From the Department of Physiology, University of
Sydney, New South Wales 2006, Australia and the
§ Hanson Centre for Cancer Research, Institute of Medical
and Veterinary Science, Frome Road, Adelaide, South Australia 5000, Australia
Received for publication, December 14, 2000, and in revised form, February 1, 2001
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ABSTRACT |
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The amiloride-sensitive epithelial
Na+ channels (ENaC) in the intralobular duct cells of
mouse mandibular glands are inhibited by the ubiquitin-protein ligase,
Nedd4, which is activated by increased intracellular Na+.
In this study we have used whole-cell patch clamp methods in mouse
mandibular duct cells to investigate the role of the C termini of the
The epithelial Na+ channel
(ENaC)1 (1) is a major
protein supporting the transepithelial transport of Na+ in
many epithelia, including the renal collecting duct, the descending colon, the salivary ducts, and the respiratory epithelium (2, 3). It
plays an important role in the regulation of blood pressure (4) and of
fluid balance across the respiratory tract (5). Activating mutations in
these channels, such as those found in the autosomal dominant form of
hereditary hypertension known as Liddle's syndrome, produce
hypertension both in humans (6) and in mouse models (7). Conversely,
inactivating mutations produce salt-sensitive hypotension and fluid
accumulation in the respiratory tract (8-10).
The C termini of each of the three subunits of ENaC contain PY
motifs that bind the ubiquitin-protein ligase, Nedd4 (11-13). Nedd4
then ubiquitinates the Na+ channel (14-16) leading to its
endocytosis (17) and degradation (16). Thus, mutation or deletion of
the PY motifs leads to increased surface expression of Na+
channels in the Xenopus oocyte expression system (15,
18-20) and in renal epithelial cells (21). Furthermore,
over-expression of wild-type Nedd4 reduces Na+ channel
activity in Xenopus oocytes (15, 22), whereas
over-expression of a Nedd4 mutant that is inactive as a ubiquitin
protein ligase leads to increased Na+ channel activity (15,
22). The binding of Nedd4 to the PY motifs in the Na+
channel has also been proposed as a prerequisite for the insertion of
the channels into the cell membrane in response to increased intracellular cyclic AMP (23).
The interaction between Nedd4 and Na+ channels appears to
be regulated. Increased intracellular Na+ triggers the
inactivation of Na+ channels by Nedd4 in Xenopus
oocytes (20, 24-26) and in mouse salivary duct cells (14, 27-29). The
importance of this regulation is evident from the finding that
Liddle's syndrome is induced by the deletion or mutation of the
PY motifs in the C termini of the We investigated this question in mouse mandibular duct cells which,
like the cells of other aldosterone-sensitive epithelia (34, 35),
express all three subunits of ENaC (3, 29) and contain an
amiloride-sensitive Na+ conductance that is activated by
aldosterone (35). This amiloride-sensitive Na+ conductance
is inhibited by increased intracellular Na+ acting via a
Nedd4-mediated mechanism (14, 29). In the present study we used
whole-cell patch clamp methods to examine whether the inclusion in the
pipette solution of fusion proteins and synthetic peptides
corresponding to the C termini of the Expression Plasmids--
The regions encoding the C termini of
human and mouse Production of GST Fusion Proteins--
Overnight cultures of
Escherichia coli DH5 Whole-cell Patch Clamping--
Isolated granular duct cells were
prepared by collagenase digestion of mouse mandibular glands from male
mice as described previously (37). 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
NMDG-glutamate, 5 mM NMDG-Cl, 1 mM
MgCl2, 10 mM HEPES, 5 mM EGTA, and
10 mM glucose. In the 70 mM Na+
pipette solution, the Na+ concentration was adjusted by
substituting sodium glutamate for NMDG-glutamate. Standard whole-cell
patch clamp techniques were used (37, 38). After establishing the
whole-cell configuration, we replaced the bath solution with one
containing 145 mM sodium 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 (37,
38). The chord conductance was measured between Pertussis Toxin--
Pertussis toxin was stored as a 111 µg/ml
stock solution. It was activated prior to use by incubation with 5 mM dithiothreitol for 15 min at 35 °C. It was then
diluted to 500 ng/ml in pipette solution to which 1 mM
nicotinamide adenine nucleotide had been added.
