1Departments of Medicine and 3Cell Biology and Physiology, University of Pittsburgh, Pittsburgh 15261; and 2Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Submitted 5 March 2003 ; accepted in final form 21 May 2003
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ABSTRACT |
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amiloride-sensitive sodium channel; dominant negative mutants
Mouse ENaC subunits are composed of between 638 and 699 amino acids and
share a common topology (3).
Each subunit has two membrane-spanning domains (M1 and M2) and adjacent
extracellular hydrophobic domains (H1 and H2) separated by a large
extracellular loop (ECL) (8),
whereas the NH2 and COOH termini of each subunit are intracellular
(7,
43,
55). The combined application
of site-directed mutagenesis and oocyte expression has yielded important
findings regarding the structure/function relationships of ENaC
(5,
32,
44,
52). Two amiloride-binding
sites have been identified within the ENaC subunits, one within the pore
region of each subunit and a second within the ECL of the -subunit
(27,
33,
46). The main selectivity
filter has been localized to a three-residue tract [(G/S)XS] within
the H2 region (or equivalently termed as pre-M2 region)
(2931,
47,
56). Mutations that alter ENaC
gating have been found in the NH2 termini
(20,
21), ECL
(50), pore regions
(48,
53) and nearby regions
(18), M2 domains
(19), and COOH termini
(12).
Limited information is available regarding the identification of domains
within the heterooligomeric Na+ channel complex that participate in
subunit-subunit interactions and promote subunit oligomerization.
Na+ channel subunits are thought to assemble in the endoplasmic
reticulum (ER) where they undergo N-linked glycosylation
(2,
7,
11,
13,
23,
40,
41,
45,
55,
65). The interactions of newly
synthesized Na+ channel subunits within the ER must be of a
sufficient affinity to promote Na+ channel assembly. A previous
study by Adams and co-workers
(2) showed that the
NH2-terminal domain of -ENaC interacted with and prevented
functional expression of full-length
-ENaC. Similarly, our
previous work indicated that the NH2-terminal domain of
-ENaC interacted with full-length
-ENaC and
prevented the expression of amiloride-sensitive currents in Xenopus
laevis oocytes (3),
whereas results published by Chalfant and co-workers
(9) showed that NH2
terminally deleted
-,
-, or
-ENaC prevented functional
expression of full-length
-ENaC. We have extended these
observations and have identified multiple domains throughout
-ENaC
that, when coexpressed with full-length
-ENaC, confer a
dominant negative phenotype. These domains may have an important role in
facilitating subunit-subunit interactions that allow for proper assembly and
functional expression of the heterooligomeric Na+ channel.
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MATERIALS AND METHODS |
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Mouse ENaC constructs. Mouse (m) -,
-, and
-ENaC cDNAs were previously cloned into pBluescript SK()
(Stratagene, La Jolla, CA) (3).
The following
-mENaC domains were generated by PCR:
-M1H1-ECL
containing S102-S568;
-M1H1 containing S102-R164;
-M1H1-cytoplasmic COOH terminus (Ct) containing S102-R164 fused to
H613-L699;
-Ct containing H613-L699;
-glutamyl transpeptidase
(GT)/ECL containing the short cytoplasmic tail and transmembrane domain of rat
-GT (M1-T28) (28) fused
to the
-subunit's extracellular loop (Y165-S568); and Tac/M2H2
containing the extracellular domain of the human interlukin-2 receptor
-subunit (Tac, M1-Q240)
(39) fused to the
-subunit's second hydrophobic and transmembrane domains (V569-R618).
