1 University Laboratory of Physiology, University of Oxford, Oxford OX1 3PT, United Kingdom; and 2 Institut für Zelluläre und Molekulare Physiologie, Universität Erlangen-Nürnberg, D-91054 Erlangen, Germany
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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 is composed of three homologous subunits:
,
, and
. Only heteromultimeric channels made of
ENaC
are efficiently expressed at the cell surface, resulting in maximally
amiloride-sensitive currents. To study the relative importance of
various regions of the
- and
-subunits for the expression of
functional ENaC channels at the cell surface, we constructed
hemagglutinin (HA)-tagged
-
-chimeric subunits composed of
-
and
-subunit regions and coexpressed them with HA-tagged
- and
-subunits in Xenopus laevis oocytes. The whole cell
amiloride-sensitive sodium current (
Iami) and
surface expression of channels were assessed in parallel using the
two-electrode voltage-clamp technique and a chemiluminescence assay.
Because coexpression of
ENaC resulted in larger
Iami and surface expression compared with
coexpression of
ENaC, we hypothesized that the
-subunit is
more important for ENaC trafficking than the
-subunit. Using
chimeras, we demonstrated that channel activity is largely preserved
when the highly conserved second cysteine rich domains (CRD2) of the
- and
-subunits are exchanged. In contrast, exchanging the whole
extracellular loops of the
- and the
-subunits largely reduced
ENaC currents and ENaC expression in the membrane. This indicates that
there is limited interchangeability between molecular regions of the
two subunits. Interestingly, our chimera studies demonstrated that the
intracellular termini and the two transmembrane domains of
ENaC are
more important for the expression of functional channels at the cell
surface than the corresponding regions of
ENaC.
epithelial sodium channel domains; chimeras; whole cell amiloride-sensitive current; surface expression
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INTRODUCTION |
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THE AMILORIDE-SENSITIVE EPITHELIAL NA+ channel (ENaC) is the rate-limiting step for sodium absorption in a variety of epithelia, including the renal collecting duct (2, 19). The appropriate regulation of this channel is essential for the maintenance of renal sodium balance and, hence, for long-term regulation of arterial blood pressure (39). Indeed, the analysis of two human genetic diseases, Liddle's syndrome and pseudohypoladosteronism type 1 (PHA-1), has provided direct evidence that molecular dysfunction of ENaC has severe effects on arterial blood pressure. Although loss-of-function mutations of ENaC cause urinary sodium loss, hyperkalemia, and low blood pressure in patients with PHA-1 (10), gain-of-function mutations in Liddle's syndrome result in increased sodium reabsorption, hypokalemia, and severe arterial hypertension (43).
ENaC is composed of three subunits called ,
, and
(7). The expression of the
-subunit in
Xenopus oocytes generated small amiloride-sensitive
currents, whereas expression of the
- and/or
-subunits generated
no currents. Coexpression of either
ENaC or
ENaC with
ENaC
resulted in three- to fivefold larger currents than those seen with
ENaC expressed on its own. When all three subunits were coexpressed,
the currents were at least 100-fold larger than those observed with
ENaC alone (7). By using a quantitative approach to
measure the surface expression of ENaC, it was demonstrated that the
increase in currents was paralleled by a similar increase in channel
surface expression (17). Because the
-subunit can
apparently form functional channels when expressed on its own, it is
thought to play a key role in pore formation. In contrast, the
- and
the
-subunits are thought to be important for efficient trafficking
of the heteromeric channel to the plasma membrane. This interpretation
is consistent with the proposed ENaC stoichiometry of two
-, one
-, and one
-subunit (
2
) (15,
29) and with biochemical studies demonstrating that
-subunits, but not
- or
-subunits, could assemble to homomeric channel complexes (11). However, it has to be acknowledged
that the stoichiometry of ENaC is still a matter of debate and that in
addition to a tetrameric model, a channel arrangement of eight or nine
subunits has been proposed (14, 44).
Amino acid sequence analysis and biochemical studies suggest that the
ENaC subunits have cytoplasmic NH2 and COOH termini, two
hydrophobic transmembrane domains (M1 and M2), and a large extracellular domain (6, 7, 37, 45). Recent studies have
focused on the function of the different domains of ENaC. The
extracellular loop contains two cysteine-rich domains (CRD1 and CRD2)
important for the efficient transport of assembled subunits to the
plasma membrane (16). The region immediately preceding the
second transmembrane domain (pre-M2) contains an amiloride-binding site
and determines ion selectivity, suggesting that it forms part of the
channel pore (41), together with the M2 domain
(18). The NH2 terminus contains two key
regions, one with an endocytic motif important for channel retrieval
from the plasma membrane (8) and a second one, just before
the M1 (pre-M1) domain, involved in channel gating (20).
