Regulation of a cloned epithelial
Na+ channel by its
- and
-subunits
Mouhamed S.
Awayda1,
Albert
Tousson2, and
Dale J.
Benos3
1 Department of Medicine and
Department of Physiology, Tulane University School of Medicine, New
Orleans, Louisiana 70112; and
2 Department of Cell Biology and
3 Department of Physiology and
Biophysics, University of Alabama at Birmingham, Birmingham, Alabama
35223
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ABSTRACT |
Using the Xenopus oocyte
expression system, we examined the mechanisms by which the
- and
-subunits of an epithelial Na+
channel (ENaC) regulate
-subunit channel activity and the mechanisms by which
-subunit truncations cause ENaC activation. Expression of
-ENaC alone produced small amiloride-sensitive currents (
43 ± 10 nA, n = 7). These currents
increased >30-fold with the coexpression of
- and
-ENaC to
1,476 ± 254 nA (n = 20).
This increase was accompanied by a 3.1- and 2.7-fold increase of
membrane fluorescence intensity in the animal and vegetal poles of the
oocyte, respectively, with use of an antibody directed against the
-subunit of ENaC. Truncation of the last 75 amino acids of the
-subunit COOH terminus, as found in the original pedigree of
individuals with Liddle's syndrome, caused a 4.4-fold
(n = 17) increase of the
amiloride-sensitive currents compared with wild-type 