COOH terminally truncated
-ENaCs were generated by placing
stop codons at H613, R564, and R583 of
-,
- and
-,
respectively. The FLAG epitope was added to the COOH terminus of the
-,
- and
-cDNAs, the V5 epitope was added to the COOH terminus of
the
-subunit and
-Ct cDNA, and the myc epitope was added to the
COOH termini of
-M1H1-ECL and
-GT-ECL cDNAs. All epitope-tagged
constructs were cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA). All
constructs were confirmed by automated DNA sequencing at sequencing facilities
at the University of Pennsylvania or at the University of Pittsburgh. cRNAs
were synthesized from the above constructs in linearized pBlue-script or
pcDNA3.1 using the T3 or T7 mMESSAGE mMACHINE kit (Ambion, Austin, TX). cRNAs
were stored at 80°C and diluted in nuclease-free water before
injection.
Oocyte preparation and injection. Oocytes were obtained from adult
female X. laevis using protocols approved by Institutional Animal
Care and Use Committees at the University of Pennsylvania and the University
of Pittsburgh. Stage V and VI X. laevis oocytes were enzymatically
defolliculated in 2 mg/ml type IV collagenase and then maintained at 18°C
in modified Barth's saline [MBS; (in mM) 88 NaCl, 1 KCl, 2.4
NaHCO3, 15 HEPES, 0.3 Ca(NO3)2, 0.41
CaCl2, and 0.82 MgSO4, as well as 10 µg/ml sodium
penicillin, 10 µg/ml streptomycin sulfate, and 100 µg/ml gentamicin
sulfate, pH 7.4]. Oocytes were microinjected with mouse -,
-, and
-ENaC cRNAs (2 ng/subunit) with or without test cRNAs (20 ng, unless
otherwise indicated) in 50 nl nuclease-free H2O. Oocytes were also
coinjected with
H613X,
R564X, and
R583X cRNAs (0.5
ng/subunit) with or without
-Ct cRNA (5 ng). Whole cell currents were
measured 12 days after cRNA injections.
Whole cell current measurements. The two-electrode voltage-clamp
technique was used to measure the amiloride-sensitive whole cell inward
currents with a DigiData 1200 interface (Axon Instruments, Foster City, CA)
and a TEV 200A Voltage Clamp Amplifier (Dagan, Minneapolis, MN). Data
acquisition and analyses were performed using pClamp 7.0 software (Axon
Instruments) with a Pentium II-based PC (Gateway 2000, N. Sioux City, SD).
Recording pipettes were pulled from borosilicate glass capillaries (World
Precision Instruments, Sarasota, FL) and filled with 3 M KCl. Pipettes with
tip resistances of 0.53 M in a 110 mM NaCl bath solution were
chosen for experiments. Oocytes were bathed in a solution containing (in mM)
96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES-NaOH, pH
7.4. All measurements were carried out at room temperature
(2225°C), and the bath solution was continuously perfused at
56 ml/min by gravity. Oocytes were typically incubated in the bath
solution for at least 5 min before the current was recorded to allow currents
to stabilize. Oocytes were clamped at 100 mV for 900 ms. Currents were
measured at 600 ms after initiation of the clamp potential. Between
recordings, the circuit was opened, allowing the oocytes to rest at the
reversal potential. To minimize the contribution of variability in expression
levels from different batches of oocytes, we measured the whole cell currents
in oocytes coinjected with
-mENaC cRNAs and in oocytes
coinjected with
-mENaC cRNAs plus an
-subunit
construct cRNA, using oocytes obtained from a single batch. Oocytes from the
two groups were clamped in an alternating manner so that the contribution of
expression time to the measured currents was similar for each group.
Measurements from repeated experiments, using oocytes from at least two frogs,
were pooled to obtain a sufficient number of observations for statistical
analysis.
Transient expression in Chinese hamster ovary cells. Wild-type
Chinese hamster ovary (CHO) cells were cultured as previously described
(4). Transient expression of
mENaC subunits, dominant negative constructs, and/or -GT was carried
out in CHO cells by infection with recombinant vaccinia (vT7CP) expressing T7
RNA polymerase, followed by transfection with expression vectors containing T7
promoters at the 5'-end of the cDNAs as previously described
(42,
64). The full-length
-GT used contained a substitution of asparagine for threonine at
position 380. This mutation prevents the autocatalytic cleavage of
-GT
and eliminates enzymatic activity without affecting processing or trafficking
(Hughey R, unpublished observations). vT7CP was a kind gift from Bernard Moss
(National Institute of Allergy and Infectious Disease, Bethesda, MD).
