver and kidney followed by brain, heart, and lung. Longer exposures (not shown) revealed relatively low but detectable levels of
transcript in spleen, skeletal muscle, and testis. It is interesting to
note that skeletal muscle, which shows strong expression of Nedd4
mRNA (22), has very low levels of KIAA0439 transcript. However,
like KIAA0439, Nedd4 expression is high in liver and kidney (22). In
addition to the tissues shown in Fig. 2, an analysis of the
GenBankTM data base suggests that the KIAA0439 transcript
is also present in the adult thymus, ovary, skin, retina, urinary
bladder, and mammary gland, as well as many embryonic tissues, because
many of the EST clones in the data base were derived from cDNA
libraries prepared from these tissues. The KIAA0439 transcript was also present in mouse mandibular gland cells (from which the mouse KIAA0439
probe was derived), which were used in the patch clamp experiments (see
below).
KIAA0439 WW Domains Bind All Three Subunits of ENaC--
To
investigate the ability of members of the Nedd4 family of proteins to
interact with ENaC subunits, a far-Western assay was used. First, the
WW domains of Nedd4-like proteins (Fig.
3) were amplified by either PCR or
reverse transcriptase-PCR and cloned into an appropriate pGEX vector.
The majority of GST-WW domain fusion proteins, except for KIAA0439 WW
domains, were present in the insoluble fraction, and therefore it was
decided to use this fraction of Escherichia coli lysates for
far-Western analysis. Equivalent amounts of GST-WW domain fusion
proteins (as determined by SDS-PAGE and Coomassie staining) (Fig.
4A)were electrophoresed by SDS-PAGE and transferred to
nitrocellulose. Filters were probed with 32P-labeled
GST-ENaC subunit fusion proteins ( WW Domains of KIAA0439 Block the Inhibitory Effect of High
Na+--
Using whole-cell patch clamp techniques, we have
previously shown that GST fusion proteins containing all three WW
domains of mouse Nedd4, together or separately, when added to the
pipette solution can overcome the inhibitory effect of high
Na+ on amiloride-sensitive Na+ current (25,
26). The GST-Nedd4-WW domain proteins thus act as dominant negative
mutants, presumably by displacing or precluding the interaction between
ENaC subunits and endogenous Nedd4. To examine a potential role for
KIAA0439 in the Na+ feedback pathway, we decided to test
whether KIAA0439 WW domains would generate a similar effect. We first
confirmed that the amiloride-sensitive Na+ current in mouse
mandibular duct cells is inhibited by increasing the Na+
concentration in the pipette solution. As shown in Fig.
5A, when the 0 mM
Na+ pipette solution was used, the addition of 100 µM amiloride to the bath solution inhibited an inwardly
rectifying current (Fig. 5D), which we have previously shown
to be Na+-selective and to have the characteristics of the
amiloride-sensitive Na+ conductance in intact salivary
ducts (38). This current was not present when the 70 mM
Na+ pipette solution was used (Fig. 5, B and
D). The inclusion in the 70 mM Na+
pipette solution of 100 µg/ml GST-KIAA0439 fusion protein containing all four WW domains completely reversed the inhibitory effect of
increased Na+ in the pipette solution (Fig. 5, C
and D). Fig. 6 summarizes the
results of the experiments in Fig. 5 together with the results of
additional control experiments. From these data it is evident that the
inclusion of 100 µg/ml GST-KIAA0439 WW domain fusion protein in the
70 mM Na+ pipette solution overcomes the
inhibition of the amiloride-sensitive Na+ current. The
effect of this fusion protein is similar to that produced by the
inclusion of a similar concentration of GST-Nedd4 WW domain fusion
protein (Fig. 6). The addition of 100 µg/ml GST-KIAA0439 WW domain
fusion protein to the 0 mM Na+ pipette
solution had no effect on the amiloride-sensitive Na+
current (Fig. 6), indicating that the effects of the fusion protein are
due to inhibition of Na+ feedback inhibition rather than to
a nonspecific stimulatory effect on the amiloride-sensitive
conductance. Under similar conditions, control GST protein has no
effect on the ENaC activity with high or low Na+ in the
pipette solution (25). The effects of GST-WW domain fusion proteins are
not due to nonspecific binding of the KIAA0439 WW domains to the PY
motifs in ENaC subunits, as individual Nedd4 WW domains fused to GST,
which can interact with ENaC PY motifs in vitro, do not
influence ENaC function in patch clamp experiments (26). Furthermore,
the GST-Nedd4 WW domain proteins, carrying a single mutated WW domain
and two WW domains in the wild-type configuration, lose their ability
to affect amiloride-sensitive ENaC current, irrespective of which of
the WW domains is mutated (26). These results can be interpreted to
suggest that WW domains of KIAA0439 act by displacing either the
endogenous Nedd4 protein or the endogenous KIAA0439 protein. As both
Nedd4 and KIAA0439 are expressed in salivary gland cells, and both
proteins are able to bind ENaC subunits, either possibility may explain
the effect seen in Figs. 5 and 6.
