Cloning and functional expression of the mouse epithelial
sodium channel
Yoon J.
Ahn,
David R.
Brooker,
Farhad
Kosari,
Brian J.
Harte,
Jinqing
Li,
Scott A.
Mackler, and
Thomas R.
Kleyman
Departments of Medicine and Physiology, University of Pennsylvania,
and Veterans Affairs Medical Center, Philadelphia, Pennsylvania
19104-6144
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ABSTRACT |
The epithelial sodium channel (ENaC) plays a major role in the
transepithelial reabsorption of sodium in the renal cortical collecting
duct, distal colon, and lung. ENaCs are formed by three structurally
related subunits, termed
-,
-, and
ENaC. We previously isolated and sequenced cDNAs encoding a portion of mouse
-,
-, and
ENaC (
-,
-, and
mENaC). These cDNAs were used to screen an oligo-dT-primed mouse kidney cDNA library. Full-length
mENaC and
partial-length
- and
mENaC clones were isolated. Full-length
-
and
mENaC cDNAs were subsequently obtained by 5'-rapid
amplification of cDNA ends (5'-RACE) PCR. Injection of mouse
-,
-, and
ENaC cRNAs into
Xenopus oocytes led to expression of
amiloride-sensitive (Ki = 103 nM),
Na+-selective currents with a
single-channel conductance of 4.7 pS. Northern blots revealed that
-,
-, and
mENaC were expressed in lung and kidney.
Interestingly,
mENaC was detected in liver, although transcript
sizes of 9.8 kb and 3.1 kb differed in size from the 3.2-kb message
observed in other tissues. A partial cDNA clone was isolated from mouse
liver by 5'-RACE PCR. Its sequence was found to be nearly
identical to
mENaC. To begin to identify regions within
mENaC
that might be important in assembly of the native heteroligomeric
channel, a series of functional experiments were performed using a
construct of
mENaC encoding the predicted cytoplasmic
NH2 terminus. Coinjection of
wild-type
-,
-, and
mENaC with the intracellular
NH2 terminus of
mENaC abolished amiloride-sensitive currents in
Xenopus oocytes, suggesting that the
NH2 terminus of
mENaC is involved in subunit assembly, and when present in a 10-fold
excess, plays a dominant negative role in functional ENaC expression.
cloning; Xenopus oocytes; structure-function relationship
 |
INTRODUCTION |
THE ORGANIZATION of plasma membrane proteins in the
epithelial cell layer of polarized cells is a necessary requirement for vectorial transport of solutes. Apical and basolateral plasma membranes
differ in protein composition and contain
Na+-selective transport proteins
that facilitate the movement of Na+ across the epithelium in a
directed fashion (5, 33, 38, 49). Epithelial
Na+ channels are expressed in
apical plasma membranes of principal cells in the distal nephron,
airway and alveolar epithelia in the lung, surface cells in the distal
colon, urinary bladder epithelia, and other tissues including ducts of
salivary and sweat glands (5, 13, 39, 50). These channels mediate
Na+ transport across polarized
epithelia (5, 17, 39, 49) and are selectively inhibited by
submicromolar concentrations of amiloride (28). Renal epithelial
Na+ channels are aldosterone
responsive and are the rate-limiting step for distal
Na+ reabsorption from the
uriniferous space. Na+ crosses the
apical surface of the cortical collecting duct principal cell via ENaC
and reenters the bloodstream via
Na+-K+-ATPase
located on the basolateral membrane. Although precise gating mechanisms
have not been fully elucidated, the up- or downregulation of ENaCs in
the collecting tubule is manifest in perturbations of total body sodium
homeostasis, extracellular fluid volume status, and blood pressure
control (5, 18, 58). ENaC gain of function mutations has been
identified in patients with Liddle's disease, a disorder characterized
by volume expansion and hypertension (24, 48); conversely, ENaC loss of
function mutations have been noted in patients with type I
pseudohypoaldosteronism, a disorder characterized by volume depletion
and hypotension (10).