The COOH terminus contains a proline-rich PPXY motif, which is believed
to be important for interaction with the ubiquitin-protein ligases
Nedd4 and Nedd4-2, promoting the endocytosis of the channel
(23, 42, 47, 48). The importance of these motifs is
illustrated in Liddle's syndrome, in which mutations and/or deletions
of the PPXY motif in - or
ENaC reduce the endocytic retrieval of
ENaC from the membrane (1, 46). This results in an
increase in the number of ENaC channels in the membrane, which causes
hyperabsorption of Na+ and hypertension (17,
24).
Interestingly, the - and
-subunits share a higher degree of amino
acid homology between them than with
ENaC (7, 51). As
mentioned above,
and
have common functional domains. However, the finding that the simultaneous presence of both subunits is required
for maximal ENaC surface expression (17) suggests that they have similar but complementary functions. The aim of the present
study was to investigate the distinct roles of the
- and
-subunits for the expression of functional ENaC channels at the cell surface.
To elucidate the relative contributions of various regions of the -
and
-subunits to ENaC trafficking, we constructed hemagglutinin (HA)-tagged
-
-chimeric subunits composed of molecular regions of
the
- and
-subunits and coexpressed them with HA-tagged
- and
-subunits in Xenopus laevis oocytes.
ENaC-mediated whole cell currents and ENaC surface expression were
assessed using the two-electrode voltage-clamp technique and a recently
established chemiluminescence assay (26, 52). Our results
demonstrate that exchanging the whole extracellular loop of the
-
and the
-subunits results in an inefficient surface expression of
the ENaC complex. On the other hand, some well-conserved regions, like
the CRD2, can be exchanged between the
- and the
-subunits, with
most of the channel activity being preserved. Moreover, our results
suggest that the intracellular termini and the two transmembrane domains of
ENaC are more important than the corresponding
ENaC regions to promote the trafficking of the ENaC complex to the plasma membrane.
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MATERIALS AND METHODS |
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Isolation of oocytes and injection of cRNA. Xenopus laevis oocytes were prepared and injected as described (27). Defolliculated stage V-VI oocytes were injected with 1 ng of cRNA of each ENaC subunit and/or chimera. Injected oocytes were kept in modified Barth's saline [in mM: 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.3 Ca(NO3)2,, 0.41 CaCl2, 0.82 MgSO4, and 15 HEPES, adjusted to pH 7.6 with Tris] containing 2 µM amiloride to reduce sodium loading of the oocytes.
Two-electrode voltage-clamp experiments.
Oocytes were studied 2 days after injection using the two electrode
voltage-clamp technique as previously described (27). Oocytes were clamped at a holding potential of 60 mV. The
amiloride-sensitive whole cell current (
Iami)
was determined by subtracting the corresponding current value measured
in the presence of 2 µM amiloride from that measured before the
application of amiloride in a NaCl solution (in mM: 95 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES, adjusted to pH
7.4 with Tris).
Surface labeling of oocytes. Experiments were performed as recently described (25, 26, 28, 52). Oocytes were incubated for 30 min in ND96 (in mM: 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, adjusted to pH 7.4 with Tris) with 1% bovine serum albumin (BSA) at 4°C to block nonspecific binding of the antibodies. Oocytes were then incubated for 60 min at 4°C with 1 µg/ml rat monoclonal anti-HA antibody (3F10, Boehringer, in 1% BSA/ND96), washed eight times at 4°C with 1% BSA/ND96, and incubated with 2 µg/ml horseradish peroxidase (HRP)-coupled secondary antibody (goat anti-rat Fab fragments; Jackson ImmunoResearch) in 1% BSA/ND96 for 40 min. Oocytes were washed thoroughly, initially in 1% BSA/ND96 (4°C, 60 min), and then in ND96 without BSA (4°C, 60 min). Individual oocytes were placed in 50 µl of Power Signal Elisa solution (Pierce, Chester, UK) and, after an equilibration period of about 10 s, chemiluminescence was quantified in a Turner TD-20/20 luminometer (Sunnyvale, CA) by integrating the signal over a period of 15 s. Results are given in relative light units (RLU).
Construction of chimeras and cRNA synthesis.