-ENaC.
This was accompanied by a 35% increase of animal pole membrane
fluorescence intensity. Injection of a 30-amino acid peptide with
sequence identity to the COOH terminus of the human
-ENaC
significantly reduced the amiloride-sensitive currents by 40-50%.
These observations suggest a tonic inhibitory role on the channel's
open probability (Po) by the COOH terminus of
-ENaC. We conclude that the changes of current observed with coexpression of the
- and
-subunits or those observed with
-subunit truncation are likely the result of
changes of channel density in combination with large changes of
Po.
oocyte expression; immunofluorescence; Liddle's syndrome; channel
activation
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INTRODUCTION |
THE GENE CODING for the
-subunit of an epithelial
Na+ channel (ENaC) was cloned from
rat colon in 1993 by Canessa et al. (5) and Lingueglia et al. (17).
Expression of this subunit by itself in
Xenopus oocytes generated
amiloride-sensitive Na+ currents.
Expression of this subunit in an in vitro translation system and its
subsequent incorporation into planar lipid bilayers also confirmed that
it was capable of producing an
Na+-selective channel (2, 11).
Despite the ability of the subunit to induce
Na+ currents in both the oocyte
and the lipid bilayer systems, the currents attributed to
-ENaC
expressed in Xenopus oocytes were smaller than those obtained in oocytes injected with total mRNA from
Na+-transporting epithelia. This
observation has lead to the cloning of two additional "auxiliary"
subunits (
and
) that, when coexpressed with the
-subunit,
cause a large increase of currents by more than 20-fold (6, 24).
The presence of ENaC homologues in many native and cultured epithelial
and nonepithelial tissues has been well documented (6-8, 14, 16,
18, 24, 25). In these tissues, the protein or message levels for all
three subunits were highly variable, such that not all tissues
expressed equal levels of these subunits and some tissues even
expressed only
-ENaC. Moreover, in some experiments, the
- and
-subunits were found to be preferentially upregulated in response to
hormones such as aldosterone (1). These observations may indicate that
the changes in "auxiliary" subunit mRNA or protein levels are
potential intrinsic mechanisms by which tissues regulate their rates of
Na+ transport.
Another mechanism by which the
- and
-subunits regulate ENaC
activity involves the COOH termini of these subunits. Mutations that
truncate the COOH termini of either the
- or the
-subunit, as
observed in Liddle's syndrome (10, 22), cause an increase in channel
activity. This was experimentally confirmed in experiments in which
- and
-ENaC were coexpressed with a truncated
-ENaC in
Xenopus oocytes and the finding of
elevated amiloride-sensitive whole cell currents compared with those
observed with the coexpression of all three wild-type subunits (21,
23).
A lack of consensus exists as to how
-subunit truncations activate
ENaC. Ismailov et al. (13) have observed that the channel purified from
Liddle's syndrome-affected human lymphocytes is constitutively
activated when incorporated into planar lipid bilayers. This activated
channel was inhibited (~25%) by application of a 30-amino acid
peptide derived from the COOH-terminal region of the
-subunit. They
concluded that the observed increase of activity is likely because of
large (4- to 5-fold) changes of open probability
(Po) and that this
increase of Po is
mediated, at least in part, by relief of the inhibitory actions of the
COOH-terminal region of the
-subunit. Snyder et al. (23) reported
that the changes of activity likely result from an increase of channel density (NT) alone. On the other
hand, a recent report by Firsov et al. (9) indicated that ENaC
activation after subunit truncation is the result of a combination of
increases in NT and
Po.
We used a combination of an immunofluorescence assay and dual-electrode
voltage recording of amiloride-sensitive ENaC currents in the
Xenopus oocyte expression system to
examine the mechanisms by which expression of
- and
-ENaC alter
the induced amiloride-sensitive currents. We also examined the
differences in plasma membrane channel levels between oocytes that
express a normal
-subunit and those that mimic Liddle's syndrome by
expression of a truncated
-subunit. Electrophysiological
measurements were carried out on the same batch of oocytes as were the
immunofluorescent measurements. We report that coexpression of the
-
and
-subunits caused a threefold increase of membrane-bound
-ENaC
levels accompanied by a 34-fold increase of amiloride-sensitive whole
cell currents. On the other hand, only a 35% increase of membrane
fluorescence levels was observed when
-ENaC was truncated.
This was accompanied by a 4.4-fold increase of whole cell current.
Moreover, intracellular injection of a peptide derived from the
-subunit COOH-terminal sequence (final concentration
~300 µM) resulted in a 40-50% inhibition of ENaC currents. We
conclude that
- and
-ENaC and the COOH termini of these subunits
affect channel activity by altering both
NT and
Po of the resulting
Na+ channel.
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MATERIALS AND METHODS |
Antibody production.
A rabbit anti-
-ENaC antibody was produced as previously described
(12). Briefly, 200 µg of bovine
-ENaC full-size fusion protein
(dissolved in 1 ml of sterile water) were injected into multiple
subcutaneous dorsal sites in a New Zealand White rabbit. This was
repeated every 3 wk until an acceptable antibody titer was obtained in
an enzyme-linked immunosorbent assay using the fusion protein as
substrate. After five boosts, the rabbit was killed and serum was
collected. Immunoglobin G (IgG) was purified from serum with the use of
protein A-coupled Sepharose beads (Bio-Rad, Richmond, CA) and was
stored at
20°C until further use. Preimmune serum was
treated in the same manner as the immune serum. This antibody
cross-reacts with the rat
-ENaC homologue, as assessed using both
immunofluorescence methods and Western blotting.
Chimera construction.
To circumvent potential problems with the coexpression of rat and human
subunits, a situation that by itself may cause changes of the magnitude
of amiloride-sensitive currents (18) and potentially influence membrane
fluorescence intensity, we used a
-subunit chimera (rat + human)
that only contained a small portion of human ENaC sequences. This
chimera was constructed by replacing the COOH-terminal region (last 125 amino acids) of the rat
-ENaC with a polymerase chain reaction
(PCR)-generated human COOH-terminal sequence, using cDNAs prepared from
peripheral blood lymphocytes either from a normotensive individual or
from an individual from the original Liddle's kindred in the
amplification procedure. The PCR primers were designed so that the
products contained an artificially introduced
Xho I (5') and
Acc I (3') sites. These products
were digested with both Xho I and
Acc I and were subsequently ligated
into rat
-ENaC (
-rENaC) contained in pSPORT that was predigested
with these two enzymes. The Xho I site
in
-rENaC was created at the corresponding site of the human
-subunit by PCR-directed mutagenesis. An extra
Acc I site in
-rENaC at nucleotide position 2 (numbered according to GenBank X77932) was removed by
restriction with Sal I followed by
blunt-end ligation.
Two different chimeras containing different human
-COOH-terminal
sequences were used: 1) a control
chimera containing normal human
-sequences
(
chim) and
2) a truncated chimera containing a
premature stop codon as it was found in the original Liddle's pedigree
that encoded a C to T mutation at Arg-564, deleting the last 75 amino
acids from the COOH terminus of the
-subunit
(
). The control chimera
was found to behave in an identical manner to the full-length rat
-subunit in both electrophysiological and immunofluorescent assays
(see RESULTS). The validity of the sequence of both chimeras was confirmed by restriction analysis and DNA
sequencing.
RNA synthesis.
RNA synthesis was as previously described (3). The plasmid containing
either
-,
-, or
-rENaC (a gift of B. Rossier) or the chimeric
constructs (a gift of Y. Oh and D. Warnock) was linearized with the
appropriate 3' restriction enzyme. Linearized plasmid DNA was
purified using the Geneclean kit (Bio 101, Vista, CA). Sense RNA was
synthesized from purified plasmid DNA using T7 RNA polymerase according
to the manufacturer's instructions (Promega, Madison, WI). RNA was in
vitro synthesized in the presence of methyl guanosine cap analog
m7G(5')ppp(5')G (NEB,
Beverly, MA) in threefold excess to GTP. This stabilizes the cRNA and
enhances its translational efficiency (15, 19). After two rounds of
phenol-chloroform extraction and ethanol precipitation, RNA was
quantitated by measuring optical density at 260 nm and
stored at
80°C.
Oocyte expression and recording.
Toads were obtained from Xenopus I (Ann Arbor, MI) and were kept in
dechlorinated tap water at 18°C. Oocytes were surgically removed
from anesthetized toads and were processed as previously described (3).
All injection volumes consisted of 50 nl of nuclease-free water
containing cRNAs at various concentrations.
-rENaC-expressing
oocytes were injected with 12.5 ng of cRNA, whereas