Transfections were carried out 30 min postinfection with lipofectamine reagent
as described by the manufacturer (Invitrogen, Carlsbad, CA). After overnight
incubation,
1 x 106 cells were solubilized at room
temperature in 300 µl of a detergent solution (50 mM Tris · HCl, 1%
Nonidet P-40, 0.4% deoxycholate, and 62.5 mM EDTA, pH 8.5) supplemented with
1% Protease Inhibitor Cocktail Set III (Calbiochem, La Jolla, CA). Insoluble
material was removed by centrifugation in a microcentrifuge at 20,000
g for 7 min at 4°C. Supernatants were recovered and incubated
overnight at 4°C after the addition of protein G-Sepharose (Sigma) and
mouse monoclonal antibodies against either Flag (Anti-Flag M2, Sigma) or Myc
(9E10, Santa Cruz Biotechnology, Santa Cruz, CA). Immunoprecipitates were
recovered by brief centrifugation, and each was washed once with 1 ml each of
1% Triton X-100 in HEPES-buffered saline (HBS; 10 mM HEPES-NaOH, pH 7.4, 150
mM NaCl), 0.01% SDS in HBS, and finally HBS. Where indicated,
immunoprecipitates were treated with N-glycosidase (PNGase) F as
described by the manufacturer (New England BioLabs, Beverly, MA). Proteins
were recovered by heating for 3.5 min at 95°C in Laemmili SDS-sample
buffer containing fresh 0.14 M
-mercaptoethanol. Samples were subject to
SDS-PAGE (all reagents were from Bio-Rad, Richmond, CA) on 7.5% Criterion gels
and then electrophoretically transferred from the gel to Immobilon-NC filters
(Millipore, Bedford, MA). The blot was blocked overnight at 4°C in PBS (8
mM sodium phosphate, 2 mM potassium phosphate, 140 mM NaCl, 10 mM KCl, pH 7.4)
containing 5% nonfat dry milk. Subsequently, the blot was incubated in PBS
containing 1% nonfat dry milk and the indicated mouse monoclonal antibody at
room temperature for 3 h. All blots were then washed extensively with PBS
followed by a 45-min incubation with horseradish peroxidase (HRP)-conjugated
goat anti-mouse IgG (KPL, Gaithersburg, MD) in PBS containing 1% nonfat dry
milk. After an extensive washing in PBS, HRP bound to proteins was detected
using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Boston,
MA) and BioMax MR film (Eastman Kodak, Rochester, NY) as directed by the
manufacturer.
mENaC surface expression in X. laevis oocytes. Full-length
-mENaC was coexpressed with or without
-M1H1-ECL in
X.laevis oocytes. The
-subunit was tagged with the V5 epitope
at its COOH terminus. The vitellin membrane was manually removed with fine
forceps and, after being washed in MBS, the oocytes were incubated twice for
20 min with 1.5 mg/ml sulfo-NHS-SS-biotin (Pierce, Rockford, IL) in MBS
containing 10 mM triethanolamine, pH 9.0, on ice. Free biotin reagent was
quenched by four 5-min washes in MBS containing 5 mM glycine. After three
washes with MBS, 15 oocytes were solubilized on ice in 300 µl of a
detergent solution {25 mM MES, pH 6.4, 200 mM NaCl, 1% Triton X-100, 60 mM
n-octyl glucoside, 0.1% SDS, 0.5% Nonidet P-40, 0.02% sodium
deoxycholate, 1% digitonin, 0.5% Tween 20, 0.02%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 2 mM Empigen
BB}. Insoluble material was removed by centrifugation for 15 min at 15,000
g at 4°C. Supernatants were recovered and incubated overnight at
4°C with immobilized streptavidin (Pierce). Streptavidin precipitates were
then washed and subjected to SDS-PAGE and immunoblot analyses as above.