The -,
-, and
-subunits of rat ENaC were extracellularly HA
tagged according to the FLAG-epitope placement into the rat ENaC
sequence (17). The HA epitope (YPYDVPDYA) was
inserted as follows:
191NSSYYPYDVPDYASSR206 in
ENaC,
135NTTSYPYDVPDYATLN141 in
ENaC, and
139EAGSYPYDVPDYAPRF154 in
ENaC. HA-tagged constructs were subcloned into the pGEMHE vector
(gifts from Dr. Blanche Schwappach, Heidelberg, Germany). To
facilitate the construction of the
-
-chimeras, unique restriction sites were created in the
- and
-subunits, resulting in the following alterations in the amino acid sequences: in
ENaC A383I, I455T and M457L (
A383I,I455T,M457L), in
ENaC G617E
and a silent mutation at Y107 (
G617E,Y107). Point
mutations were generated using the QUICKCHANGE XL site-directed
mutagenesis protocol (Stratagene, La Jolla, CA). To confirm that the
point mutations did not affect channel function, the mutant subunits
were expressed in oocytes and
Iami and
channel surface expression were determined. In the same batch of
oocytes,
Iami averaged 1.21 ± 0.18 µA
(n = 7) in
control oocytes, 1.32 ± 0.20 µA
(n = 7) in
A383I,I455T,M457L
oocytes, 1.33 ± 0.12 µA (n = 7)
G617E,Y107 oocytes, and 1.29 ± 0.21 µA
(n = 7) in
A383I,I455T,M457L
G617E,Y107 oocytes.
Similarly, the chemiluminescence signals obtained in the surface
expression assay averaged 16.1 ± 3.1 RLU (n = 10) in
-control oocytes, 15.9 ± 2.8 RLU (n = 10) in
A383I,I455T,M457L
oocytes, 16.8 ± 2.4 RLU
(n = 10)
G617E,Y107 oocytes, and
15.9 ± 3.0 RLU (n = 10) in
A383I,I455T,M457L
G617E,Y107 oocytes.
Thus the
A383I,I455T,M457L and
G617E,Y107
were deemed suitable for the construction of chimeras; they were used
throughout the experiments instead of the wild-type subunits, and, for
simplicity, we refer to them as "
" and "
". The name and
exact amino acid constitution of each chimera (E) is shown in Table
1. Table
2 indicates the percent amino acid identity of the swapped regions. All constructs were verified by DNA
sequencing. Capped cRNAs from linearized cDNAs were synthesized in
vitro using the T7 mMESSAGEmMACHINE (Ambion, Austin, TX), according to
the provider's instructions.
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Western blot analysis. Oocytes were homogenized, separated by SDS electrophoresis, and transferred to nitrocellulose filters. Primary rat anti-HA monoclonal antibody (100 ng/ml) and secondary peroxidase-conjugated goat anti-rat antibody (160 ng/ml) were diluted in TBS-blocking solution. Detection was performed with the enhanced luminol reagent NEN (NEN, Boston, MA).
Data analysis.
To summarize data from different batches of oocytes and to account for
the batch-to-batch variability of overall expression levels, individual
Iami and surface expression values were
normalized to the average
Iami of at least
five control
ENaC oocytes from the same batch and to the
average surface expression of at least 10 control oocytes,
respectively.
Iami and surface expression were assessed in 14 different batches of oocytes for
-,
-, and
ENaC and in at least two different batches of
oocytes for each chimera. Data are given as mean values ± SE,
n indicates the number of oocytes, and N
indicates the number of different batches of oocytes used. Significance
was evaluated by the appropriate version of Student's
t-test using values from individual oocytes.
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RESULTS |
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ENaC travels to the membrane more efficiently than the
ENaC.
Expression of
ENaC in Xenopus laevis oocytes
resulted in
Iami averaging 5.60 ± 0.79 µA (n = 70, N = 14) at a holding
potential of
60 mV and also in high chemiluminescence signals in the
surface expression assay consistent with previously reported data
(25, 26, 28). In contrast,
Iami
and surface expression levels were very low in
ENaC- or
ENaC-expressing oocytes (Fig. 2C). Normalized to the
values obtained in oocytes expressing
ENaC,
Iami averaged 1.1 ± 0.2% (n
= 70, N = 14) or 3.1 ± 0.4% (n = 70, N = 14) in oocytes expressing
ENaC or
ENaC,
respectively. Similarly, the surface expression averaged 1.5 ± 0.2% (n = 144, N = 14) in
ENaC oocytes and
3.0 ± 0.4% (n = 146, N = 14) in
ENaC
oocytes (Fig. 2C). Interestingly, both the
Iami and the surface expression values for
ENaC were significantly higher than those for
ENaC (P
< 0.001). These results confirm the well-known finding that all
three ENaC subunits are required to form a fully functional channel
(7, 17). In addition, they suggest that the
-heteromer travels more efficiently to the plasma membrane than
the
-heteromer.