-rENaC-expressing oocytes were injected with 2.5 ng of each
cRNA. Both concentrations have been shown to result in subsaturating
levels of ENaC expression (21). Injected oocytes were incubated at
18°C for 2-3 days until their recording or processing for
immunofluorescence measurements. All recordings were performed at
19-21°C.
In experiments that required the direct injection of peptide, oocytes
were first impaled with the injecting electrode and then impaled with
the two recording microelectrodes as previously described (3). The
injecting electrode was filled with the appropriate peptide. The tip of
the electrode was filled with air (~10-nl volume) so that the peptide
did not leak into the oocyte immediately after impalement. This allowed
us to determine a control period with a relatively constant baseline
before the injection of any material into the oocyte. A 30-amino acid
peptide with a sequence identical to that of the COOH-terminal region of the human
-ENaC and a 26-amino acid peptide of random sequence were both synthesized by Research Genetics (Huntsville, AL),
followed by high-performance liquid chromatography purification. The
10-amino acid uncharged peptide was synthesized in-house as
described by Ismailov et al. (13). All peptides were dissolved in water
at a stock concentration of 5 mM. Attempts to use other peptides (a
charged 10-amino acid peptide; see Ref. 13) were unsuccessful because
these peptides were dissolved in dimethyl sulfoxide (DMSO) and resulted
in DMSO concentrations that were incompatible with the oocyte
expression system.
Solutions and chemicals.
The pH of all oocyte recording solutions was adjusted to 7.5. Oocytes
were defoliculated in Ca2+-free
Ringer of the following composition (in mM): 84 NaCl, 1 MgCl2, 2 KCl, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). The oocyte culture medium was one-half strength L-15
medium (Sigma, St. Louis, MO) supplemented with 15 mM HEPES and 0.5%
of a 10,000 U/ml solution of penicillin-streptomycin (GIBCO, Grand
Island, NY). The recording medium ND-96 contained (in mM) 96 NaCl, 1 MgCl2, 2 KCl, 1.8 CaCl2, and 5 HEPES. Oocytes were
continuously perfused with solution at the rate of 2 ml/min or ~2
chamber volumes/min.
Data acquisition and analysis.
The program pCLAMP version 5.5 (Axon Instruments, Foster City, CA) was
used for data acquisition and analysis as previously described (3).
Oocyte holding voltage was clamped to 0 mV at all times except during
acquisition of whole cell currents at various voltages. During periods
of data acquisition, the membrane voltage was stepped for 450 ms from
100 mV to +80 mV in increments of 20 mV. Currents at
100
mV were summarized from the averaged value of the last five current
points at the end of each voltage episode. By convention, inward flow
of cations is designated as inward current (negative current), and all
voltages are reported with respect to ground or bath. All data are
reported as means ± SE.
Oocyte fixation.
All procedures were carried out at room temperature except where noted.
Oocytes were fixed in 3% formaldehyde (EM grade; Tousimis, Rockville,
MD) in ND-96 for 2 h at room temperature. Fixed oocytes were partially
dehydrated by incubation in ND-96 containing 95% ethanol for 45 min,
with a new solution change every 15 min. This was followed by complete
dehydration in 100% ethanol, followed by xylene for 45 min, each with
three bath solution changes. Oocytes were then infiltrated with liquid
paraffin at 60°C for 1 h with two solution changes and were allowed
to cool before sectioning. To facilitate sectioning, paraffin-embedded
samples were reembedded in larger blocks. These blocks were sectioned
with a microtome at a section thickness of ~5 µm. Sections were air
dried, followed by an overnight incubation in a 60°C oven.
Sections were deparaffinized by a 15-min incubation in xylene, with
three solution changes. Sections were subsequently rehydrated by a
series of 15-min incubations in ethanol ranging from 100 to 0% in
ND-96. This was followed by a 15-min postfixation in 3% formaldehyde.
Immunofluorescence.
Postfixed sections were incubated in
tris(hydroxymethyl)aminomethane-NaCl (pH 8.2, 220 mosM)
for 30 min to quench any unreacted aldehydes. Nonspecific
immunoreactivity was blocked using 20% normal goat serum (NGS; Sigma)
in ND-96 for 15-30 min. Sections were then incubated for 1 h with
either the primary antibody (anti
-bENaC antibody)
or with preimmune IgG at the same final concentration of 0.2 mg/ml in
20% NGS. Unbound primary antibody was washed away with four solution
changes within 15 min. Sections were blocked with 5% NGS in ND-96 for
15 min and were then incubated with the secondary antibody for 1 h at
37°C at a final concentration of 0.05 mg/ml in 5% NGS in ND-96.
This antibody was a goat anti-rabbit IgG conjugated to fluorescein
isothiocyanate (Boehringer Mannheim). After 1 h, sections were washed
in ND-96 and then were mounted in 0.1%
p-phenylenediamine in 9:1 glycerol to
ND-96.
Photography and fluorescence quantification.
Micrographs were prepared with a Leitz Orthoplan fluorescence
microscope equipped with a Vario-orthomat II camera system and a
digital photometer. The microscope was outfitted with epifluorescence and phase-contrast optics. Images were recorded on Kodak T-Max 400 film
or Ektachrome p1600 (Kodak, Rochester, NY) push processed to 800 ASA.
High-contrast prints were made on polycontrast III RC glossy paper
using a no. 4 contrast filter (Kodak) on a Beseler CB7 enlarger (East
Orange, NJ). All experimental and negative control trials were
photographed with the same exposure times.
Fluorescence intensity was measured using a digital photometer attached
to the microscope. The photometer was standardized using the InSpeck
Green (490- and 515-nm absorption and emission wavelengths,
respectively) microscope image intensity calibration kit (Molecular
Probes, Eugene, OR). Fluorescence intensity of individual calibration
beads was measured by spot metering. The reciprocity compensation
control was adjusted so that the measured bead intensity would most
closely match the standard relative bead intensity set by the
manufacturer. With the camera set for spot metering, the zoom lens was
adjusted to 5×, a 50× fluotar objective was swiveled into
place, and the intensity of a 30-µm segment of cell membrane surface
was measured. All measurements were done in triplicate and were
averaged and treated as a single value. Values of fluorescence
intensity were >90% accurate as assessed from the average triplicate
variability. Reported values were corrected for background cytoplasmic
fluorescence because it was not possible to eliminate its contribution.
We assumed that this background signal contributed equally to the raw
data because it was observed in all groups of oocytes, including those treated with the secondary antibody alone.
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RESULTS |
Effect of coexpression of
- and
-ENaC.
Previous reports have indicated that expression of
-ENaC produces
currents that are much smaller than those observed with 

