Regulation of the epithelial
Na+ channel by intracellular
Na+
Mouhamed S.
Awayda
Department of Medicine and Department of Physiology, Tulane
University School of Medicine, New Orleans, Louisiana 70112
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ABSTRACT |
The hypothesis that the intracellular
Na+ concentration
([Na+]i)
is a regulator of the epithelial
Na+ channel (ENaC) was tested with
the Xenopus oocyte expression system
by utilizing a dual-electrode voltage clamp.
[Na+]i
averaged 48.1 ± 2.2 meq (n = 27)
and was estimated from the amiloride-sensitive reversal potential.
[Na+]i
was increased by direct injection of 27.6 nl of 0.25 or 0.5 M
Na2SO4.
Within minutes of injection,
[Na+]i
stabilized and remained elevated at 97.8 ± 6.5 meq
(n = 9) and 64.9 ± 4.4 (n = 5) meq 30 min after the
initial injection of 0.5 and 0.25 M
Na2SO4,
respectively. This increase of
[Na+]i
caused a biphasic inhibition of ENaC currents. In oocytes injected with
0.5 M
Na2SO4
(n = 9), a rapid decrease of inward
amiloride-sensitive slope conductance
(gNa) to 0.681 ± 0.030 of control within the first 3 min and a secondary, slower
decrease to 0.304 ± 0.043 of control at 30 min were observed.
Similar but smaller inhibitions were also observed with the injection
of 0.25 M
Na2SO4.
Injection of isotonic
K2SO4
(70 mM) or isotonic
K2SO4
made hypertonic with sucrose (70 mM
K2SO4-1.2
M sucrose) was without effect. Injection of a 0.5 M concentration of
either
K2SO4,
N-methyl-D-glucamine (NMDG) sulfate, or 0.75 M NMDG gluconate resulted in a much smaller initial inhibition (<14%) and little or no secondary decrease. Thus
increases of
[Na+]i
have multiple specific inhibitory effects on ENaC that can be
temporally separated into a rapid phase that was complete within 2-3 min and a delayed slow phase that was observed between 5 and 30 min.
epithelial sodium channel; Xenopus
oocytes; inhibition; autoregulation; intracellular sodium concentration
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INTRODUCTION |
IT HAS LONG BEEN RECOGNIZED that macroscopic rates of
Na+ transport across epithelia
display saturation kinetics with increasing extracellular
Na+ concentration
([Na+]o)
and that inhibition of Na+ entry
by various means causes a stimulation of channel activity (12,
22). Because the single-channel current saturates at much
higher [Na+] than the
macroscopic current, it is inferred that intrinsic regulatory processes
that cause inhibition of the open probability and/or channel density
must exist (29, 33). Lindemann (19) classified these types of intrinsic
regulation of the epithelial Na+
channel into self-inhibition and feedback inhibition. Self-inhibition is thought to reflect direct interaction between the channel and external Na+. On the other hand,
feedback inhibition or autoregulation may be mediated via the indirect
actions of Na+ and second
messengers on the Na+ channel
(32).
Fuchs et al. (9) provided the initial evidence for intrinsic regulation
of the Na+ channel by luminal
[Na+] in the
short-circuited, K+-depolarized
epithelium of frog skin. They found that the apical membrane
Na+ permeability
(PNa) was
inhibited within seconds after increasing apical
[Na+]. These effects
were observed in the absence of detectable changes of membrane voltage
(Vm) and
presumably intracellular Na+
concentration
([Na+]i)
and were attributed to self-inhibition of the apical
Na+ channel by
[Na+]o.
Kroll et al. (17) arrived at a similar conclusion for the epithelial
Na+ channel expressed in
Xenopus oocytes, where
PNa was found to inversely vary with
[Na+]o,
with no apparent correlation with
[Na+]i.
Palmer et al. (24) arrived at a similar conclusion by utilizing a whole
cell patch clamp of rat cortical collecting tubules (CCT). These
investigators found that decreasing
[Na+]i
by decreasing pipette
[Na+] by substitution
with K+ did not affect the whole
cell currents. However, changes of
[Na+]o
were found to be accompanied by changes of channel activity, indicating
that extracellular Na+ is
responsible for inhibiting the Na+ channel.
Another intrinsic regulatory process of feedback inhibition or
autoregulation is observed after inhibition of apical membrane Na+ entry (1, 6, 7, 11, 20, 27,
28). This process exhibits a longer time course than self-inhibition
and is thought to be mediated via second messengers that may involve
protein kinase C (PKC) (10, 20) and potential interactions with the actin cytoskeleton (6). Data from Komwatana et al. (16) also indicate
that
[Na+]i
inhibited channel activity via an indirect mechanism that involves G
proteins. Unfortunately a time course of the effect of
[Na+]i
on channel activity could not be obtained because these studies were
carried out by using the whole cell patch clamp mode on cells dialyzed
with the pipette contents. In a follow-up study these authors (4)
concluded that the regulation of the
Na+ channel in salivary glands by
[Na+]i
also involves the ubiquitin ligase protein Nedd4.
Data indicating that the cloned epithelial
Na+ channel (ENaC) is also
regulated by Na+ have been
recently accumulating. Ishikawa et al. (14) have reported that ENaC
transfected into Madin-Darby canine kidney cells is inhibited by
increasing
[Na+]i
in excised inside-out membrane patches, indicating a likely direct
effect of
[Na+]i
on this channel. Kellenberger et al. (15) have also reported that ENaC
expressed in Xenopus oocytes is
inhibited by increasing [Na+]i.