Anti-V5 antibody (Invitrogen) was used to detect the ENaC
-subunit.
Statistical analysis. Data are expressed as means ± SE. Unpaired Student's t-tests were used to assess significance. P < 0.05 was considered significant.
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RESULTS |
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We observed that coexpression of -ENaC with
-M1H1-ECL also inhibited functional Na+ channel expression
(Fig. 3A). Further
experiments were carried out to determine whether
-ECL, independently
of
-M1H1, had a role in conferring a dominant negative phenotype. To
ensure proper orientation of the ECL within the ER lumen, we generated a
fusion protein composed of the membrane-spanning domain of a control type 2
integral membrane protein,
-GT, fused to
-ECL (
-GT-ECL).
Coexpression of
-ENaC with
-GT-ECL resulted in an
inhibition of functional Na+ channel expression
(Fig. 3B). In
contrast, coexpression of
-ENaC with either the
-ECL (lacking a signal-anchor sequence) or full-length
-GT did
not inhibit functional channel expression
(Fig. 3, C and
D).
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-ECL has six consensus sites for N-linked glycosylation
(7). To confirm that the ECL
within the
-M1H1-ECL or
-GT-ECL constructs was properly
transferred into the ER lumen, we transiently expressed myc epitope-tagged
-M1H1-ECL or
-GT-ECL in CHO cells. The expressed constructs were
immunoprecipitated from cell lysates, and N-glycans were cleaved by treatment
with PNGase F. Both
-M1H1-ECL and
-GT-ECL displayed a shift to a
lower apparent molecular weight after treatment with PNGase F
(Fig. 4), confirming the
addition of N-glycans to these polypeptides and indicating that the ECLs
within these constructs were present within the ER lumen.
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Previous studies have shown that residues within M2 and H2 form the pore of
the channel
(2931,
36,
37,
4749).
We examined whether -H2M2 conferred a dominant negative phenotype. The
NH2-terminal ectodomain of a control type 1 integral membrane
protein, the human interleukin-2 receptor
-subunit (commonly referred
to as Tac), was fused to
-H2M2 (Tac-
-H2M2) to ensure proper
orientation of
-H2M2 in the ER membrane. Coexpression of
-ENaC with Tac-
-H2M2 inhibited functional
Na+ channel expression (Fig.
5A), whereas full-length Tac did not confer a dominant
negative phenotype (Fig.
5B).
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We also observed that coexpression of -Ct with full-length
-ENaC inhibited the expression of functional
Na+ channels (Fig.
6A). In contrast, coexpression of ENaC subunits lacking
their COOH termini with
-Ct did not alter functional Na+
channel expression (Fig.
6B), suggesting that direct interactions exist between
ENaC intracellular domains. As simultaneous overexpression of several proteins
may overload the translation machinery of a cell, leading to a false dominant
negative phenotype, we performed similar experiments with an unrelated
channel, ROMK. The expression of ROMK channels was not inhibited when
full-length ROMK was coexpressed with either
-Ct or
-M1H1
(Fig. 6C), suggesting that
multiple domains within the
-subunit confer a dominant negative
phenotype, presumably by specifically associating with full-length ENaC
subunits and preventing the assembly of tetrameric channels composed of
full-length subunits.
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Experiments were performed to examine whether -M1H1-ECL binds to
full-length ENaC subunits with sufficient affinity to allow for
coimmunoprecipitation.
-M1H1-ECL with a COOH-terminal myc tag was
transiently coexpressed with full-length, COOH-terminal FLAG-tagged
-,
-, or
-mENaC, or full-length
-GT (control) in CHO cells.
