RAPID COMMUNICATION
Antisense oligonucleotides against the
-subunit of ENaC decrease
lung epithelial cation-channel activity
Lucky
Jain1,2,
Xi-Juan
Chen1,
Bela
Malik2,3,
Otor
Al-Khalili2,3, and
Douglas C.
Eaton2,3
Departments of 1 Pediatrics and
3 Physiology and
2 The Center for Cell and
Molecular Signaling, Emory University School of Medicine, Atlanta,
Georgia 30322
 |
ABSTRACT |
Amiloride-sensitive
Na+ transport by lung epithelia
plays a critical role in maintaining alveolar
Na+ and water balance. It has been
generally assumed that Na+
transport is mediated by the amiloride-sensitive epithelial
Na+ channel (ENaC) because
molecular biology studies have confirmed the presence of ENaC subunits
,
, and
in lung epithelia. However, the
predominant Na+-transporting
channel reported from electrophysiological studies by most laboratories
is a nonselective, high-conductance channel that is very different from
the highly selective, low-conductance ENaC reported in other tissues.
In our laboratory, single-channel recordings from apical membrane
patches from rat alveolar type II (ATII) cells in primary culture
reveal a nonselective cation channel with a conductance of 20.6 ± 1.1 pS and an
Na+-to-K+
selectivity of 0.97 ± 0.07. This channel is inhibited by
submicromolar concentrations of amiloride. Thus there is some question
about the relationship between the gene product observed with
single-channel methods and the cloned ENaC subunits. We have employed
antisense oligonucleotide methods to block the synthesis of individual
ENaC subunit proteins (
,
, and
) and determined the effect of
a reduction in the subunit expression on the density of the
nonselective cation channel observed in apical membrane patches on ATII
cells. Treatment of ATII cells with antisense oligonucleotides
inhibited the production of each subunit protein; however,
single-channel recordings showed that only the antisense
oligonucleotide targeting the
-subunit resulted in a significant
decrease in the density of nonselective cation channels. Inhibition of
the
- and
-subunit proteins alone or together did not cause any
changes in the observed channel density. There were no changes in open
probability or other channel characteristics. These results support the
hypothesis that the
-subunit of ENaC alone or in combination with
some protein other than the
- or
-subunit protein is the major
component of lung alveolar epithelial cation channels.
alveolar type II cells; epithelial sodium channel; single-channel
recording
 |
INTRODUCTION |
ACTIVE Na+
transport across the pulmonary epithelium drives liquid from the lung
lumen to the interstitium, with subsequent absorption of the fluid into
the vasculature (1, 22, 23, 27). In the lung,
Na+ reabsorption is a two-step
process. The first step is passive movement of
Na+ from the lumen across the
apical membrane into the cell through Na+-permeable ion channels. The
second step is active extrusion of Na+ from the cell across the
basolateral membrane into the serosal space. Active amiloride-sensitive
Na+ transport by lung epithelia
has been demonstrated by several investigators (10, 19, 21, 25). Also,
all three subunits,
,
, and
, of the amiloride-sensitive
Na+ channels that have been
described in other
Na+-transporting epithelia have
also been identified in airway epithelial cells (4, 5, 24, 26, 31).
Because of this observation, several investigators (1, 13, 20, 22, 23,
31) have suggested that the epithelial
Na+ channel (ENaC) subunits known
to be responsible for Na+
transport in other epithelial tissues must also be responsible for
Na+ transport in the lung.
Consistent with this idea is the observation by Hummler et al. (13)
that genetically knocking out the
-subunit of ENaC leads to
defective lung liquid clearance and premature death in perinatal mice.
Thus there appears to be direct evidence that, in vivo, the ENaC
constitutes the limiting step for
Na+ absorption in epithelial cells
of, at least, the fetal lung. Furthermore, when observed in other
epithelial tissues or if ENaC subunits are expressed in oocytes, single
ENaCs are low-conductance (4-5 pS), highly sodium-selective
channels. In contrast, most laboratories studying lung ENaCs report
nonselective cation channels [Na+-to-K+
permeability ratio
(PNa/PK) = 1] with a high conductance (10-45 pS) that are quite
different from the highly selective, low-conductance ENaCs. In fetal
distal lung epithelial cells from 20-day-gestation rat fetuses, Orser
et al. (28) observed, using symmetric solutions and inside-out
recordings, single channels with a conductance of 23 ± 1.1 pS and a
PNa/PK
of 0.9. These channels were blocked by amiloride applied to the apical
side of the membrane. Marunaka (18) has described an
Na+-permeable, nonselective cation
channel with a linear current-voltage relationship and a single-channel
conductance of 26.9 ± 0.8 pS in the fetal distal lung epithelium.