The previous work by Abriel et al. (28) using
Xenopus oocytes in which Xenopus KIAA0439 and rat
ENaC proteins were coexpressed demonstrated that the wild-type KIAA0439
protein causes a dose-dependent decrease in
amiloride-sensitive Na+ current. Conversely, a Cys
Although there are clear-cut differences in the levels of transcript in
different tissues, both Nedd4 and KIAA0439 are expressed in many of the
same tissues, suggesting that Nedd4 and KIAA0439 may be functionally
redundant. Alternatively, Nedd4 and KIAA0439 may function in different
tissues in response to different regulatory signals. Neither of these
possibilities can be ruled out as yet, as all of the data currently
available on the function of Nedd4 (and KIAA0439) in ENaC regulation
are derived from indirect experiments using (i) overexpression of the
wild-type or catalytically inactive Nedd4/KIAA0439 in
Xenopus oocytes and (ii) GST-WW domain proteins or
polyclonal antibodies in whole-cell patch clamp configuration. To
establish the relative contribution of Nedd4 and KIAA0439 in ENaC
regulation, it will be necessary in the future to utilize a cell system
in which the activity of one of the genes is ablated, for example, by
gene knock-out technology.
,
, and
subunits. The carboxyl terminus of each
ENaC subunit contains a PPxY, motif which is
believed to be important for interaction with the WW domains of the
ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in
Liddle's syndrome, where mutations delete or alter the
PPxY motif of either the
or
subunits, has been
proposed to result in increased ENaC activity. Here we present evidence
that KIAA0439 protein, a close relative of Nedd4, is also a potential
regulator of ENaC. We demonstrate that KIAA0439 WW domains bind all
three ENaC subunits. We show that a recombinant KIAA0439 WW domain
protein acts as a dominant negative mutant that can interfere with the
Na+-dependent feedback inhibition of ENaC in
whole-cell patch clamp experiments. We propose that KIAA0439 and Nedd4
proteins either play a redundant role in ENaC regulation or function in
a tissue- and/or signal-specific manner to down-regulate
ENaC.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,
, and
subunits, which are thought to assume a multimeric structure at the
membrane (5, 6). The carboxyl terminus of each ENaC subunit contains a
PPxY sequence (the PY motif), which when mutated or deleted
in either the
or
ENaC subunits leads to Liddle's syndrome, an
autosomal dominant form of hypertension (7-11). Therefore, mutating
just one PY motif from a single subunit of the multimeric ENaC complex
is sufficient to lead to a disease phenotype. In in vitro
systems, identical mutations to those that cause Liddle's syndrome
have been shown to increase amiloride-sensitive Na+ current
(8, 11-15). This increase is believed to result from the presence of
increased numbers of active Na+ channels in the cell
membrane (12, 16-18), although an increase in channel open
probability may also contribute (12, 17, 19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,
, and
ENaC subunits
were PCR-amplified2 and cloned into either BamHI
or BamHI/EcoRI sites of pGEX-2TK. GST fusion
proteins were produced and purified as described previously (25,
26).