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CRD2 substitution chimeras.
The CRD2 domain is highly conserved between the ENaC subunits and is
thought to play an essential role in the efficient transport of
assembled ENaC channels to the plasma membrane (16). Thus we wondered whether functional ENaC channels could be formed when the
CRD2 domains were swapped between - and
-subunits. Figure 2A illustrates chimeras E9 and
E10, in which the
ENaC CRD2 was substituted by the
ENaC CRD2 and
vice versa.
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Extracellular loop substitution chimeras.
In the next pair of chimeras, the complete extracellular loop, except
the pre-M2 domain of ENaC, was substituted by the corresponding loop
of
ENaC (E1) and vice versa (E2) (Fig.
3A). As shown in Fig.
3B, the
Iami and the surface
expression resulting from the coexpression of each chimera with either
or
were very low and similar to the values obtained for
and
alone.
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M2 and COOH termini substitution chimeras.
Chimeras E7 and E8 (Fig. 4A)
had the pre-M2 domain, the M2 domain, and the COOH terminus of ENaC
substituted by the corresponding regions of
ENaC (E7) and vice versa
(E8). In
E8 oocytes,
Iami and surface
expression averaged 16.5 ± 1.5% (n = 10, N
= 2) and 28.7 ± 4.6% (n = 19, N = 2),
respectively (Fig. 4B). This indicates that E8 can at least
partially substitute for
ENaC. In
E7 oocytes,
Iami and surface expression averaged
75.9 ± 10.7% (n = 10, N = 2) and 86.9 ± 4.7% (n = 23, N = 2), respectively, reaching
similar levels as observed in
ENaC oocytes. This suggests that
the E7 chimera can almost fully substitute for the function of the
-subunit.
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NH2 termini and M1 substitution chimeras.
In chimeras E5 and E6 (Fig.
6A), the NH2
terminus and M1 domain of ENaC were substituted by the corresponding
regions of
ENaC (E5) and vice versa (E6). In
E5 oocytes,
Iami and surface expression averaged
52.3 ± 11.5% (n = 10, N = 2) and 53.3 ± 10.1% (n = 20, N = 2), respectively. This
indicates that E5 can replace
ENaC function to a large extent (Fig.
6B). In contrast, in
E6 oocytes,
Iami and surface expression were very low and
not significantly different from the value measured in
oocytes
(Fig. 6B). Hence, although the NH2 terminus and
the M1 region of
can be substituted by the corresponding regions of
, the reverse substitution fails to produce functional channels.
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NH2 and COOH termini substitution chimeras.
Both the NH2 and COOH termini of - and
-subunits have
been shown to be important for the retrieval/endocytosis of the ENaC channels from the plasma membrane (8, 42). We speculated that chimeras with both intracellular
- or
-termini substituted by the corresponding
- or
-termini, respectively, may still be
efficiently expressed at the plasma membrane when coexpressed with
or
. These chimeras are illustrated in Fig.
7A, and are named E13 and E14.
Figure 7B shows that the
Iami and
the surface expression of each chimera when coexpressed with either
or
were very low and similar to the values obtained for
and
alone. Therefore, it is apparently not possible to
efficiently transport an ENaC complex to the plasma membrane when all
intracellular termini are contributed by the
- or
-subunits. This finding, in combination with the results in Figs.
4 and 6, suggests that at least one of the intracellular termini must
be contributed from
and at least another one from
in order for
the ENaC complex to be transported to the plasma membrane.
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DISCUSSION |
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In this study, we used a chimera approach to investigate the
relative importance of several domains of the - and
-subunits to
promote the expression of functional ENaC channels in the plasma membrane of Xenopus laevis oocytes. The main findings of the
present study are the following: 1) replacing the
extracellular loop of
ENaC by that of
ENaC or vice versa results
in a very inefficient surface expression of ENaC, 2) the
CRD2 region and the
CRD2 region can efficiently substitute for
each other, 3) the M2 and COOH terminus of
ENaC have a
more potent role than the corresponding regions of
ENaC in the
assembly/trafficking of the ENaC complex and its expression at the
plasma membrane, and 4) the NH2 terminus and the
M1 region of
ENaC are more important for the formation of a
functional ENaC channel than the corresponding regions of
ENaC.