Feng et al. (8) and Yue et al. (34) have also recently described
nonselective cation channels in apical cell-attached and inside-out
patches from adult rat alveolar type II (ATII) cells. The channels have
a
PNa/PK of 1, are voltage independent, and are inhibited by amiloride. In
apical membrane patches on adult rat ATII cells in primary culture,
Jain et al. (15) have also observed nonselective cation channels similar in conductance and ion selectivity to the ones described by Orser et al. (28). Thus the predominant
Na+-permeable channels observed
electrophysiologically in both adult and fetal lung cells appear to be
nonselective cation channels (although some details of channel
characteristics appear to differ between preparations; for a review,
see Ref. 20). Although the exact role that these channels play in vivo
is unclear, electrophysiological considerations and studies from
epithelial cells from other organs suggest a role for this channel in
Na+ transport in the lung. For
example, Tohda et al. (30) showed that these channels may play a role
in the increased reabsorption of fluid by alveolar epithelia in
response to
-agonist stimulation.
The apparently conflicting information obtained from
electrophysiological and molecular biological experiments caused us to investigate the relationship of ENaC to the nonselective cation channels in lung epithelial cells. Antisense oligonucleotides targeting
individual subunits were used to manipulate the expression of the
respective subunit proteins, and the patch-clamp technique was used to
characterize the resulting single channels. Our results suggest that
the
-subunit is responsible for the nonselective lung epithelial
cation channels in rat lung epithelia.
 |
METHODS |
Type II pneumocyte isolation and
culture. ATII cells were isolated by
enzymatic digestion of lung tissue from adult Sprague-Dawley rats
(100-250 g) with the use of published techniques (15). Briefly,
the rats were anesthetized with pentobarbital sodium and heparinized
(100 U/kg). ATII cells were digested by tracheal installation of
elastase (0.4 mg/ml). The lung tissue was minced in DNase (1 mg/ml) and
filtered sequentially through 100- and 20-mM nylon mesh. The cells thus
obtained were collected, centrifuged, and seeded onto glass coverslips
(~2 × 105
cells/cm2) in Dulbecco's
modified Eagle's medium-Ham's F-12 medium containing 5% fetal calf
serum and antimicrobial agents and supplemented with
L-glutamine and sodium
bicarbonate. Cells were incubated in 90% air-10%
CO2 and used for patch-clamp
studies between 24 and 96 h after being plated on glass coverslips. No
significant difference in
Na+-channel activity or
characteristics was observed during this time period. Cell viability
(>90%) and ATII cell purity (>95%) has been previously
established in our laboratory (15).
Solutions and drugs. All solutions
were made with deionized water and then passed through a 0.2-µm
filter (Gelman Sciences, Bedford, MA) before use. The bath and pipette
solutions used in the cell-attached mode contained (in mM) 140 NaCl, 1 MgCl2, 1 CaCl2, 5 KCl, and 10 HEPES, pH 7.4 with 2 N NaOH. The contents of the bath and pipette solutions were
varied as appropriate for specific protocols. All chemicals were
obtained from Sigma (St. Louis, MO).
Procedure for single-channel
recordings. Patch-clamp experiments were carried out at
room temperature. The pipettes were pulled from filamented borosilicate
glass capillaries (TW-150, World Precision Instruments) with a
two-stage vertical puller (Narishige, Tokyo, Japan). The pipettes were
coated with Sylgard (Dow Corning) and fire polished (Narishige). The
resistance of these pipettes was 5-8 M
when filled with the
pipette solution. We used the cell-attached configuration for most of
our studies because in this configuration, the cytoplasmic constituents
remain intact, thus allowing us to study the role of cytoplasmic second
messengers in the regulation of ion-channel activity. Inside-out
patches were also used to determine the selectivity of the channel and to determine whether the effects of agents were directly on the channel
or mediated by a signaling cascade. After formation of a
high-resistance seal (>50 G
) between the pipette and the cell membrane, channel currents were sampled at 5 kHz with a patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, CA) and filtered at 1 kHz with an eight-pole, low-pass Bessel filter. Data were
recorded by a computer with pCLAMP 6 software (Axon Instruments).