The C Termini of Identification of the Motif Responsible for the
Feedback-independent Inhibitory Action of the C Terminus of
We confirmed these findings by using synthetic peptides corresponding
to the final 10 amino acids of mouse Identification of the Motif Responsible for the
Feedback-independent Inhibitory Action of the C terminus of
The experiments with GST fusion proteins suggested that the region
between 10 and 19 residues from the C terminus of mouse The Basis for the Feedback-independent Inhibitory Activity of Human
A Peptide Including the The C Termini of This study has shown that peptides and fusion proteins containing
the PY motifs of On the other hand, we found that the C terminus of As explained in the Introduction, the WW1 domains of mouse and human
Nedd4 do not bind any of the subunits of ENaC (11, 29). In the case of
mouse Nedd4, which has only three WW domains, this raises the question
of whether WW2 and WW3 bind to all three subunits of the
Na+ channel or to only a subset of them. The present
results demonstrate that WW2 and WW3 bind only to the The other major finding of our study is that the exogenous C termini of
both the Consistent with our present findings, it has been reported previously
that deletion of the C-terminal 8 amino acids of The mechanism by which the C termini of the -,
-, and
-subunits of ENaC in mediating this inhibition. We
found that peptides corresponding to the C termini of the
- and
-subunits, but not the
-subunit, inhibited the activity of the
Na+ channels. This mechanism did not involve Nedd4 and
probably resulted from the exogenous C termini interfering
competitively with the protein-protein interactions that keep the
channels active. In the case of the C terminus of mouse
-ENaC, the
interacting motif included
Ser631,
Asp632, and
Ser633. In the C terminus of
mouse
-ENaC, it included
Ser640. Once these motifs
were deleted, we were able to use the C termini of
- and
-ENaC to
prevent Nedd4-mediated down-regulation of Na+ channel
activity. The C terminus of the
-subunit, on the contrary, did not
prevent Nedd4-mediated inhibition of the Na+ channels. We
conclude that mouse Nedd4 interacts with the
- and
-subunits of
ENaC.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
- or
-subunits (6, 30-33). Yet
little is known of the mechanism by which Nedd4 binds to ENaC. WW1, one
of the three WW domains present in mouse Nedd4, does not bind any of
the subunits of the epithelial Na+ channel (29), and the
same observation has been made for the WW1 domain of human Nedd4 (11).
Given that all three subunits of the Na+ channel,
,
,
and
, contain PY motifs in their C termini, this finding raised the
question of whether the remaining two WW domains in mouse Nedd4 bind
all three subunits of the Na+ channel indiscriminately or
whether they bind specifically to only two of them. The available
information that might have resolved this question is contradictory.
Thus, although no cases of Liddle's syndrome have been reported to be
caused by the deletion or mutation of the PY motif in the
-subunit
(4), deletion of the C terminus of the
-subunit does lead to
increased ENaC activity in Xenopus oocytes (33).
-,
-, and
-subunits of
the epithelial Na+ channel could prevent down-regulation of
Na+ channel activity in mouse mandibular duct cells. We
found that the C termini of
- and
-ENaC, but not of
-ENaC, are
able to prevent inhibition of Na+ channel activity by
raised intracellular Na+.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-,
-, and
-ENaC subunits were polymerase chain
reaction-amplified and cloned into either BamHI or
BamHI/EcoRI sites of pGEX-2TK. Mouse C terminus
-ENaC-10, mouse C terminus
-ENaC-10, and mouse C terminus
-ENaC-19 were polymerase chain reaction-amplified and cloned into
the BamHI/EcoRI sites of pGEX-2TK. Human
-ENaC PPNA, human
-ENaC S637G, and mouse
-ENaC PPRA and S643G
mutants were generated by quick-change mutagenesis (Stratagene). Mouse
-,
-, and
-ENaC subunit cDNAs were cloned from mouse
mandibular duct cells as previously described (29) and found to be
identical with the published sequences (36). Human a-,
-, and
-ENaC cDNA clones were the gift of Dr. M. Welsh (University of Iowa).
harboring the appropriate GST
expression plasmid were diluted 1/50, incubated for 2 h at
37 °C, induced with 1 mM isopropyl
-D-thiogalactoside, and incubated for an additional
5 h at 37 °C. Bacterial cell pellets were resuspended in
phosphate-buffered saline, lysed by sonication, and clarified by
centrifugation at 10,000 × g for 10 min.