-ENaC
expression. As shown in the representative example in Fig.
1, the whole cell currents and the
amiloride-sensitive currents were much smaller in oocytes expressing
-ENaC alone. This was observed despite a fivefold higher
concentration of RNA in oocytes injected with the
-subunit by
itself.

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Fig. 1.
Expression of - and -rat epithelial
Na+ channel (rENaC) with -rENaC
causes a marked increase of amiloride-sensitive whole cell currents.
Representative whole cell currents
(Im) in oocytes expressing
-rENaC (A) and   -rENaC
(C) and corresponding changes of
these currents with 10 µM amiloride
(B and
D). Currents were recorded in both
groups of oocytes for 30 min (until a stable value was observed),
followed by addition of 10 µM amiloride for 10 min.
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The values of whole cell currents in water-injected oocytes and in
oocytes expressing
-rENaC and 

-rENaC before and after amiloride treatment are summarized in Table
1. The ratio of whole cell currents at
100 mV was 16-fold higher in oocytes expressing 

-rENaC
than in those expressing
-rENaC. Moreover, the ratio of the
amiloride-sensitive currents was 34-fold higher. Consistent with
previous reports (18, 26), water-injected oocytes exhibited amiloride-sensitive currents. These currents averaged
3 nA.
These values of endogenous currents are small compared with the
microampere-level currents observed with expression of all three
subunits but are important to note because of the presence of a
Xenopus ENaC homologue (20) and the
levels of spontaneous membrane fluorescence in water-injected oocytes.
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Table 1.
Whole cell currents in Xenopus oocytes expressing
-rENaC and   -rENaC and their
subsequent inhibition by amiloride
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With the assumption that the conductance of the channel formed by the
-subunit alone and that of the channel formed by all three subunits
are similar (see DISCUSSION), the
differences of whole cell currents imply a 34-fold difference of
channel activity, or
NT · Po
(the product of total NT and the
single-channel Po). Because currents are very low in oocytes expressing
-ENaC and because of the large size of oocytes, it would be difficult to determine the origin of the differences between these two channels using patch-clamp analysis. Instead, we resorted to an
immunofluorescence approach to estimate differences of membrane ENaC
content in oocytes expressing these two channel complexes.
Whole oocytes, because of their thickness, are difficult to examine
intracellularly by routine fluorescence microscopy. The protocol using
paraffin embedding and sectioning (see MATERIALS AND
METHODS) circumvented this problem and yielded the
best overall oocyte preservation. Figure 2
shows a low-magnification view of paraffin-embedded oocytes, as imaged
by phase contrast microscopy. The absence of large membrane or
cytoplasmic gaps was taken as an indication that paraffin embedding did
not disrupt the overall distribution of cytosolic and membrane-bound
proteins.

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Fig. 2.
Formaldehyde fixation and paraffin embedding preserve oocyte structure.
Representative phase-contrast image of formaldehyde-fixed,
paraffin-embedded oocytes (see MATERIALS AND
METHODS for details). Note absence of any large gaps in
cytosol or plasma membrane. Also note dark-pigmented segment of
membrane (animal pole) and light-pigmented segment (vegetal pole).
Scale bar, 500 µm.
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Under the conditions stated in MATERIALS AND
METHODS, the protein A-purified, preimmune IgG did not
exhibit any membrane surface immunoreactivity with control noninjected
oocytes (data not shown) or with oocytes expressing 

-rENaC
(Fig. 3). There was an appreciable amount
of yolk-related fluorescence that was observed in these oocytes.
Moreover, this nonspecific fluorescence was always limited to the
cytoplasm and was not observed at the plasma membrane. As such, this
limited our studies to an examination of the specific fluorescence at
the plasma membrane.