Functional role of extracellular domains of the - and
-subunits of ENaC.
The inability of the chimeras E1 and E2 to increase the surface
expression of
and
, respectively, suggests that the
extracellular loops of both the
- and the
-subunits have to be
simultaneously present to allow a fully functional ENaC channel complex
to form. An alternative interpretation is the possibility that chimeras with an inability to increase surface expression do not fold properly due to some misfit between the extracellular, intracellular, and transmembrane domains and that this causes their retention in the ER
(i.e., an "intrasubunit" problem rather than an "intersubunit" problem). At present, the functional role of the large extracellular loops of ENaC and the relative importance of the
-,
-, and
-subunit loops are still poorly understood (5). It is
possible that a distinct glycosylation pattern of the
- and
-subunits is essential for the efficient assembly of the channel and
its translocation to the plasma membrane. It has also been suggested
that the extracellular loops may contain important target sites for
ENaC-activating proteases (12, 50). Our study provides
evidence that the extracellular loops of the
- and
-subunits each
have unique and distinct functional features because both loops are
required for proper channel function.
Preferential role of the -subunit in the trafficking of ENaC.
A series of results suggests that several domains of the
ENaC have a
more potent role in trafficking the channel complex to the plasma
membrane than the corresponding regions of
ENaC. First, expression
of
E5 resulted in surface expression and
Iami values that were substantially greater
than those obtained with
, whereas
E6 failed to increase
expression levels above those observed with
. Hence, the
NH2 terminus and the M1 region of
can successfully
replace the corresponding regions of the
-subunit, whereas the
reverse substitution fails to produce fully functional channels.
Second,
E7 produced higher
Iami and
surface expression values than
E8. This indicates that the
combined pre-M2 region, M2 domain, and COOH terminus of
ENaC can
substitute for the corresponding regions of
ENaC more efficiently
than the combined pre-M2 region, M2 domain, and COOH terminus of
ENaC can substitute for the corresponding regions of
ENaC. Third,
E8, which essentially represents a duplication of most of the
-subunit except the pre-M2 region, M2 domain, and the COOH terminus
of E8 that are from
ENaC, can be transported efficiently to the
plasma membrane. This is not the case with
E7 representing a
similar duplication of most of the
-subunit, indicating once more
the primacy of the
-subunit over the
-subunit in promoting the
formation of ENaC channel complexes that can efficiently traffic to the
plasma membrane.
Physiological significance of the present results.
Interestingly, the BFA experiments demonstrated that E7 and
E8 have a rapid retrieval rate similar to that of
ENaC controls. This result is of significance because it indicates that the
PPXY motifs of the
- and
-COOH termini are recognized equally
well from the endocytic machinery (i.e., Nedd4/Nedd4-2). As a
result, channel complexes are internalized efficiently even in subunit
arrangements in which there are two
ENaC PPXY motifs and not a
single
ENaC PPXY motif present and vice versa. Therefore, the
COOH-terminal PPXY motifs are apparently functionally interchangeable between the
- and the
-subunits. This conclusion is in good agreement with a recent study suggesting that each of the second and
the third WW motifs of Nedd4 binds both to
- and
-COOH termini (21). It is probably the absence of any preference of the
binding of these two WW motifs to the
- and
-COOH termini that
makes the COOH termini interchangeable between the two subunits and, on
the other hand, also explains why truncation of both
- and
-COOH
termini has an additive effect regarding the hyperactivity of the
channel (40).
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ACKNOWLEDGEMENTS |
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We thank Dr. S. J. Tucker for expert advice regarding the construction of the chimeras.
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FOOTNOTES |
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This work was supported by grants from the Wellcome Trust and the National Kidney Research Fund. A.-A. Konstas is a recipient of a National Kidney Research Fund studentship.
Address for reprint requests and other correspondence: C. Korbmacher, Institut für Zelluläre und Molekulare Physiologie, Universität Erlangen-Nürnberg, Waldstr. 6, D-91054 Erlangen, Germany (E-mail: christoph.korbmacher{at}physiologie2.med.uni-erlangen.de).
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.
10.1152/ajpcell.00385.2002
Received 26 August 2002; accepted in final form 23 September 2002.
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