Glutathione-Sepharose (Amersham Pharmacia Biotech) was incubated with
the cleared lysate for 60 min at room temperature, after which the
beads were washed three times with phosphate-buffered saline. Fusion
protein was eluted with glutathione buffer according to the
manufacturer's protocol. Protein concentration was measured using a
BCA kit (Pierce). Fusion proteins were routinely checked by
electrophoresis of 3 µg of the protein on a 12% SDS-polyacrylamide
gel, which was then stained by Coomassie Blue (data not shown). In
accordance with our usual practice, for the protein concentrations
quoted in this paper, compensation was made for the presence of
contaminating proteins (29).
80 mV and the
zero-current potential of the amiloride-sensitive current (37). The
results are expressed as the mean ± S.E. Statistical significance
was assessed using Student's unpaired t test. All
experiments were performed at 20-22 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-ENaC and
-ENaC, but Not
-ENaC, Reduce
Na+ Channel Activity Independently of Feedback
Inhibition--
Prior to examining the effect of the C-terminal fusion
proteins (Fig. 1) on Na+
feedback inhibition of the Na+ channels, we checked whether
they affected Na+ channel activity when the
Na+ feedback system is inactive. We did so by including
300 µg of each GST fusion protein ml in the zero
Na+ pipette solution and then observing the effect of the
fusion protein on the amiloride-sensitive Na+ current. To
our surprise, we found that both the fusion protein containing the C
terminus of human
-ENaC and that containing the C terminus of mouse
-ENaC inhibited the amiloride-sensitive Na+ current
(Fig. 2). The fusion protein containing
the C terminus of mouse
-ENaC, however, did not have this effect
(Fig. 2).
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Fig. 1.
Amino acid sequences of the C termini used in
this study. The PY motifs present in -,
-, and
-ENaC are
underlined. The SH3-binding domain present in
-ENaC is
indicated by shading. The residues that are identical
between the human and mouse proteins are shown in bold. *,
termination codon.
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Fig. 2.
The C termini of -
and
-ENaC inhibit the amiloride-sensitive
Na+ conductance. This figure shows the effects on the
chord conductance of the amiloride-sensitive Na+ current of
including in the zero Na+ pipette solution the GST fusion
proteins of the C termini of
-,
-, or
-ENaC at a concentration
of 300 µg/ml. The number of experiments is shown in
parentheses.
-ENaC--
We first investigated the basis of the inhibitory
activity of the C terminus of
-ENaC. We found that a fusion protein
containing the C terminus of human
-ENaC, in which the PY motif
(PPNY) had been mutated (PPNA) so that it no longer interacted with
Nedd4 (29), was still inhibitory (Fig.
3). Furthermore, inclusion of the C
terminus of mouse
-ENaC in the pipette solution together with a
dominant negative mutant of ubiquitin (K48R), which we have previously
shown to block inhibition of Na+ channels by increased
intracellular Na+ in these cells (14), failed to prevent
the inhibition produced by the C terminus of mouse
-ENaC. Because
inclusion of the K48R mutant of ubiquitin in zero Na+
pipette solution is not itself inhibitory (14), we concluded that the
inhibition produced by the C terminus
-ENaC does not involve
ubiquitination of the Na+ channel. Deletion of 10 amino
acids from the C terminus of the mouse
-ENaC fusion protein,
however, prevented the inhibitory effect of the fusion protein (Fig.
3), suggesting that the inhibitory motif is present in the last 10 amino acids of the
-subunit.
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Fig. 3.