The epithelial Na+ channel was
initially cloned from rat distal colon (7, 9, 30, 59). An expression
cloning technique led to the identification of one cDNA, termed
ENaC, whose cRNA induced expression of amiloride-sensitive
Na+ currents in
Xenopus oocytes (7, 30). However,
Na+ current levels were
considerably lower than expected. Two subsequent cDNA clones were
isolated on the basis of their ability to complement
ENaC cRNA in
the expression of amiloride-sensitive
Na+ currents in
Xenopus oocytes, and these were termed
ENaC and
ENaC (9). Coinjection of the three cRNA species into
Xenopus oocytes led to expression of
Na+ channels with characteristics
nearly identical to those observed in
Na+ channels of renal cortical
collecting tubules and in A6 cells (9, 23, 35). The three subunits
share limited (~30% to 40%) sequence similarity, suggesting that
they are derived from a common ancestral gene. ENaCs have subsequently
been cloned from human lung and kidney (31, 32, 59),
Xenopus kidney (36), avian distal
intestine (21), and bovine kidney (
-subunit) (16). We have isolated
cDNA clones encoding mouse
-,
-, and
ENaC and have examined
their tissue distribution and functional characteristics. To begin to
identify regions within
mENaC that might be important in assembly of
the native channel, a deletion construct of
mENaC encoding the
cytoplasmic NH2 terminus was
synthesized. We report that coinjection of wild-type
-,
-, and
mENaC with the intracellular amino terminus of
mENaC inhibited
expression of amiloride-sensitive currents in oocytes, suggesting that
the NH2 terminus of
mENaC is
involved in subunit assembly.
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MATERIALS AND METHODS |
Reagents were purchased from Sigma Chemical (St. Louis, MO), unless
otherwise specified. For molecular biology protocols, standard
procedures were followed (2, 41, 57). DNA sequencing and
oligonucleotide syntheses were performed by the University of
Pennsylvania DNA Core Facility.
Generation of probes and library
screen. cDNAs partially encoding mouse
-,
-, and
ENaC were isolated as previously described (6). These cDNAs were
radiolabeled by random priming (Prime-It II random primer labeling kit;
Stratagene, La Jolla, CA) with [
-32P]dATP (ICN,
Costa Mesa, CA). A mouse kidney cDNA library cloned into the Lambda
Uni-ZAP XR vector (Stratagene) and transformed bacteria were plated,
and colonies were transferred to Hybond-N nylon filters (Amersham,
Arlington Heights, IL). For primary screening, the filters were
hybridized overnight to the labeled probes in hybridization solution
[6× standard saline citrate (SSC), 20 mM NaH2PO4,
0.5% (wt/vol) SDS, and 500 µg/ml denatured, sonicated salmon sperm
DNA] at 55°C. Filters were washed with 2× SSC + 0.1% SDS at room temperature, twice with 1× SSC + 0.1% SDS at
50°C, and once with 0.1× SSC + 0.1% SDS at 50°C. Filters
were exposed overnight to Kodak X-Omat AR film at
70°C.
Positive colonies on duplicate filters were selected and rescreened.
Colonies were isolated by sib selection, subjected to
plasmid rescue, excision to determine insert size, and partial
nucleotide sequencing of the 5' end of the inserts.
Generation of full-length
- and
mENaC subunits.
As no full-length
mENaC or
mENaC clones were obtained in the
library screen, 5'-rapid amplification of cDNA ends
(5'-RACE) PCR using gene-specific antisense primers based on
known sequence (5'-TGGAAGACATCCAGAGATTG-3' for
- and
5'-CCACCAGTTTCTTCGACTCAT-3' for
mENaC) was performed to
obtain the missing upstream fragments of the
- and
mENaC
subunits. PCR conditions were as follows: initial denaturation at
94°C for 2 min, 30 amplification cycles (94°C for 10 s,
55°C for 20 s, 68°C for 2 min), and final elongation at
68°C for 10 min. The PCR products were subcloned into pCR2 (Invitrogen, Carlsbad, CA). The upstream (PCR products) and downstream (cDNAs from library screen) fragments of
- and
mENaC were ligated following restriction enzyme digestion to generate the full-length
-
or
mENaC. Full-length clones were sequenced in both directions by
the method of Sanger et al. (42).
Cloning of
NH2-terminal
mENaC and
ectodomain
mENaC.