Current-amplitude histograms were made from stable continuously
recorded data, and the open and closed current levels were determined
from least-squares fitted Gaussian distributions. We used the product
(NPo) of the
number of channels (N) times the
open probability
(Po) as a
measure of the activity of the channels within a patch. This product
could be calculated from the single-channel recording without making
any assumptions about the total N in a
patch or the Po
of a single channel
|
|
where
T is the total recording time,
i is the number of channels open, and
ti is the
recording time during which i channels are open. Current-amplitude histograms provide the clearest
demonstration of multiple current levels. The total
N in a patch was estimated by
observing the number of peaks in a current-amplitude histogram over the
entire duration of the recording period. The
Po of the channels was calculated with FETCHAN in pCLAMP 6 and with locally developed software (17).
RT-PCR and Western blotting.
Semiquantitative RT-PCR of total RNA obtained from rat ATII cells was
performed with subunit-specific primers based on published sequences.
PCR reactions were run in the linear range of amplification, with
glyceraldehyde-3-phosphate dehydrogenase amplified as standard for
comparison. These primers amplified a single fragment of the expected
size for each RNA [579 bp for rat (r)
-ENaC, 548 bp for
-rENaC, and 558 bp for
-rENaC]. The PCR products were not
seen in control reactions without RNA. The sequences of the amplified
fragments are identical to the published sequences (31). The sequences
of the primers are given in Table 1.
To determine whether protein for each of the subunits was present, we
used Western blot experiments to confirm the presence of protein for
all three subunits in ATII cells in primary culture. Polyclonal
antibodies were produced by identifying unique antigenic 20-amino acid
sequences in each subunit. The peptides were produced in Emory's
microchemical facility and sent to Lofstrand Laboratories where the
peptides were coupled to KLH and used to immunize at least two rabbits
with each peptide. This provided at least one anti-peptide antibody
specific for each subunit.
 |
RESULTS |
Characteristics of single nonselective cation channels
in the apical membrane of ATII cells. Jain et al. (15)
and others (18, 28) have previously reported that the predominant
Na+-permeable channel observed in
cell-attached patches on the apical membrane of ATII cells with 140 mM
NaCl in the bath and pipette is a high-conductance (20.6 ± 1.1 pS)
channel (Fig.
1A).
The single-channel current has a linear current-voltage relationship
with no detectable rectification (Fig.
1B). The pipette potential at which
current polarity reversed was estimated to be
37 mV. Because the
resting membrane potential of alveolar epithelium has previously (28) been shown to be approximately
30 to
40 mV, the reversal
potential appears to be close to 0 mV, which would be expected for a
nonselective cation channel.

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Fig. 1.
Characteristics of nonselective cation channel.
A: single-channel recordings of a
nonselective cation channel from a cell-attached patch in alveolar type
II cell. C, closed state. Nos. on right, mV.
B: current-voltage relationship showing that
unit conductance of channel is 20.6 ± 1.1 pS and that
current-voltage relationship is linear, with no detectable
rectification. Vp, membrane potential.
C: amplitude histogram of patch in
A showing that open probability is
~0.2.
|
|
Ion selectivity was determined with excised inside-out patches and
solutions of varying ionic compositions. The channel had a similar
permeability to Na+ and
K+
(PNa/PK = 0.97 ± 0.07; n = 7 patches). The
channel Po was
decreased by amiloride (0.1-1 mM) applied to the extracellular
side (i.e., in the micropipette). The
Po of untreated
channels was 0.31 ± 0.01 vs. 0.03 ± 0.01 after 100 nM amiloride
(P < 0.01;
n = 7 patches). More than one current
level was observed in 85.7% of the active patches.
Functional importance of
Na+-permeable
nonselective cation channels and their relationship to ENaC.
We examined the expression of mRNA for the three subunits of the ENaC
by RT-PCR experiments. Semiquantitative RT-PCR of total RNA obtained
from rat type II cells was performed with subunit-specific primers.
PCRs were run in the linear range of amplification, with glyceraldehyde-3-phosphate dehydrogenase amplified as a standard for
comparison. These primers amplified a single fragment of the expected
size for each RNA (579 bp for
-rENaC, 548 bp for
-rENaC, and 558 bp for
-rENaC; Fig.
2A).
The PCR products were not seen in the control reactions without RNA.
The sequences of the amplified fragments are identical to the published
sequences (31). Thus ATII cells, at least, have an RNA message for all
of the ENaC subunits. To determine whether protein for each of the
subunits was present, we used Western blot experiments to confirm the
presence of protein for all three subunits in ATII cells in primary
culture (Fig. 2B). These results
show that even under conditions when the predominant
Na+-permeable channel is the
nonselective cation channel, there is an easily detectable message for
all ENaC subunits and protein for the
- and
-subunits (we have
yet to perform Western blot experiments for the
-subunit). These
results, of course, in no way prove that the nonselective cation
channel is related to ENaC.