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Fig. 3.
Specificity of -subunit antibody used in present studies. This
antibody did not show any appreciable immunoreactivity in oocytes
expressing either of the - or -subunits
(A and
B, respectively). Moreover, preimmune
serum did not cross-react with any of the 3 subunits
(C). Arrowheads indicate position of
plasma membrane. Scale bar, 50 µm.
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Before assessment of the differences between oocytes expressing the
-subunit alone and those expressing all three subunits, it was
important to determine by immunofluorescence whether this antibody
showed any appreciable cross-reactivity with the
- and
-subunits.
Two representative oocytes that have been incubated with the
anti-
-ENaC antibody and are expressing
- and
-rENaC, respectively, are shown in Fig. 3, A
and B. There was no detectable membrane-associated fluorescence in these oocytes. Given the
observation by Firsov et al. (9) of similar membrane-bound expression
levels of
- and
-ENaC compared with
-ENaC when expressed by
themselves in Xenopus oocytes (see
Fig. 1B in Ref. 9), we can surmise that our antibody was likely specific for the
-subunit over the
-
and
-subunits. This is not altogether surprising given the low
homology at the amino acid level among the three subunits (6, 18).
An example of membrane immunofluorescence in water-injected oocytes and
those expressing
-rENaC and 

-rENaC is shown in Fig.
4. Membrane fluorescence in control oocytes
was almost absent. The oocyte shown in Fig.
4A was intentionally chosen to reflect the highest amount of membrane fluorescence observed in this group. This membrane-associated fluorescence was still much smaller than that
observed in oocytes expressing
-rENaC and 

-rENaC, shown in
the two typical examples in Fig. 4, B
and C, respectively. A punctate
pattern of membrane staining was the typical observation, indicating a
possible grouping or clustering of this
Na+ channel in the membrane. As
seen from the accompanying bright-field images, these examples of
membrane segments originated from the animal pole of the oocyte,
because the dark-pigmented background allowed a better delineation of
membrane fluorescence. A very similar pattern of membrane-associated
fluorescence was observed in the vegetal pole.

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Fig. 4.
Membrane-surface fluorescence is increased in oocytes expressing all 3 ENaC subunits. Control oocytes (A)
exhibited a small amount of endogenous membrane labeling.
-rENaC-expressing oocytes exhibited higher membrane
immunofluorescence (B) that is
further increased by expression of - and -subunits
(C).
Right panels show corresponding
phase-contrast images. Note discrete and punctate staining of plasma
membrane in all 3 examples. Conditions are same as in Fig. 3.
Arrowheads indicate position of plasma membrane. Scale bar, 50 µm.
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Values of membrane fluorescence in all three groups of oocytes are
summarized in Table 2. These values are in
arbitrary units and are inversely related to the exposure times and
directly related to the fluorescence intensity. Moreover, these values
are corrected for the unavoidable contribution of background
cytoplasmic fluorescence.
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Table 2.
Relative plasma membrane fluorescence intensity of Xenopus oocytes
expressing -rENaC and   -rENaC
probed by an anti- -subunit antibody
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To determine whether there was any differential partitioning of
-rENaC and 

-rENaC to either the animal or vegetal poles, data from both parts of the membrane were summarized. The values of
fluorescence intensity in control oocytes were not different from
either hemisphere. However, it is clear that there was a higher
distribution of the Na+ channel in
the animal pole in the experimental groups of oocytes. This was
especially evident in oocytes expressing 

-rENaC, in which the
fluorescence intensity attributed to exogenous ENaC (corrected for the
values measured in control oocytes) was 2.3-fold higher in the animal
pole. The differences in membrane fluorescence intensity between the
two hemispheres in
-ENaC and 

-ENaC-expressing oocytes
cannot be attributed to differences in the contribution of background
fluorescence, because values of membrane autofluorescence were the same
in both hemispheres.
Oocytes expressing the
-subunit alone exhibited higher fluorescence
intensity than control oocytes. This was observed in both the animal
and vegetal poles but was more pronounced in the animal pole. Oocytes
expressing 

-rENaC exhibited higher fluorescence intensity than
oocytes exhibiting
-rENaC alone. Likewise, the differences were more
pronounced between these two groups of oocytes in the animal pole,
where the amount of staining in 

-rENaC-expressing oocytes was
3.1-fold higher than that in
-rENaC oocytes. Thus these values alone
cannot account for the change of amiloride-sensitive macroscopic
currents that were >30-fold. Instead, it is likely that a combination
of increase of NT and of
Po best explains the increase of current with
- and
-subunit expression.
Effect of truncation of the
-subunit.
The COOH termini of the
- and
-subunits also control ENaC
activity, whereby truncation of the COOH terminus of these subunits has
been reported to stimulate ENaC currents when expressed in Xenopus oocytes. To examine the
mechanisms by which
-subunit truncation causes
Na+ hyperabsorption and Liddle's
syndrome, we expressed the truncated
-subunit
with
- and
-rENaC. As shown in the representative example in Fig.
5, truncation of the terminal 75 amino
acids of the
-subunit construct caused a large activation of whole
cell currents over those observed with the nontruncated
-subunit
rat-plus-human construct
chim.
The majority of whole cell currents in both oocytes was blocked by 10 µM amiloride. Moreover, as observed in Fig.
6, there were no detectable differences at
the level of whole cell currents or amiloride sensitivity (data not
shown) between the channels formed with wild-type
-subunits and
those formed with
chim.