-32P]ATP in a buffer containing
20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 12 mM MgCl2, and 1 mM dithiothreitol
for 60 min at 4 °C. Beads were washed five times in
phosphate-buffered saline, and labeled protein was eluted with
glutathione buffer. To prepare WW domain protein filters, ~1 µg of
each induced GST fusion protein lysate was resolved on SDS-PAGE gels
and transferred to nitrocellulose membrane (Schleicher & Schuell).
Membranes were blocked in Hyb 75 (33) and then hybridized with
,
, or
ENaC 32P-labeled protein probes for 4 h at
4 °C in Hyb 75. Membranes were washed three times in Hyb75 and
exposed to x-ray film.
80 mV and the zero current potential of the amiloride-sensitive Na+ current. Results are presented as
the mean ± S.E. Statistical significance was assessed using
Student's unpaired t test. All experiments were performed
at 20-22 °C.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Nedd4 and KIAA0439 are two separate proteins
in mammals. A, phylogenetic relationship tree of
different human members of the Nedd4 family. B, a
phylogenetic tree showing the relative homology of Nedd4 and KIAA0439
orthologs. The sequences for various Nedd4-like proteins were derived
from the GenBankTM data base. The accession numbers
for various entries are as follows: human Nedd4, P46934; mouse Nedd4,
P46935; rat Nedd4, S70642; Drosophila Nedd4, AAF49328; human
KIAA0439, BAA23711; Xenopus KIAA0439/Nedd4, CAA03915; human
WWP1, AAC51324; human WWP2, AAC51325; human Smurf1, AAF08298; human
Smurf2, AF301463; human AIP4, CAC09387; human KIAA0322,
BAA20780. Mouse KIAA0439 protein sequence was derived from a number of
overlapping EST sequences in the GenBankTM data base.
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Fig. 2.
Northern blot analysis of Nedd4 and KIAA0439
expression in mouse tissues. A mouse multiple tissue
poly(A)+ RNA blot (CLONTECH) was
hybridized to a 1.2-kb mouse KIAA0439 cDNA probe that covers the
region of the gene encoding the four WW domains. The gene was expressed
as an ~4.0-kb transcript. The figure shows an overnight exposure of
the blot. In longer exposures, low levels of transcript were clearly
present in the spleen, skeletal muscle, and testis. The hybridization
of the blot with other gene probes indicated equal poly(A)+
RNA loading in all lanes (data not shown).
,
, or
) and exposed to
x-ray film. As identical results were obtained when either the human or
mouse ENaC proteins were used as probes, only one set of data (using
human ENaC proteins) is shown in Fig. 4.
The
ENaC subunit bound with equal strength to the WW domains of human Nedd4 and KIAA0439 and only weakly to the WW domains of KIAA0322,
Smurf1, WWP2/AIP2, AIP4, and Itch (Fig. 4B). Similar results
were obtained when an identical filter was probed with
ENaC
(Fig. 4C).
ENaC also bound strongly to both human
Nedd4 and KIAA0439 but not at all to WW domains from other Nedd4-like proteins (Fig. 4D). These results show that in
vitro, ENaC subunits bind with strong affinity to the WW domains
of human Nedd4 and KIAA0439 but weakly or not at all to the other Nedd4
family members tested. The number of WW domains present in these fusion
proteins is unlikely to impact upon the results described here as
WWP2/AIP2, AIP4, and Itch all had the same number of WW domains (four)
to human Nedd4 and KIAA0439. This observation suggests that
in vivo, KIAA0322, Smurf1, WWP2/AIP2, AIP4, and Itch are
unlikely to target ENaC for ubiquitination. It raises the possibility,
however, that like Nedd4, KIAA0439 also regulates the activity of
ENaC.
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Fig. 3.