Coimmunoprecipitation was observed when full-length ENaC subunits were
immunoprecipitated, and subsequent immunoblots were probed for
-M1H1-ECL (Fig.
7A). We also observed coimmunoprecipitation when
-M1H1-ECL was immunoprecipitated, and subsequent immunoblots were
probed for full-length ENaC subunits (Fig.
7B).
-M1H1-ECL did not coimmunoprecipitate with
full-length
-GT (Fig.
7A). We examined whether the dominant negative phenotype
observed with coexpression of
-M1H1-ECL was associated with an
inhibition of surface expression of Na+ channels. Full-length
-ENaC were coexpressed with or without
-M1H1-ECL in
X. laevis oocytes. The
-subunit was tagged with the V5 epitope
at its COOH terminus. The vitellin membrane was removed, and surface proteins
were labeled by treatment with membrane-impermeant sulfo-NHS-SS-biotin.
Oocytes were homogenized/solubilized, and biotinylated (i.e., cell surface)
proteins were precipitated with streptavidin-agarose. Precipitated proteins
were subjected to SDS-PAGE and immunoblot analyses to detect the ENaC
-subunit using anti-V5 antibody. Coexpression of
-ENaC and
-M1H1-ECL in X. laevis oocytes
was associated with a marked reduction in surface expression of
-ENaC
(Fig. 8). We also examined
whole cell expression of selected dominant negative constructs in oocytes
coinjected with
-ENaC cRNAs and either
-M1H1-ECL,
-GT-ECL, or
-Ct cRNA to confirm that these constructs were
expressed in oocytes. Figure 9
illustrates that
-M1H1-ECL,
-GT-ECL, and
-Ct were readily
detected by immunoprecipitation followed by immunoblotting.
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DISCUSSION |
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Our work indicates that multiple -subunit domains, when coexpressed
with full-length
-ENaC, confer a dominant negative
phenotype. These domains include 1) the cytoplasmic NH2
terminus (3); 2)
-M1H1; 3)
-M1H1-ECL; 4) the ECL itself when
fused to a type 2 signal-anchor sequence to facilitate ER translocation;
5)
-H2M2, when properly oriented in the ER membrane by fusion
to a type 1 start transfer sequence; and 6)
-Ct. Furthermore,
the cellular localization of an ENaC domain (cytoplasm or ER lumen) was an
important factor in determining whether it conferred a dominant negative
phenotype. For example, the ECL only conferred a dominant negative phenotype
when translocated into the ER lumen by fusion to a signal-anchor sequence
(
-M1H1 or
-GT). Similarly, the COOH terminus conferred a
dominant negative phenotype when expressed in the cytoplasm but did not
inhibit functional expression when translocated into the ER lumen by fusion to
a signal-anchor sequence (
-M1H1). The mislocalization of the COOH
terminus within the
-M1H1-Ct construct might hinder interactions
between
-M1H1 with full-length ENaC subunits, which could prevent M1H1
from inhibiting functional ENaC expression. Alternatively, exposure of the
normally cytoplasmic COOH terminus to the lumen of the ER may have induced the
rapid degradation of the
-M1H1-Ct before
-M1H1 could interact
with full-length subunits.
Interactions of any of the dominant negative constructs with full-length
-,
-, or
-ENaC could inhibit functional ENaC expression by
causing the sequestration of full-length subunits in the ER, by interfering
with the function of channels at the plasma membrane, or by inducing the
ER-associated degradation of full-length channel subunits associated with the
dominant negative constructs in nonfunctional channel complexes. Dominant
negative-induced degradation of full-length channel subunits was observed in
the suppression of
-ENaC currents mediated by the overexpression of
truncated
-subunits (2)
and also in the suppression of Kir 4.1 currents by the coexpression of members
of the Kir 3.0 family (61). We
observed that the
-M1H1-ECL dominant negative construct was associated
with a marked reduction in surface expression of the full-length
-subunit, when coexpressed with full-length
- and
-subunits, suggesting that overexpression of the
-M1H1-ECL
construct led to the sequestration and/or degradation of full-length channel
subunits in the ER; however, other possible explanations such as enhanced
degradation in post-ER compartments cannot be excluded.