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Fig. 5.
Expression of a truncated -subunit
( ) causes a marked
increase of whole cell currents. Representative whole cell currents in
oocytes expressing
 chim -rENaC
(A) and
  -rENaC
(B).
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Fig. 6.
Whole cell currents in oocytes coexpressing control chimeric
-subunits (B) were
indistinguishable from those observed in oocytes coexpressing rat
-subunit (A).
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Amiloride-sensitive currents averaged
1.48 ± 0.25 µA
(n = 20) in wild-type 

-rENaC
and
1.39 ± 0.31 µA (n = 14) in control chimeric

chim
-rENaC. Thus it
appears that the channel formed with the control nontruncated
rat-plus-human chimera behaved in an identical manner to that observed
with the
-subunit of rat origin. On the other hand, currents in
oocytes expressing the truncated chimeric
-subunit were much larger
than control and averaged
6.50 ± 0.92 µA
(n = 17). This resulted in a 4.4-fold activation of amiloride-sensitive current and is consistent with observations from other laboratories (21, 23).
As shown in the representative example in Fig.
7, the membrane fluorescence intensity in
an oocyte expressing


-rENaC was
similar to that of an oocyte expressing

chim
-rENaC. Moreover, the
pattern and magnitude of membrane fluorescence were indistinguishable between 

-ENaC- and

chim
-ENaC-expressing
oocytes, and the values from these two control groups were combined. To
obtain a better estimate of small changes of membrane fluorescence
intensity, we limited our analysis to the animal pole of the oocytes,
where the largest signal was observed. In this hemisphere and after we
corrected for the background fluorescence signal of control water-injected oocytes, oocytes expressing 

-rENaC or

chim
-rENaC exhibited
intensity values of 6.28 ± 0.03 (n = 50), whereas those expressing


-rENaC exhibited
a fluorescence intensity of 8.45 ± 0.09 (n = 50). Thus
-subunit truncation
resulted in a 35% increase of fluorescence intensity compared with
oocytes without the truncated
-subunits. The differences between
these two groups of oocytes were essentially similar in the vegetal pole in that there was only a small change observed between oocytes with a normal
-subunit and
(data not shown). This
finding indicates that the activation of ENaC observed with Liddle's
syndrome is the result of a combination of small changes of
NT and larger changes of
Po.

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Fig. 7.
Truncation of -subunit COOH terminus results in a small increase of
Na+ channel membrane-associated
immunofluorescence. Membrane fluorescence in control oocytes was
essentially absent (C).
Membrane fluorescence intensity was slightly higher in oocytes
expressing truncated -subunit (A)
compared with oocytes expressing chimeric nontruncated
-subunit (B). This small increase
of signal alone could not explain 4.4-fold increase of
amiloride-sensitive currents (see Fig. 5 and associated text). In same
group of oocytes there were no detectable differences between
expression of - and -rENaC with chimeric -subunit or full rat
-subunit (compare, for example, Fig.
4C and Fig.
5B). Arrowheads point to cell
surface. Bar, 50 µm.
|
|
To further demonstrate the importance of the COOH terminus of the
-subunit in the regulation of ENaC activity, we injected a 30-amino
acid peptide corresponding to the human
-subunit COOH terminus into
either the 
chim
-rENaC- or
the


-rENaC-expressing oocytes (Figs. 8 and
9). Thirty to forty nanoliters of the
30-amino acid peptide solution (5 mM) were injected into the oocytes,
resulting in an approximate final concentration of 300-400 µM in
the oocyte cytosol (assuming that an average oocyte cytosol volume is
500 nl). Injection of this 30-amino acid peptide into oocytes caused an
inhibition of whole cell currents within minutes. This inhibition reached a plateau at ~8-10 min in both groups of ENaC-expressing oocytes (data not shown). On a paired basis, i.e., comparing the values
of amiloride-sensitive currents from the same oocyte before and 10 min
after the injection of the peptide, we observed a 38% decrease of
current
in 
-rENaC-expressing oocytes (from
5.65 ± 1.11 to
3.52 ± 0.73 µA,
n = 10). A similar ratio of inhibition
was observed in

chim
-rENaC-expressing
oocytes as currents decreased by 47% of control from
1.35 ± 0.24 to
0.72 ± 0.14 µA (n = 12).