The cDNA residues encoding the
NH2-terminal domain of
mENaC
(corresponding to amino acid residues M1-F81) were PCR amplified and
subcloned into pBS SK(
) (Stratagene). cDNA encoding the
ectodomain of
mENaC (corresponding to amino acid residues Y166-P568)
was PCR amplified and subcloned into pBK-CMV (Stratagene). Sequences were confirmed by Sanger dideoxynucleotide sequence analysis.
Northern blots. A commercial mouse
multiple tissue Northern blot containing equal quantities (2 µg) of
purified poly(A)+ RNA in each lane
was used (Clontech, Palo Alto, CA) to examine mENaC tissue
distribution. cDNA
fragments1
of
ENaC (G1365-T1755),
ENaC (T811-C1018),
ENaC (G958-C1205), and mouse
-actin were 32P
labeled (14) and individually hybridized with the membrane overnight at
50°C following the manufacturer's protocol. The blots were washed
at high stringency (final wash, 0.1× SSC at 65°C) and exposed
to film or to a phosphor screen for imaging (Molecular Dynamics,
Sunnyvale, CA).
Oocyte expression and
electrophysiology. cRNAs were generated from mENaC cDNA
inserts in pBS SK(
) or pBK-CMV vector using a T3 cRNA synthesis
kit (m-MESSAGE MACHINE; Ambion, Austin, TX) following the
manufacturer's protocol. Prior to transcription all cDNA constructs
were linearized by restriction digestion with Xho I except for
mENaC where
Kpn I was used. Oocytes were isolated from Xenopus laevis females (Nasco,
Fort Atkinson, WI), and stage V-VI oocytes were selected for
collagenase treatment following standard protocols (57). Oocytes were
injected with full-length mouse
-,
-, and
ENaC cRNAs at a
concentration of 2 ng · subunit
1 · oocyte
1.
NH2-terminal
ENaC cRNA was
injected at concentrations of 20 ng, 6 ng, or 2 ng per oocyte. All
cRNAs were injected in a volume of 50 nl/oocyte. Following injection,
oocytes were incubated at 18°C in modified Barth's saline
[88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 15 mM HEPES, 0.3 mM
Ca(NO3)2,
0.41 mM CaCl2, 0.82 mM
MgSO4, 10 µg/ml penicillin, 10 µg/ml streptomycin sulfate, 100 µg/ml gentamycin sulfate, and 10 µg/ml nystatin; pH 7.2] and then assayed 18-48 h
postinjection. In selected experiments, oocytes were incubated in a
low-Na+ modified Barth's saline
(the 88 mM NaCl was replaced with 88 mM KCl) to prevent the cells from
loading with Na+ prior to voltage
clamping. Whole cell currents were measured using the two-electrode
voltage clamp technique (TEV) at a holding potential of
100 mV
(with reference to bath) for 500 ms and then 450 ms at 0 mV. During
recordings, oocytes were bathed in a sodium gluconate buffer [100
mM sodium gluconate, 2 mM KCl, 1.8 mM
CaCl2, 10 mM HEPES, 5 mM
BaCl2, 10 mM tetraethylammonium
chloride (TEA-Cl2), pH
7.2]. Cation selectivity measurements were performed in sodium gluconate or potassium gluconate (100 mM potassium gluconate, 2 mM KCl,
1.8 mM CaCl2, 10 mM HEPES, 5 mM
BaCl2, and 10 mM
TEA-Cl2, pH 7.2) buffers.
Amiloride-sensitive currents were determined by subtracting currents
measured in oocytes perfused with sodium gluconate (or potassium
gluconate) buffers supplemented with 100 µM amiloride from baseline
currents in sodium gluconate (or potassium gluconate) buffers. TEV was
performed under continuous flow (~4 ml/min) of buffers.
Single-channel recordings were performed in the cell-attached
configuration. All data were collected at room temperature and were
filtered at 300 Hz. The applied voltage to the membrane patch
represents the voltage deflection from the resting membrane potential.
Inward Na+ currents were
represented by downward deflections in single-channel recordings.
Measurements of single-channel conductance were performed with a buffer
containing 100 mM NaCl, 1.8 mM
CaCl2, 2 mM KCl, and 10 mM HEPES,
pH 7.2, in the patch pipette and in the bath. Statistical analyses were
performed with pCLAMP software (Axon Instruments) or MatLab (MathWorks).