These results, together with previous reports (2, 3, 9), suggest the presence of multiple sites of intersubunit association within the heterooligomeric Na+ channel complex and are in agreement with studies demonstrating that other oligomeric ion channels have multiple sites of intersubunit interactions. For example, Tu et al. (60) used a dominant negative approach and demonstrated that multiple sites within the central core of Kv1.3 were involved in intersubunit association. Similarly, multiple sites of interaction have been demonstrated in the homooligomerization of Kir 1.1a channels (35) and in the heterooligomerization of Kir3.1 and Kir3.2 (66). These domains included the NH2-terminal, core (i.e., M1 through M2), and COOH-terminal domains.
The ECL of each ENaC subunit contains 16 conserved cysteine residues,
suggesting a complex organization of the loop structures. Mutations of the
first, sixth, eleventh, and twelfth cysteine residues have been shown to lead
to reduced surface expression and channel activity of rat
-ENaC in oocytes
(17). In addition, whole or
partial deletion of the
-ECL resulted in no functional expression of
-mENaC in oocytes (Sheng S and Kleyman TR, unpublished
observations), and it was reported that even a six-residue deletion within the
-ECL led to no detectable current or surface protein expression
(31). These observations are
consistent with an expectation that the ECL has a role in ENaC assembly.
Similarly, it is expected that
-M1H1 and
-H2M2 would have a role
in subunit assembly, as the assembly of multiple transmembrane domains
requires helix-helix interactions to ensure proper packing
(1,
15,
62). However, the role of the
COOH termini in ENaC assembly is less clear, as truncation of the entire COOH
terminus of the
- or
-subunit in fact increases channel activity
and cell surface density (52).
While the
-Ct confers a dominant negative phenotype on functional ENaC
expression, it should be noted that a domain that confers a dominant negative
phenotype may be involved in, but not required for, subunit assembly. It is
possible that the mechanism of inhibition of functional ENaC expression by
-Ct may differ from that observed with the other constructs. For
example,
-Ct may confer a dominant negative phenotype by preventing
interactions with accessory proteins that facilitate functional channel
expression.
Truncations of ENaC subunits have been described in patients with autosomal
recessive pseudohypoaldosteronism type I
(6,
10). Bonny et al.
(6) reported that coexpression
of an -subunit truncated just before the pore region with full-length
and
allowed for functional ENaC expression in X.
laevis oocytes. However, the onset of expression was delayed 48 h and the
magnitude of the expressed current was markedly reduced with respect to
wild-type
-ENaC. The truncated
-subunit was not
expressed with full-length
-ENaC to test for a dominant
negative effect. It is tempting to speculate that heterozygotes expressing
both truncated and full-length
-ENaC subunits may have reduced levels
of Na+ channel expression due to a dominant negative effect on
channel assembly.
Transgenic overexpression of dominant negatives has become a useful tool
for the in vivo study of ion channel function. For example, London and
colleagues (38) have used the
overexpression of a Kv1.1 dominant negative construct to disrupt Shaker-like
K+ channel function in the hearts of transgenic mice. Beyond
providing insight into -ENaC domains that may be important in
Na+ channel assembly, these data may prove useful for a dominant
negative knockout of ENaC function in cell lines and animals.
In summary, our results indicate that multiple domains throughout
-ENaC, when coexpressed with
-ENaC, confer a
dominant negative phenotype. We propose that these domains may play an
important role in facilitating and stabilizing subunit-subunit interactions
that allow for the proper assembly and functional expression of the
heterooligomeric ENaC.
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DISCLOSURES |
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FOOTNOTES |
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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.
* J. B. Bruns and B. Hu contributed equally to this work.
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REFERENCES |
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