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Fig. 8.
Representative effect of peptide injection into ENaC-expressing
oocytes. After establishment of a control period, peptide was injected
using a third pipette into
 chim -rENaC-
(A) and
  -rENaC
(B)-expressing oocytes, and whole
cell currents were measured every minute for 10 min. Inhibitory effect
of peptide reached a plateau at 7-10 min, and data shown for
inhibition of  chim -rENaC-
(C)
and  -rENaC
(D)-expressing oocytes are at
plateau time point. E and
F demonstrate effect of 10 µM
amiloride on  chim - and
  -rENaC,
respectively.
|
|

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Fig. 9.
Summary of inhibitory effects of peptide injection in ENaC-expressing
oocytes. In all groups, amiloride-sensitive whole cell currents
(Iamil) are
summarized at a holding voltage of 100 mV. hC30, 30-mer
peptide derived from last 30 amino acids of human -subunit.
|
|
Conducted as a control for the peptide experiments, injection of
isotonic KCl or a 10-amino acid peptide corresponding to the last 10 amino acids of the rat
-subunit COOH terminus, with its four
positively charged amino acids substituted with neutral glycine
(NH2-MGSGSGVGAI-COOH), did not result in any appreciable change of whole cell currents (n = 8;
see Fig. 10). Moreover, injection of the
random 26-amino acid peptide
NH2-NAHNFPLDLASGEQAPVALTAPAVNG-COOH resulted in a small
(<10%) inhibition of whole cell currents
(n = 6). Thus, despite the presence of
multiple charged residues, this 26-mer failed to display the same
inhibitory effects as the more specific 30-mer with sequence identity
to the
-subunit COOH terminus.

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Fig. 10.
Lack of effect of 10-amino acid uncharged peptide on currents in
ENaC-expressing oocytes. After establishment of a control period,
peptide was injected as described. A:
currents in a representative oocyte expressing
  -rENaC were
unchanged after 10 min of injection of this peptide
(B). Data representative of 8 such
experiments. Lack of an inhibitory action of this peptide was also
observed in
 chim -rENaC-expressing
oocytes (n = 6).
|
|
 |
DISCUSSION |
We used a rabbit primary antibody directed against a nearly full-size
-ENaC fusion protein as previously described (12) to determine the
changes of
-subunit density at the plasma membrane subsequent to
coexpression with full-length
- and
-subunits or after
coexpression with a full-length
-subunit and a truncated
-subunit. We conclude that the changes of whole cell currents produced by expression of
-subunits with
- and
-subunits are likely the result of a combination of a threefold change of
NT and a 10-fold change of
Po. Likewise, the
increase of current observed with
-subunit truncation is
also attributed to a combination of a 1.35-fold increase of
NT and approximately threefold
increase of Po. In both
cases, it appears that the potential changes of NT are small in comparison to the
potential changes of
Po.
Because this antibody is directed against a full-length fusion protein,
it is expected that it would recognize a complete and functional
-subunit as well as a partially degraded and electrically nonfunctional
-subunit. This is not unique to this antibody, and
this possibility could be envisioned with any site-specific and even
monoclonal antibody. This is a shortcoming of determining the changes
of NT with antibodies irrespective
of their specific recognition site in that an electrically silent or
partially degraded channel could still contain antigenic sites.
However, the issues of changes of
NT and
Po cannot be easily
answered using patch- clamp methodologies, because translation of
changes of channel activity
(NT · Po)
in multiple-channel patches to changes of either NT or
Po is very difficult.
This situation is further exacerbated in
low-Po patches and in
cases in which the channel activity varies enormously, as with oocytes
expressing the
-subunit alone and those expressing


-subunits. Moreover, this situation is also complicated by the
potential presence of subconductance states (11) that render the task
of determining channel number in multiple-channel patches a very
formidable task. Therefore, the methods used in the current report
represent an alternative approach to estimate changes of
NT.
It is unlikely that any observed differences in fluorescence intensity
result from differences in the affinity of the channel to the antibody.
This could be a potential pitfall if the antibody were recognizing a
single antigenic site. However, because this is a polyclonal antibody
recognizing multiple antigenic sites, it is unlikely that the affinity
of the antibody to all of these sites is altered in the same manner.
Moreover, the relatively high concentration of antibody used in
immunofluorescence detection assays is likely to represent a saturating
concentration that is relatively unaffected by small changes of the
antigen-antibody binding rate constants or affinity.
Does
-subunit alone form a functional channel?
We have previously reported that
-ENaC by itself is capable of
forming an amiloride-sensitive Na+
channel when incorporated into planar lipid bilayers (2). These
experiments were carried out with
-rENaC obtained by way of an in
vitro translation system. Because the control reaction contained no
channel activity, it was straightforward to attribute the channel
activity to the newly translated channel protein that contained the
-subunit alone. Moreover, in mammalian cells transfected with
-rENaC alone, an amiloride-sensitive
Na+ channel is observed (14),
indicating that
-ENaC alone can form a functional channel. The
observation that there is membrane-bound distribution of
-rENaC when
expressed by itself in oocytes may be indicative of its potential to
form a channel. This is especially true given the finite ionic current
observed in oocytes expressing the
-subunit. This current is
probably not a result of the presence of endogenous
- and
-ENaC,
because injection of
- or
-ENaC individually or of
- and
-ENaC together in the absence of
-ENaC does not result in any
current over that observed in water-injected oocytes.
Mechanism by which
- and
-subunits
increase current.
The origin of the changes of current simplifies to changes in
NT,
Po, or Na+
single-channel conductance
(gNa).
It seems unlikely that the increase of macroscopic current is the
result of an increase of gNa for the
channel formed with
-,
-, and
-subunits, because we have
previously reported that the single-channel conductance is actually
higher in the absence of
- and
-subunits when tested in planar
lipid bilayers (11). A similar observation of a relatively high-conductance channel formed by
-rENaC was made by Kizer et al.
(14) who, after transfection of mammalian fibroblasts with the
-subunit cDNA, observed a 24-pS channel that displayed short burstlike activity punctuated by long closed intervals. These observations of higher
gNa between
-
and 