Statistics. Results are expressed as
means ± SE. Statistical significance was determined by Student's
t-test.
 |
RESULTS |
Cloning of full-length ENaC subunits.
Labeled probes for
-,
-, and
mENaC were synthesized as
previously described (6) and used to screen mouse kidney
oligo-dT-primed cDNA lambda libraries. Primary screening yielded 8 positive colonies for
mENaC, 9 positive colonies for
mENaC, and
12 positive colonies for
mENaC. All
mENaC, 2
mENaC, and 11
mENaC colonies were successfully isolated by sib selection,
subjected to plasmid rescue, and excision to determine insert size,
followed by partial nucleotide sequencing of the 5' end of the
inserts. Two full-length
mENaC clones were isolated
(clones B1 and
B2), but no full-length
mENaC or
mENaC clones were obtained (9). The
mENaC clone with the longest insert (clone A2) apparently lacked
978 residues of the 5' end of the open-reading frame compared
with rat
ENaC (9). The
mENaC clones with the longest inserts
(clones G5 and
G11) appeared to lack 428 and 388 residues, respectively, at the 5' end of the open-reading frame
compared with
rENaC (9). Clones A2,
B1, and
G5, and
G11 were sequenced. The sequence of
G11 was ~1 kb longer than both the
mENaC clone G5 and
rENaC in the
3'-untranslated region (data not shown). On the basis of sequence
analysis, this 1-kb addition likely represented a cloning artifact.
5'-RACE was performed to obtain cDNAs encoding the 5'
regions of
- and
mENaC. Sequence analyses confirmed that we had
obtained the 5' regions of
- and
mENaC. The RACE products
and the partial-length
- and
mENaC cDNAs were subjected to
restriction digestion and ligation to obtain full-length
- and
mENaC cDNAs.
The full-length mENaC clones were sequenced. The deduced amino acid
sequences of the mouse
-,
-, and
ENaCs were 95%, 96%, and
97% identical to rat
-,
-, and
ENaC, respectively, and 87%,
88%, and 88% identical to human
-,
-, and
ENaC. Sequence comparisons illustrate the high degree of identity between rat and
mouse ENaC, as depicted in Fig. 1. Of
particular interest was a 15-nucleotide insertion present in
mENaC
encompassing residues T399-C413, which is absent in rat.
This insertion did not occur in close proximity to defined rat genomic
intron-exon junctions (55) and likely did not arise as a result of
alternative mRNA splicing. PCR analysis of mouse genomic DNA using
primers flanking the 15-bp insertion (sense
5'-GATGTTCTAGACAGTACACCTCGGAAA-3'; antisense 5'-
AAATCCCACCAGTTTCTTCGACTCATG-3') was performed. The 235-bp PCR
product was sequenced and confirmed that the 15-bp insertion
represented a species-specific phenomenon at the genomic level.



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Fig. 1.
Deduced amino acid sequences of mouse -, -, and -subunits of
the epithelial sodium channel (ENaC). Sequence comparisons with rat
ENaCs (7, 9) are included. Amino acid identity is indicated by a dash.
Putative membrane-spanning domains (M1 and M2) are indicated by bold
underscore.
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Tissue distribution. Tissue
distribution of
-,
-, and
mENaC was examined by Northern blot
analyses. As expected, all three mENaCs were expressed in mouse lung
and kidney, with the
-,
-, and
mENaC probes recognizing mRNAs
of 3.7, 2.6, and 3.2 kb, respectively (Fig.
2).
mENaC recognized two mRNA species in
mouse liver, of ~9.8 and 3.1 kb. Hybridization to liver mRNAs was
observed under high-stringency conditions, but the signal was
relatively weak. These results suggested that
mENaC is expressed in
liver. To further confirm this observation, 5'-RACE PCR using a
3' gene-specific primer
(5'-CGGAACCTGTGCAGTAACATGATGAG-3') and a 5' adaptor
primer was performed on liver cDNA (Clontech). A PCR product of 702 bp was obtained. Its sequence was nearly identical to
mENaC (sequence not shown), providing additional evidence that
mENaC or an
mENaC isoform is expressed in mouse liver.

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Fig. 2.