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Fig. 2.
RT-PCR and Western blot demonstrate presence of epithelial
Na+ channel (ENaC) subunits.
A: RT-PCR is capable of amplifying
appropriate size cDNA for each subunit.
B: Western blot demonstrates presence
of - and -subunits. -Subunit has also been demonstrated in
alveolar type II cells by others (21a).
|
|
Antisense oligonucleotides against
-,
-, and
-subunits of ENaC decrease the
corresponding protein. To address directly the
relationship between the nonselective cation channel and ENaC, we used
antisense oligonucleotides directed against the sequences around the
translation start site of each subunit. Cultured cells were exposed to
oligonucleotides overnight. Using quantitative densitometry of Western
blots, we found that in cells treated overnight with antisense
oligonucleotides, the corresponding protein was more than twofold
lower. Figure 3 shows results for the
- and
-subunits. We also used RT-PCR to examine the mRNA levels after
oligonucleotide treatment. Contrary to our expectations, we found that
mRNA levels for the subunits were unchanged or even slightly increased
after oligonucleotide treatment. We believe that this represents
transcriptional control of ENaC mRNA in response to reduced
Na+ transport as described for
several other gene products (11, 12, 16).

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Fig. 3.
Antisense oligonucleotide induced inhibition of ENaC subunit
expression. Top: Western blot analysis
(n = 1) for -subunit
(A) and -subunit
(B) of ENaC in rat alveolar type II
cells treated with sense and antisense oligonucleotides.
Bottom: densitometric estimation of
protein concentration (conc) for corresponding subunit. Expression of
all subunits was significantly reduced by treatment with antisense
oligonucleotides.
|
|
Antisense oligonucleotide against
-subunit of ENaC decreases lung epithelial
cation-channel activity. Because antisense
oligonucleotides reduce the expression of ENaC subunit proteins, we
also determined the effects of antisense oligonucleotides on the
expression of cation channels by using the patch-clamp technique to
measure single-channel activity in ATII cells treated with
oligonucleotides. Cultured ATII cells were exposed to oligonucleotides
for 24 h. Addition of the antisense oligonucleotide to the
-subunit
to the culture medium produced a marked reduction in the number of apical membrane cell-attached patches with cation channels (Fig. 4) compared with those exposed to the sense
oligonucleotide as a control. In 62 patches on cells that had been
treated with sense oligonucleotides, 69.4% of the patches had one or
more nonselective cation channels; in 63 patches treated with antisense
oligonucleotides, only 34.9% of the patches contained a nonselective
cation channel (with the z-test for
statistical ratios, the probability of observing such different
frequencies is <0.0001). On the other hand, similar experiments with
antisense oligonucleotides targeting the
- or
-subunits did not
alter the channel number. There were no significant changes in
Po or other
channel characteristics
[Po of
channels from
-sense-treated cells = 0.183 ± 0.03 (n = 42 patches) vs. 0.257 ± 0.05 (n = 21 patches) in
-antisense-treated cells;
Po of channels
from
-sense-treated cells = 0.239 ± 0.037 (n = 30 patches) vs. 0.227 ± 0.045 (n = 29 patches) in
-antisense-treated cells;
Po of channels
from
-sense-treated cells = 0.148 ± 0.02 (n = 40 patches) vs. 0.141 ± 0.03 (n = 34 patches) in
-antisense-treated cells; none of the treatments produced a
significant difference]. We thought that the
- or
-subunit
might substitute for each other in formation of the cation channel so
we simultaneously inhibited both the
- and
-subunits with
antisense oligonucleotides. This treatment also did not affect the
level of expression and functional characteristics of the channels.

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Fig. 4.
Treatment with antisense oligonucleotide directed against -subunit
of ENaC produced a significant reduction in number of apical membrane
cell-attached patches with cation channels. Addition of either - or
-antisense oligonucleotide separately or together or sense
oligonucleotide for any of the subunits (as a control) did not change
cation-channel activity.
|
|
These results suggest that the lung epithelial cation channel belongs
to the ENaC family of Na+ channels
and is formed from the
-subunit protein alone or the
-subunit
with some hitherto unidentified subunits other than either the
- or
-subunit.