Localization of the inhibitory domains of the
C termini of -ENaC. The effects are shown
on the chord conductance of the amiloride-sensitive Na+
current of the inclusion in the zero Na+ pipette solution
of 300 µg/ml GST-
-ENaC fusion protein in which the PY motif had
been mutated to PPNA, of a mixture of 300 µg/ml control GST-
-ENaC
fusion protein and 300 µg/ml K48R mutant ubiquitin, or of 300 µg/ml
GST-
-ENaC fusion protein in which the final 10 amino acids of
-ENaC had been deleted. The number of experiments is
shown in parentheses, and the chord conductance observed
with the control zero Na+ pipette solution is shown as a
broken line.
-ENaC (Fig.
4A). We found that the
inclusion of 20 µM
C10 peptide in the zero Na+ pipette solution inhibited the amiloride-sensitive
Na+ current almost completely (Fig. 4B). We then
investigated whether the action of the peptide was mediated by a
pertussis toxin-sensitive G protein, because these are known to inhibit
amiloride-sensitive Na+ channels (37, 39). We found,
however, that the inclusion of activated pertussis toxin in the peptide
solution did not prevent its inhibitory action on the Na+
channel (Fig. 4B). Finally, we investigated the effects of
peptides based on
C10 in which residues that may have been involved
in the inhibitory motif were substituted by glycine (Fig.
4C). Initially we examined two peptides in which we had made
double substitutions. One of these,
E636G/I638G, in which we
replaced the glutamate and isoleucine residues at positions 8 and 10 of
the peptide (Fig. 4A) to target a possible PDZ-binding
domain at the C terminus (40), retained its inhibitory activity (Fig.
4C). The other peptide,
D632G/S633G (Fig. 4A),
was without inhibitory activity (Fig. 4C). We further found
that the inhibitory effect of the
C10 peptide was completely
abolished by substituting glycine for the serine residues at positions
3 (peptide
S631G in Fig. 4A) or 5 (peptide
S633G in
Fig. 4A). Substituting glycine for the aspartate residue at
position 4 (
D632G in Fig. 4A), which lies between the two
serine residues, reduced (p < 0.05), but did not
abolish (p < 0.05) the inhibitory action of the
peptide (Fig. 4C).
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Fig. 4.
Investigation of the inhibitory motif in the
C terminus of -ENaC. A, amino
acid sequences of the synthetic peptides used in this study.
B, effects of 20 µM
C10
peptide and of a mixture of 20 µM
C10 peptide with 500 ng/ml activated pertussis toxin on the chord conductance of the
amiloride-sensitive Na+ current when added to zero
Na+ pipette solution. C, effects of the
synthetic peptides described in panel A on the chord
conductance of the amiloride-sensitive Na+ current when
added to the zero Na+ pipette solution. In
panels B and C, the number of
experiments is given in parentheses, and the mean chord
conductance of the amiloride-sensitive Na+ current observed
using the control zero Na+ pipette solution is shown as a
broken line.
-ENaC--
We then investigated the basis of the inhibitory action
of the C terminus of mouse
-ENaC. We found that mutation of the PY motif of mouse
-ENaC (PPRA) did not alter the inhibitory activity of
the fusion protein (Fig. 5), indicating
that the inhibition was not caused by activation of the feedback
inhibitory system mediated by Nedd4. In contrast to our findings with
the C terminus of mouse
-ENaC, deletion of the terminal 10 amino
acids of mouse
-ENaC did not remove the inhibitory effect of this
fusion protein on the Na+ channel (Fig. 5). Only when we
removed 19 amino acids from the C terminus of mouse
-ENaC did the
fusion protein cease to inhibit the amiloride-sensitive Na+
current (Fig. 5).
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Fig. 5.
Localization of the inhibitory domains of the
C termini of -ENaC. The effects are shown
on the chord conductance of the amiloride-sensitive Na+
current of the inclusion in the zero Na+ pipette solution
of 300 µg/ml GST-
-ENaC fusion protein in which the PY motif had
been mutated to PPRA, 300 µg/ml GST-
ENaC fusion protein in which
the final 10 amino acids of
-ENaC had been deleted, or 300 µg/ml
GST-
ENaC fusion protein in which the final 19 amino acids of
-ENaC had been deleted. The number of experiments is
shown in parentheses, and the chord conductance observed
with the control zero Na+ pipette solution is shown as a
broken line.