Mouse -, -, and ENaC tissue distribution. A mouse multiple
tissue Northern blot containing equal quantities (2 µg) of
poly(A)+ RNA per lane was
hybridized consecutively with
32P-labeled -, -, or
mENaC or mouse actin probes as described in the
MATERIALS AND METHODS. Bound probe was
visualized by autoradiography or phosphorimager. All three mENaCs were
expressed in mouse lung and kidney, with the -, -, and mENaC
probes recognizing mRNAs of 3.7, 2.6, and 3.2 kb, respectively. Sk
muscle, skeletal muscle.
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Functional expression of mENaCs. The
Xenopus oocyte expression system was
used to examine the functional properties of mouse ENaCs. Whole cell
amiloride-sensitive currents obtained in oocytes injected with
-,
-, and
mENaC cRNAs by the TEV technique are illustrated in Figs.
3 and 4. Amiloride inhibited the
Na+ current with an
IC50 of 103 ± 16 nM (Fig. 3,
n = 7). The
Na+-to-K+
selectivity ratio was greater than 80:1 at a holding potential of
100 mV (Fig. 4,
n = 5). Analyses of
Na+ channel characteristics by
cell-attached patch clamp revealed long open and closed times (on the
order of seconds) (Fig. 5) and a linear
current/voltage relationship with a slope conductance of 4.7 pS (Fig.
6, n = 4-6). These functional characteristics of mouse ENaCs are in
agreement with the characteristics of the cloned rat, human, and
X. laevis ENaCs (9, 31, 36).

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Fig. 3.
Expression of mouse -, -, and ENaC in oocytes: amiloride
dose-response relationship. Oocytes were injected with -, -, and
mENaC cRNAs and maintained in modified Barth's saline. Currents
were measured in oocytes bathed in sodium gluconate in presence of
increasing concentrations of amiloride using the two-electrode voltage
clamp technique with a holding potential of 100 mV. Currents
shown were normalized to the current measured in absence of amiloride.
IC50 for amiloride was 103 ± 16 nM (n = 7).
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Fig. 4.
Expression of mouse -, -, and ENaC in oocytes: whole cell
current/voltage
(I/V)
relationships and cation selectivity. Oocytes were injected with -,
-, and mENaC cRNAs and maintained in a
low-Na+ modified Barth's
solution. Currents were measured while varying the holding potential
between 100 and +60 mV (20 mV steps), using the TEV technique.
Oocytes were bathed in a buffer containing either sodium gluconate
( ) or potassium gluconate ( ) in presence or absence of 100 µM
amiloride. Amiloride-sensitive currents are shown. Currents were
normalized to the value obtained at a holding potential of 100
mV in Na+ bath
(n = 5).
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Fig. 5.
Expression of mouse -, -, and ENaC in oocytes: single-channel
recording. Analyses of Na+
channels by cell-attached patch clamp.
Na+ channels present in the patch
exhibited long (>1 s) open (O1,
O2) and closed (C) states.
Pipette solution contained 100 mM NaCl. A 120-mV potential was
applied to the patch.
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Fig. 6.
Expression of mouse -, -, and ENaC in oocytes: single-channel
I/V
relationship. Oocytes were injected with -, -, and mENaC cRNAs
and maintained in modified Barth's saline. Currents were measured
while varying the holding potential between 120 and +60 mV
(20-mV steps), using a cell-attached patch clamp. Pipette and oocyte
bath solutions contained 100 mM NaCl. A linear
I/V
relationship was observed. Slope conductance was 4.7 pS. Reversal
potential was ~0 mV, suggesting that the oocytes were loaded with
Na+ prior to patch clamp analyses;
n = 4-6 different oocytes for
each data point.
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Amino-terminal
mENaC has a dominant
negative effect on ENaC expression in Xenopus oocytes.
We examined whether coexpression of the
NH2-terminal domain of
mENaC
with wild-type
-,
-, and
mENaC would inhibit the formation of
functionally competent channels in
Xenopus oocytes. Coinjection of
wild-type
-,
-, and
mENaC cRNAs with a 10-fold excess (weight
basis) of
mENaC NH2-terminal
cRNA in Xenopus oocytes inhibited
amiloride-sensitive current compared with injection with wild-type
cRNAs alone (Fig. 7,
n = 20). Furthermore, this effect was
dose dependent. A partial inhibition of amiloride-sensitive current was
observed at coinjection ratios of 3:1 and 1:1 (Fig. 7,
n = 6). Oocytes coinjected with
wild-type mENaC cRNAs and a 10-fold excess of a control cRNA encoding
for the ectodomain of
mENaC produced
Na+-selective currents
commensurate with those seen in oocytes injected with wild type alone
(Fig. 8, n = 7). These data suggest that the
NH2 terminus of
mENaC is
involved in subunit assembly, and when present in a 10-fold excess, has
a dominant negative role in functional ENaC expression in
Xenopus oocytes.