 |
DISCUSSION |
Whole tissue experiments on a variety of epithelial tissues including
the lung imply a simple picture of
Na+ channels. This picture is
consistent with tissue reabsorption of
Na+ and, at a single-channel
level, implies that the channels should be highly selective for
Na+ over
K+, should open and close
infrequently, and should be blocked by low concentrations of the drug
amiloride (2). Indeed, channels with such properties can be observed in
several epithelial preparations (9), but examination of single channels
in several Na+-transporting
epithelial cells suggests a more complicated picture of
amiloride-blockable channels. Single-channel studies (3, 20)
have identified at least four different
amiloride-blockable channels with either high selectivity, low
selectivity, or no selectivity for
Na+ over
K+ in the apical membranes of a
variety of cultured and native epithelial cells. These channels differ
in their ion selectivity, unitary conductance, and other
characteristics, but they all have been proposed to play some role in
Na+ absorption. Benos et al. (3)
recently compiled and plotted selectivity versus conductance of
epithelial Na+ channels from the
published literature and showed the wide variability in these
characteristics. Few of these reported channels match the
characteristics of the prototypical native amiloride-sensitive Na+ channel that has a low
conductance (4-6 pS) and high selectivity for
Na+
(PNa/PK > 10). In fact, only one study (6) has reported a channel fitting
this description from the lung. In this study, the investigators
recorded a 4-pS amiloride-sensitive channel from outside-out patches
excised from apical membrane. The channel was highly selective for
Na+
(PNa/PK > 10). However, no other laboratory has subsequently reported
observing this channel in lung epithelia on a consistent basis. For
epithelial cells in culture, culture conditions are apparently the
primary determinant of the frequency with which these different channel
types are found in the apical membrane (7, 33), but ultimately, the
observed variations in single-channel properties must be due to
variations in subunit stoichiometry, variations in subunit types
combining to form channels, or posttranslational modifications of the
subunit proteins.
Molecular basis for Na+ transport in lung
epithelial cells.
The differences in the properties of
Na+-permeable channels make a
determination of the molecular basis for
Na+ transport an important issue.
Several investigators and experiments with transgenic
animals (1, 13, 20, 21, 22, 23) have suggested that
-,
-, and
-ENaC subunits known to be responsible for
Na+ transport in other epithelial
tissues are also responsible for Na+ transport in the lung. This
would be consistent with the observation that rat adult and fetal lungs
have been shown to express all three subunits of the ENaC (13).
However, electrophysiological measurements suggest that if ATII cells
do express ENaCs, then the functional characteristics are quite
different from the highly selective, low-conductance (4-5 pS)
ENaCs observed in other tissues.
In this study, we have used antisense oligonucleotides targeting each
of the three subunits to manipulate the expression of the respective
protein and the patch-clamp technique to study the changes resulting
from these manipulations in single-channel characteristics. Our results
support the hypothesis that nonselective lung epithelial cation
channels are primarily composed of the
-subunit. This is the first
evidence to show that nonselective cation channels belong to the ENaC
family of channels. The different cation channels may represent
different forms of ENaC, with at least one protein (
-subunit) in
common. This conclusion is consistent with the work of Hummler et al.
(13), who showed that inactivating the mouse
-ENaC
gene leads to early death of newborns due to defective neonatal lung
liquid clearance.
-ENaC knockout completely abolished
amiloride-sensitive Na+ transport
in the airway epithelia, resulting in respiratory distress in neonates
and death within 40 h of birth. Furthermore, Canessa et al. (5) and
Ismailov et al. (14) have shown that the
-subunit alone can form
fully functional amiloride-sensitive
Na+ channels, whereas the
- and
-subunits individually or in combination cannot. In fact, Kizer et
al. (16a) have recently shown that expression of the
-subunit of the ENaCs from osteoblasts into a null cell line (LM
TK
) resulted in a nonselective cation channel
(PNa/PK = 1.1 ± 0.1) with a conductance of 24.2 ± 1.0 pS. These results are consistent with our findings that inhibition of the
-subunit reduces cation-channel number, whereas inhibition of the
- and
-subunits does not.
In summary, our results support the hypothesis that expression of the
-subunit of ENaC alone or in combination with some protein other
than the
- or
-subunit protein is the major component of lung
epithelial cation channels.
 |
ACKNOWLEDGEMENTS |
This work was supported by American Lung Association Grant RG-133-N
(to L. Jain), National Institute of Diabetes and Digestive and Kidney
Diseases Grants R01-DK-37963 and P05-DK-50268 (to D. C. Eaton), and the
Center for Cell and Molecular Signaling (Emory University School of
Medicine, Atlanta, GA).
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: L. Jain, Dept.
of Pediatrics, Emory Univ. School of Medicine, 2040 Ridgewood Dr., NE,
Atlanta, GA 30322 (E-mail: ljain{at}emory.edu).
Received 13 January 1999; accepted in final form 11 March 1999.
 |
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