-ENaC
contained the inhibitory motif. We therefore investigated whether a chemically synthesized peptide corresponding to this region
(
mC9 in Fig.
6A) was inhibitory when
included in the zero Na+ pipette solution, and we
found that it was (Fig. 6B). Substituting the three serine
residues (
Ser640,
Ser643, and
Ser644) in this peptide by glycines (
Ser
Gly)
removed this inhibitory activity (
S-G in Fig.
6B). We then found that a peptide in which Ser640 was substituted by glycine
(
S640G in Fig. 6A) was without
inhibitory activity (Fig. 6B), whereas a peptide in which
serines 643 and 644 are replaced by glycines
(
S643G/S644G in Fig. 6A) was
still inhibitory (Fig. 6B). Thus, serine 640 is required for
the inhibitory activity of the C terminus of mouse
-ENaC.
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Fig. 6.
Investigation of the inhibitory motif in the
C terminus of mouse -ENaC. A,
amino acid sequences of the synthetic peptides used in this study.
B, effects of the synthetic peptides described in
panel A on the chord conductance of the amiloride-sensitive
Na+ current when added to the zero Na+ peptide
solution. In panel B, the number of experiments
is given in parentheses, and the mean chord conductance of
the amiloride-sensitive Na+ current observed using the
control zero Na+ pipette solution is shown as a
broken line.
-ENaC--
Serine 640 in mouse
-ENaC is not present in human
-ENaC where it is replaced by an arginine residue (Fig.
7A). We thus expected that a
GST fusion protein corresponding to the C terminus of human
-ENaC
would not be inhibitory when included in the zero Na+
pipette solution (Fig. 7A). We found, however, that it did
inhibit the amiloride-sensitive Na+ current (Fig.
7B). We thus investigated the effect of mutating the sole
serine in the C terminus of human
-ENaC to glycine (
hS637G). We
found that this mutant no longer inhibited the amiloride-sensitive Na+ current when included in zero Na+ pipette
solution (Fig. 7B). Mutation of this serine in the C terminus of mouse
-ENaC, however, did not remove the inhibitory activity of the C terminus of mouse
-ENaC (Fig. 7B). This
was consistent with our earlier findings using chemically synthesized peptides (see above and Fig. 6B).
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Fig. 7.
The C terminus of human
-ENaC inhibits the amiloride-sensitive
Na+ conductance. A, sequences of the fusion
proteins used in Fig. 8B. B, the effects on the
chord conductance of the amiloride-sensitive Na+ current of
including in the zero Na+ pipette solution the GST fusion
proteins shown in panel A at a concentration of 300 µg/ml.
The number of experiments is given in
parentheses, and the mean chord conductance of the
amiloride-sensitive Na+ current observed using the control
zero Na+ pipette solution is shown as a broken
line.
-ENaC PY Motif Blocks Na+
Feedback Inhibition--
As outlined in the Introduction, we wished to
determine whether mouse Nedd4 interacts with all three subunits of the
epithelial Na+ channel or just two of them. We decided to
do this by investigating whether inclusion in the patch pipette of GST
fusion proteins of the C termini of
-ENaC,
-ENaC, or
-ENaC
(Fig. 1) could block the inactivation of Na+ channels
induced by raised intracellular Na+, which in mouse
mandibular duct cells is known to be mediated by Nedd4 (14). This
experimental strategy was based on the idea that Nedd4 in salivary duct
cells would bind exogenous PY motifs, which would then prevent its
normal interaction with, and inhibition of, Na+ channels.
We tested this idea by examining whether a synthetic peptide that
included the PY motif of
-ENaC (Fig.
8A) could block inhibition of
the Na+ channels by increased intracellular
Na+. As is evident from Fig. 8B, it does so with
a half-maximal concentration of 0.3 µM.
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Fig. 8.
Use of a peptide containing the PY motif of
mouse -ENaC to prevent inhibition of
Na+ channels by cytosolic Na+.