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Fig. 7.
NH2-terminal mENaC cRNA
coinjected with wild-type mENaC cRNAs inhibits amiloride-sensitive
Na+ currents in
Xenopus oocytes in a dose-dependent
fashion. Xenopus oocytes were
coinjected with wild-type (wt) -, -, and mENaC (2 ng · subunit 1 · oocyte 1)
cRNAs alone or in conjunction with an excess of the
NH2-terminal (nt) mENaC cRNA
construct (cRNA subunit weight ratios of 10:1, 3:1, or 1:1), as
described under MATERIALS AND METHODS.
Whole cell TEV assays were performed 18-48 h postinjection.
Na+ currents were measured at a
holding potential of 100 mV in absence or presence of 10 µM
amiloride. Normalized amiloride-sensitive currents are illustrated.
Values were normalized to the amiloride-sensitive current measured in
oocytes expressing wild-type -, -, and mENaC alone.
Coinjection of wild-type mENaC cRNAs with a 10-fold excess of the
mENaC NH2-terminal cRNA in
Xenopus oocytes almost completely
abolished amiloride-sensitive current (0.10 ± 0.04, P < 0.001, n = 20) compared with oocytes injected
with wild-type mENaCs alone (1 ± 0.10, n = 20). Coinjection of wild-type
mENaC cRNAs with a 3-fold excess (3:1 injection) or equal amounts (1:1
injection) of the mENaC
NH2-terminal cRNA significantly
reduced amiloride-sensitive currents (3:1 injection, 0.26 ± 0.08, P < 0.001, n = 6; 1:1 injection, 0.46 ± 0.08, P < 0.005, n = 6).
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Fig. 8.
mENaC ectodomain cRNA coinjected with wild-type mENaC cRNAs does not
inhibit amiloride-sensitive Na+
currents in Xenopus oocytes.
Xenopus oocytes were coinjected with
wild-type -, -, and mENaCs (wt) cRNAs alone or in conjunction
with a 10-fold excess of mENaC ectodomain (ecto) cRNA. Whole cell
TEV assays were performed 18-48 h postinjection.
Na+ currents were measured at a
holding potential of 100 mV in absence or presence of 10 µM
amiloride. Normalized amiloride-sensitive currents are depicted. Values
were normalized to the amiloride-sensitive current measured in oocytes
expressing wild-type -, -, and mENaC alone. Oocytes coinjected
with wild-type mENaC cRNAs and mENaC ectodomain cRNA expressed
currents (0.86 ± 0.15, n = 7)
commensurate with those injected with wild-type mENaC alone (1 ± 0.20; P = 0.6, n = 7).
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DISCUSSION |
The epithelial sodium channel is a heteroligomeric protein comprising
three homologous subunits,
-,
-, and
ENaC (9). We obtained
full-length cDNAs encoding mouse
-,
-, and
ENaC. The deduced
amino acid sequences of the mouse ENaCs are nearly identical to rat
-,
-, and
ENaC, respectively (Fig. 1) (7, 9). Dagenais et al.
(12) have reported a partial cDNA clone of mouse
ENaC, corresponding
to amino acid residues H445-L558 of our full-length mouse
ENaC.