-ENaC would tend to underestimate the changes of
NT or
Po that are needed to
account for the observed increase of whole cell currents and thus would not affect the present conclusions.
Another possibility to consider is that a different number of
-subunits are needed to form a functional channel in the presence of
the
- and
-subunits. This situation is impossible to evaluate without knowledge of the subunit stoichiometry of the channel. It would
seem unlikely that the differences in the number of subunits can be
large enough to account for the almost 10-fold difference between
macroscopic current activation (34-fold) and membrane fluorescence
activation (3- to 4-fold).
Truncated
-subunit.
Truncation of the
-subunit caused a 4.4-fold increase of current.
Data from the ENaC channel reconstituted into planar lipid bilayers
(13) and from patch clamping of
Xenopus oocytes expressing ENaC (21,
23) are in agreement that the single-channel conductance is unaltered.
This activation of current must then be mediated via an increase of
NT or
Po. Our observations
are consistent with the conclusion that only a small increase of the
-subunit density is observed with
-subunit truncation. The
remaining 3- to 3.5-fold increase in activation must be attributed to
changes of Po. These conclusions are in partial agreement with the recent report by Firsov
et al. (9), who used a similar approach to that used in this report
except that they used a monoclonal tag-specific antibody. The degree of
activation attributed to a potential increase of
NT is in disagreement between the
two reports (35 vs. 93%). However, the absolute values of these
numbers are complicated with the potential presence of electrically
silent channels, the potential differences in the stoichiometry between
the wild-type and Liddle's channel, and other problems associated with
quantification of membrane immunofluorescence (see above).
Peptide block.
To understand the mechanisms by which the truncation in the
-subunit
brings about the elevated whole cell currents, we carried out
experiments with injection of
-subunit COOH-terminal peptide. Consistent with previous reports (4, 13), we demonstrated that ENaC can
be blocked by a 30-amino acid
-subunit COOH-terminal peptide. Nearly
equal percentages of inhibition with injections that resulted in
an approximate intracellular concentration of 300 µM of this
peptide were observed for both the

-rENaC- and

chim
-rENaC-expressing
oocytes (see Fig. 9). This finding has several implications. First, it
supports the conclusion that a large fraction of the elevated currents
observed in


-rENaCexpressing oocytes results from an increase in
Po. Second, this
finding further documents the importance of the COOH terminus of the
- (and
-) subunit in controlling ENaC activity in part by a
direct blocking of normal ENaC currents. The observations of similar
values of peptide block of


-rENaC and

chim
-rENaC in combination
with the specificity of this peptide to block currents (see
RESULTS) argue in favor of the
conclusion that we were injecting a saturating concentration of this
peptide. Thus the observed 40-50% inhibition with this peptide
may represent the maximal in vivo inhibition that can be exerted
directly by the COOH terminus of the
-subunit on ENaC.
Conclusions.
We conclude that coexpression of the
- and
-subunits with the
-subunit increases NT and
Po. Likewise,
truncation of the
-subunit COOH terminus also increases
NT and
Po. Our data indicate that the majority of changes are mediated via effects on the
Po of the channel.
However, it is possible that the contribution of changes of
NT and
Po to channel
activation may be different in various native ENaC-expressing tissues,
because other local factors may determine the activity of the channel
complex. In comparing immunolocalization data with functional assays,
our work offers a convenient approach for assessing the relative
importance of changes in channel surface expression and
Po in regulating Na+ channel activity.
 |
ACKNOWLEDGEMENTS |
We thank David L. Stetson (The Ohio State University) for helpful
suggestions with the fixation procedures and Cathy Guy for help in
typing the manuscript. We also thank Bernard Rossier (University of
Lausanne) for the gift of ENaC and Young Oh and David Warnock (University of Alabama at Birmingham) for the truncated
-ENaC constructs.
 |
FOOTNOTES |
This work was initiated while M. S. Awayda was a fellow at the
University of Alabama at Birmingham and was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-37206
and DK-07545 to D. J. Benos and by a Louisiana American Heart Grant-In
Aid to M. S. Awayda.
Address for reprint requests: M. S. Awayda, Dept. of Medicine, SL35,
Tulane Univ. School of Medicine, 1430 Tulane Avenue, New Orleans, LA
70112.
Received 5 June 1997; accepted in final form 21 August 1997.
 |
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