A, amino acid sequence of the synthetic peptide. The PY
motif is shown in bold. B, concentration-response curve for
the effect of the peptide on the chord conductance of the
amiloride-sensitive Na+ current when the pipette contained
the high (70 mM) Na+ solution. The
number of experiments is shown in parentheses.
For ease of comparison, the chord conductance of the
amiloride-sensitive Na+ current measured using the zero
Na+ pipette solution is shown as a broken
line.
- and
- but not
-ENaC Inhibit Regulation
of Na+ Channels by Increased Intracellular
Na+--
We then proceeded to investigate whether the ENaC
C termini interrupted control of the Na+ channels by
intracellular Na+. We found that we could prevent the
inhibitory action of the 70 mM Na+ pipette
solution by including in it the C terminus of mouse
-ENaC with the
last 10 amino acids deleted (Fig. 9). To
confirm that this effect was due to the PY motif in this construct, we
also investigated the effect of this construct following mutation of the tyrosine in the PY motif to an alanine residue (
mENaC-10 PY-mut). We have previously shown that this mutation eliminates the
binding of the C terminus of mouse
-ENaC to Nedd4 (29). We found
that the mutant construct no longer prevented feedback inhibition of
the amiloride-sensitive Na+ current (Fig. 9). We also found
that the C terminus of mouse
-ENaC with the last 19 amino acids
deleted prevented the inhibition of the amiloride-sensitive
Na+ current by raised intracellular Na+ (Fig.
9). Once again, mutation of the construct by substitution of the
tyrosine in the PY motif by alanine to eliminate its capacity to bind
Nedd4 (29) rendered the fusion protein incapable of preventing
Na+ feedback inhibition (Fig. 9). Finally, inclusion of the
C terminus of mouse
-ENaC in the 70 mM
Na+ pipette solution failed to prevent the down-regulation
of the amiloride-sensitive Na+ current (Fig. 9).
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Fig. 9.
The C termini of -
and
- but not
-ENaC
inhibit Na+-dependent feedback. Effects on
the chord conductance of the amiloride-sensitive Na+
current of the inclusion of the ENaC C termini fusion proteins (300 µg/ml) in the 70 mM Na+ pipette solution. The
number of experiments is given in parentheses,
and the mean chord conductance of the amiloride-sensitive
Na+ current observed using the control zero Na+
pipette solution is shown as a broken line.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
- and
-ENaC prevent inhibition by increased intracellular Na+ of the amiloride-sensitive
Na+ current in mouse mandibular duct cells.
Na+-feedback inhibition of Na+ channels in
mouse mandibular duct cells has previously been shown to be blocked by:
(i) antibodies directed against mouse Nedd4, (ii) a mutant of mouse
Nedd4 containing the WW domains but no HECT (ubiquitin-protein ligase)
domain, and (iii) a dominant negative mutant of ubiquitin (14, 29),
indicating that the Na+ feedback inhibition is mediated by
Nedd4, which is expressed in these cells (14). Thus, the present
findings show that the interaction of mouse Nedd4 with
amiloride-sensitive Na+ channels can be blocked by PY
motifs derived from the
- and
-subunits of ENaC. Presumably these
act by competing with the PY motifs in the native
- and
-subunits
of ENaC, thus preventing the normal interaction of Nedd4 with the
Na+ channels.
-ENaC was unable
to block Na+ feedback regulation of the amiloride-sensitive
Na+ current. This fusion protein is evidently unable to
compete with the endogenous Na+ channels for binding of
Nedd4. Given that the
-subunits of rat, human, and mouse ENaC have
all been shown to bind Nedd4 in vitro (11, 12, 29), this
indicates that in vivo there must be some constraint on the
interaction of the PY motif of the
-subunit of the channel with
Nedd4. Consistent with this possibility is the report that the C
terminus of
-ENaC is bound to
-spectrin in Madin-Darby canine
kidney cells (41). The binding of
-spectrin is mediated by an SH3
(Src homology 3)-binding domain that overlaps the PY motif in
-ENaC
(Fig. 1) and is not present in
- or
-ENaC.