Schematic representation of the regions of
Nedd4-like proteins that were fused to GST for recombinant protein
production in E. coli. The Nedd4 family of
proteins contain an amino-terminal C2 domain, 2-4 WW domains, and a
carboxyl-terminal Hect domain. The WW domain-containing regions of
Nedd4-like proteins that were cloned into pGEX vectors for fusion
protein production are underlined.
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Fig. 4.
Varying binding specificities of
,
, and
ENaC for the WW domains of different Nedd4-like
proteins. A, Coomassie-stained gel of GST WW domain
fusion proteins. The first lane on the
left contains GST alone; molecular mass markers in kDa are
indicated on the right side of the gel. B,
C, and D are far-Western blots of the proteins
probed with 32P-labeled human
,
, or
ENaC protein
probes, respectively. Identical data were obtained when mouse
,
,
or
ENaC proteins were used as probes (not shown).
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Fig. 5.
Na+ feedback inhibition of the
amiloride-sensitive Na+ current and the effects of
GST-KIAA0439 WW fusion protein. The whole-cell currents in
salivary duct cells studied with: A, 0 mM
Na+ pipette solution (n = 5); B,
70 mM Na+ pipette solution (n = 5); C, 70 mM Na+ pipette solution
plus 100 µg/ml GST-KIAA0439 WW fusion protein (n = 6). For each set of experiments we show representative whole-cell
recordings taken prior to and following the addition of 100 µM amiloride to the bath solution, together with the
steady-state I-V relations. D, shows
the steady-state I-V relations of the amiloride-sensitive
current measured using each of the three pipette solutions ( , 0 mM Na+;
, 70 mM Na+;
, 70 mM Na+ plus GST-KIAA0439 WW),
calculated by subtraction of the I-V relation when the bath
solution contained amiloride from the control I-V
relation.
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Fig. 6.
A GST-KIAA0439 WW domain protein restores the
inhibitory effects of high Na+ in mouse mandibular duct
cells. The effects of adding either a GST-Nedd4 WW domain fusion
protein or a GST-KIAA0439 WW domain fusion protein (both at 100 µg/ml) on chord conductance of the amiloride-sensitive
Na+ current are shown. The addition of either the GST-Nedd4
WW domain protein or the GST-KIAA0439 WW domain protein completely
overcomes the inhibition of amiloride-sensitive Na+ current
by the inclusion of 70 mM Na+ in the pipette
solution.
Ser
mutant of KIAA0439 resulted in a 2-6-fold increase in
amiloride-sensitive Na+ current by increasing the number of
channels on the cell surface (28). Our studies with mammalian KIAA0439,
combined with those of Abriel et al. (28), strongly suggest
that KIAA0439, like Nedd4, is a regulator of ENaC. Furthermore,
mammalian KIAA0439 is also likely to regulate the activity of the PY
motif-containing cardiac voltage-gated Na+ channel, which
has recently been shown by Abriel et al. (39) to be
regulated by Xenopus KIAA0439 protein in frog oocytes.
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ACKNOWLEDGEMENTS |
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We thank Drs. T. Nagase, N. A. Jenkins, and C. A. Ross for providing cDNA clones for various Nedd4-like proteins.
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FOOTNOTES |
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* This work was supported by grants from the Australian National Heart Foundation and the National Health and Medical Research Council.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.
¶ Equal senior authors.
Fellow of the Medical Foundation of the University of Sydney.
** Wellcome Trust Senior Fellow in Medical Science. To whom correspondence should be addressed: Hanson Centre for Cancer Research, Inst. of Medical and Veterinary Science, P. O. Box 14, Rundle Mall, Adelaide, SA 5000, Australia. Fax: 61-8-8222-3139; E-mail: sharad.kumar@imvs.sa.gov.au.
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.C000906200
2 Primer sequences are available upon request.
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ABBREVIATIONS |
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The abbreviations used are: ENaC, epithelial sodium channel; PY motif, PPxY sequence; GST, glutathione S-transferase; NMDG, N-methyl-D-glucamine; EST, expressed sequence tag; I-V relation, Current-voltage relation; kb, kilobase pair; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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