There are three residues that differ from our sequence near the extreme
NH2 and COOH termini, which may
reflect sequence polymorphisms. Regions within ENaCs that have defined
functions are conserved. For example, each subunit has two putative
membrane-spanning domains (M1 and M2) that are amphipathic and
predicted to assume an
-helical structure (Fig. 1). A predominantly
hydrophobic domain, previously termed H2 by Canessa and coworkers (9),
precedes the second membrane-spanning domain of each subunit and may
contribute to the channel pore and selectivity filter (9, 37), as
mutations within this region alter cation selectivity,
amiloride-sensitivity, and single-channel conductance (29, 37, 44, 45,
60). The region between the two hydrophobic membrane-spanning domains
comprising approximately two-thirds of the mass of each subunit is
extracellular (8, 52). Sequence analysis of this large extracellular
domain reveals multiple N-linked glycosylation sites (6, 12, and 5 N-linked glycosylation sites for
-,
-, and
-subunits,
respectively) and cysteine-rich domains, features conserved between the
three subunits as well as other members of the
ENaC/mec/deg superfamily (11, 19). The relatively short
cytoplasmic NH2- and COOH-termini
have consensus sites for phosphorylation by protein kinase A and
protein kinase C. Recent studies support the notion that
phosphorylation of the
- and
-subunits of the channel may have a
role in the regulation of the channel by forskolin, insulin,
aldosterone, and phorbol esters (47). Proline-rich regions and PXY
motifs have defined roles in the binding of
-spectrin
and the ubiquitin ligase Nedd4 to the channel (40, 54). A
gating domain has been identified within the
NH2 terminus of the
-subunit
(22). These regions are conserved within the mouse ENaCs reported here.
Several groups have reported that the subunit stoichiometry of ENaC is
2,
1,
1
(15, 29), although one group has reported a subunit
stoichiometry of
3,
3,
3
(51). When the three subunits are expressed in
Xenopus oocytes, they oligomerize to
form a channel with properties similar to that observed in native
tissues (9, 19, 31). We also observed that mouse ENaCs, when expressed in Xenopus oocytes, are highly
selective for sodium (Na+
>>> K+) (Fig. 4),
demonstrate slow gating kinetics (long open and closed times on the
order of several seconds) (Fig. 5), low single-channel conductance (4.7 pS) (Fig. 6), and are blocked by amiloride with a
Ki of 103 nM
(Fig. 3).
Liver
mENaC gene product may serve
as an amiloride-sensitive
Na+
channel.
Mouse ENaC mRNA was expressed in sodium-absorptive epithelia (Fig. 2),
including kidney and lung. Mouse
ENaC, like human
ENaC (31), was
expressed in liver. Interestingly, the
- and
-subunits were not
detected in liver. The expression of the
-subunit alone raises the
question of whether functional Na+
channels are expressed in the liver, either composed solely of
-subunits or a heteroligomer of
-subunits with
related ENaC subunits that have yet to be identified.
Na+-conductive pathways have an
important role in hepatocyte volume regulation (61). Rat hepatocytes in
confluent primary cultures respond to hypertonic stress with a
considerable increase in cell membrane
Na+ conductance. These adaptations
(the regulatory volume increase, the increases in
Na+ conductance and intracellular
Na+ concentration, as well as the
activation of
Na+-K+-ATPase)
were completely blocked by 10 µM amiloride (61). At this
concentration, amiloride had no effect on osmotically induced cell
alkalinization via
Na+/H+
exchange (61). These data imply that an amiloride-sensitive Na+ channel is expressed in
hepatocytes and serves as the conduit for
Na+ influx. Activation of
Na+ channels, in conjunction with
activation of
Na+-K+-ATPase,
results in increases in intracellular
K+ and
Na+, and these are the major ionic
mechanisms responsible for regulatory volume increases in
hepatocytes. Our observation that
mENaC was expressed in liver
suggested that
ENaC (and possibly as yet unidentified related
subunits) may function as a mechanosensitive cation channel and play an
important role in hepatocyte cell volume regulation.
The question of whether ENaC is a mechanosensitive ion channel has been
examined by several laboratories. Differing results have been reported.
Channels composed of all three ENaC subunits were mechanosensitive when
expressed in lipid bilayers (25). Xenopus oocytes expressing
-,
-,
and
ENaC responded to cell swelling with either no change (3) or a
decrease (26) in Na+ conductance
and responded to cell shrinkage with an increase (26) or decrease (3)
in Na+ conductance.
Na+ channels in the collecting
tubule responded in a variable manner to an increase in membrane
tension by altering the hydrostatic pressure in a patch pipette (34);
an increase in open probability was reported in 6 of 22 patches.
Although controversy exists in the literature as to the effects of
changes in cell volume on functional
-,
-, and
ENaC
expression, these data do not exclude the possibility that channels
formed by
-subunits alone are mechanosensitive.