- and
-subunits of the channel in vivo. Given that we have
previously shown that the sites recognized by WW2 and WW3 in ENaCs from
mouse salivary cells are distinct and are able to distinguish between
WW2 and WW3 (29), it would thus appear likely that the WW2 of
Nedd4 binds
-ENaC and the WW3 of Nedd4 binds
-ENaC or vice versa.
From the present data we cannot distinguish these possibilities.
- and
-subunits of the Na+ channel inhibit
Na+ channel activity. As argued below, this probably
occurs because the exogenous C termini compete with the Na+
channels to interact with a protein, perhaps a kinase, that is required
to maintain channel activity. In the case of the C terminus of mouse
-ENaC, this motif is located in the final 10 amino acids and
requires the presence of both of the serine residues present in this
peptide,
Ser631 and
Ser633, as well as
the aspartate residue between them,
Asp632. In the case
of the C terminus of mouse
-ENaC, the motif is in the region between
10 and 19 residues from the C terminus, and requires the presence of
only one of the three serine residues present in this region,
Ser640. We further found that the C terminus
of human
-ENaC inhibits the amiloride-sensitive Na+
current. This inhibition requires the presence of the serine
Ser637, the only serine present in the C terminus of
human
-ENaC. Interestingly, the corresponding serine in mouse
-ENaC is not involved in inhibiting the amiloride-sensitive
Na+ current (Fig. 7).
-ENaC increases
Na+ channel activity in Xenopus oocytes (21),
although a subsequent study in which the final 10 amino acids were
deleted from
-ENaC failed to observe any stimulation (33). It has
also been reported that the constitutively activated Na+
channels obtained from lymphocytes of subjects with Liddle's syndrome
can be blocked by the application of peptides that include the final 10 amino acids of the C terminus of
-ENaC (42). This inhibition was
attributed to the marked negative charge of the terminal decapeptide of
-ENaC, because a peptide in which the four negatively charged
residues found in it had been substituted by uncharged residues was
inactive (42). The four negative charges mutated in the lymphocyte
studies included, however,
Asp632, which we
have shown in the present study to be required for the inhibitory
activity of the C terminus of
-ENaC. Furthermore, we have found that
mutation of
Glu636 to an uncharged residue does not
impair the inhibitory activity of the C-terminal peptide of
-ENaC
(42).
- and
-C
subunits inhibit the Na+ channels is unclear. For the C
termini of both
-ENaC and
-ENaC, we can exclude any role for
binding of Nedd4. In the case of
-ENaC, we can also exclude any role
for ubiquitination or pertussis toxin-sensitive G proteins, which are
known to inhibit Na+ channels in these and other cell types
(14, 37, 39). Finally, the small size of the inhibitory regions of the
C termini make it unlikely that they are forming a
-pleated sheet
and hence blocking the channel pore (43). Given the requirement for the presence of serine residues in the C termini of both
-ENaC and
-ENaC and the report that these regions are constititutively phosphorylated in Madin-Darby canine kidney cells (44), it is tempting
to postulate that the addition of exogenous
-ENaC or
-ENaC C
termini to the cytosol may be interfering in the phosphorylation processes required to maintain the channels in an active state. Consistent with this possibility is the observation that the C terminus
of
-ENaC, which in the present studies is without inhibitory activity, is not phosphorylated in vivo (44).
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FOOTNOTES |
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* This project was supported by the National Health and Medical Research Council of Australia, the National Heart Foundation of Australia, and the Australian Kidney Foundation.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.
¶ Wellcome Trust Senior Fellow in Medical Science.
These authors contributed equally to this work.
** Fellow of The Medical Foundation of the University of Sydney. To whom correspondence should be addressed: Dept. of Physiology (F-13), University of Sydney, NSW 2006, Australia. Fax: 61-2-9351-9926; E-mail: davidc@physiol.usyd.edu.au.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M011273200
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ABBREVIATIONS |
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The abbreviations used are: ENaC, epithelial Na+ channels; PY motif, PPXY sequence; GST, glutathione S-transferase; NMDG, N-methyl-D-glucamine.
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