The biophysical properties of Na+
channels composed of
-subunits differ from
-,
-, and
ENaC
with regard to single-channel conductance and cation selectivity (9,
31, 32). Application of a hydrostatic pressure gradient across lipid
bilayers increases the open probability of
ENaC (4). When expressed
in fibroblasts,
ENaCs were activated in response to increases in the
negative hydrostatic pressure applied to patch pipettes (27). These
data suggest that
-subunits may oligomerize to form channels that are mechanosensitive and participate in hepatocyte volume regulation.
Functional effect of coexpression of
NH2-terminal
mENaC
with wild-type
Na+
channels.
The putative cytoplasmic domains of each ENaC subunit comprise a small
fraction of the total mass of the channel protein and contain regions
critical for ENaC activity. For example, regulatory motifs in the COOH
termini of
- and
ENaC including proline-rich domains
and tyrosine-based internalization signals have been implicated in ENaC
regulation and protein-protein interactions. Mutations or deletions of
internalization signals or sites of interaction with Nedd4 have been
associated with increases in functional ENaC activity (43, 46, 53, 54).
A recent study suggested that the
NH2 terminus of
hENaC
participates in subunit-subunit interactions and in subunit assembly
(1). Studies of other ion channels, such as voltage-gated
K+ channels, suggest that
NH2 termini are important sites of
subunit-subunit interaction (56). We examined whether the
NH2 terminus of
mENaC might be
a site of subunit-subunit interactions. Our data suggest that the
NH2 terminus of
mENaC may serve
as an intersubunit association domain, as coexpression of the
NH2 terminus of
mENaC with
wild-type
-,
-, and
mENaC subunits in
Xenopus oocytes inhibited
amiloride-sensitive current. The mechanisms of suppression of ENaC
expression are unclear, but the data are consistent with a disruption
in normal assembly by formation of a heteroligomeric complex between
the NH2 terminus of
mENaC and
wild-type
-,
-, and/or
mENaC subunits.
Type 1 pseudohypoaldosteronism (PHA1) is a disorder exhibiting
Mendelian inheritance that is characterized by salt wasting, metabolic
acidosis, and hyperkalemia. Our data suggest that the NH2-terminal domain of
ENaC
functions as a dominant negative mutant when coexpressed with wild-type
subunits, perhaps by inhibiting assembly of functional
Na+ channels. Our dose-response
experiments highlight the fact that inhibition of ENaC function was
most profound when an excess of NH2-terminal
mENaC was
expressed with wild-type
mENaC (Fig. 7). Genotyping and
single-strand chain polymorphism analysis of several autosomal
recessive PHA1 kindreds have identified frame-shift mutations within
human
- and
ENaC prior to the first transmembrane domain
resulting in truncated subunits (10). To date, ENaC mutations associated with autosomal dominant kindreds have not been described (20). It is interesting to speculate that heterozygotes expressing both
truncated and wild-type
hENaC (or
hENaC) may have reduced levels
of ENaC expression. However, the PHA1 phenotype might be observed only
under conditions of stress, such as in the immediate postpartum period,
in states of profound salt/water depletion, and in association with
increased potassium intake.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Bruce Stanton for providing sequence information for
the upstream portion of
mENaC.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants
DK-51391, DK-54354, HL-07027, and DK-07006. Y. J. Ahn was a recipient
of postdoctoral fellowship awards from the American Heart Association,
Southeastern Pennsylvania Affiliate, and from the Cystic Fibrosis
Foundation. B. J. Harte was a recipient of a medical student research
award from the American Heart Association.
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. §1734 solely to indicate this fact.
1
Refer to GenBank accession numbers AF112185,
AF112186, and AF112187 for full nucleotide sequence. cDNA nucleotide residues referred to in this manuscript are numbered from the initiation ATG of the open-reading frame.
Address for reprint requests and other correspondence: Y. J. Ahn, Renal
Electrolyte Hypertension Division, Univ. of Pennsylvania, 700 Clinical
Research Bldg., 422 Curie Boulevard, Philadelphia, PA 19104-6144 (E-mail: yahn{at}mail.med.upenn.edu).
Received 17 December 1998; accepted in final form 24 March 1999.
 |
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