These investigators increased
[Na+]i
by increasing
[Na+]o
and estimated the magnitude of
[Na+]i
from the membrane reversal potentials. However, their methods could not
completely differentiate the effects of
[Na+]o
and
[Na+]i
on gNa.
Nevertheless, these reports indicate that ENaC is inhibited by
Na+ and that this is likely a
property of the cloned channel itself.
In this study the regulation of ENaC expressed in
Xenopus oocytes by
[Na+]i
was examined to determine whether changes of
[Na+]i
in the absence of changes of
[Na+]o
can affect this channel in intact cells. The effect of a general increase of intracellular ions on ENaC activity was also assessed. Experiments were carried out with the 

-subunit of rat
ENaC-expressing oocytes voltage clamped to 0 mV to
eliminate the effects of changing Vm
subsequent to altering
[Na+]i.
[Na+]i
was increased by direct injection of
Na+ into intact oocytes, thus
circumventing issues of loss of cell signaling by cell dialysis and
further avoiding changes of
[Na+]o.
Increases of
[Na+]i
caused a biphasic inhibition of ENaC. A rapid initial phase was
observed within seconds and is consistent with a direct inhibition of
ENaC by
[Na+]i.
A secondary and slower inhibition that continued to 30 min was also
observed during a phase in which the
[Na+]i
was elevated but constant. Injection of hypertonic nonionic solutions
did not affect ENaC activity, whereas injection of ionic solutions that
do not contain Na+ (cations or
anions) caused a much smaller rapid inhibition in the absence of an
appreciable secondary inhibition at 30 min.
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MATERIALS AND METHODS |
Oocyte isolation and injection.
Toads were obtained from Xenopus Express (Beverly Hills, FL) and were
kept in dechlorinated tap water at 18°C. Conditions for oocyte
removal, processing, injection, and cRNA synthesis were as previously
described (3). Injected oocytes were incubated at 18°C for 1-3
days until the recordings were made. All recordings were performed at
19-21°C.
Two procedures were utilized for the direct injection of solutions. In
the first, oocytes were impaled with the injecting electrode at the
beginning of the experiment and the injections were followed by
impalement of the oocytes with the two recording microelectrodes as
previously described (3). A small air bubble was initially drawn into
the tip of the electrode to minimize leakage of its contents into the
oocyte cytoplasm. In the second, oocytes were impaled with the
injecting electrode immediately before the injection of its contents.
No differences between the results of these two procedures were found.
All injections were limited to 27.6 nl to avoid membrane disruption.
Some oocytes that were injected with the 0.5 M salt swelled and lysed
within the 30-min experimental period. This lysis phenomenon was
previously described in connection with volume injections of ~180 nl
into ENaC-expressing oocytes and a >33% decrease of external
solution osmolarity (3). To circumvent this problem, the external
perfusion solution was changed during the initial 5 min after injection
to one that also contained 50 or 75 mM sucrose. Furthermore, each
oocyte was visually inspected to determine its volume status. A small
degree of cell swelling was preferred because it assured that there was
no small but undetectable cell shrinking. This was an important
criterion because cell swelling is without immediate or long-term
effects on ENaC in contrast to cell shrinking, which causes a slow
inhibition of gNa
(3).
Solutions and chemicals.
All solutions and chemicals were as described by Awayda and Subramanyam
(3). Amiloride was a gift from Merck Sharp & Dohme (Rahway, NJ). All
other chemicals were of the highest grade and were obtained from Sigma
Chemical (St. Louis, MO). Solution
Na+ content was measured with a
flame photometer (model 443; Instrumentation Laboratory, Watertown, MA).
Dual-electrode clamp.
Whole cell currents were recorded and analyzed as described by Awayda
and Subramanyam (3) with a TEV-200 two-electrode voltage clamp (Dagan
Instrument, Minneapolis, MN). In most experiments the bath was perfused
with solution at the rate of 6 ml/min, or ~4 chamber volumes/min. In
some experiments a smaller volume chamber that allowed an exchange rate
of ~12 chamber volumes/min was used. No differences between the
results of experiments in either chamber were detected. Values for
gNa were
calculated from the amiloride-sensitive current-voltage
(I-V) relationship between
100 and
80 mV as described by Awayda et al. (2). By
convention the inward flow of cations is designated as inward current
(negative current), and all voltages are reported with respect to
ground or bath. Except where noted, all data are reported as means ± SE.
Intracellular Na+ activity was
estimated from the fit of the I-V
relationship to the Goldman equation (13):
INa = PNa · F ·
· A · ([Na]i
[Na]o · e
)/(1
e
)
where
= Vm · F/R · T,
and F, R, T and A are Faraday's
number, gas constant, absolute temperature, and area, respectively.
An activity coefficient for Na+ of
0.778 was used (18). Data were fit in the voltage range of
100
to
20 or
40 mV. Data were fit by using the least-squares
minimization fitting subroutine in SigmaPlot (Jandel Scientific, San
Rafael, CA); an oocyte membrane area of 0.15 cm2 was assumed (16).
Statistical analysis was carried out by using the paired Student
t-test where appropriate. Significance
was determined at the 95% confidence level
(P < 0.05).
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RESULTS |
Effects of increasing
[Na+]i.
ENaC-expressing oocytes were incubated in media that contained 81 mM
[Na+]o,
and the whole cell currents were recorded 1-3 days later in Ringer
containing 100 mM Na+.
[Na+]i,
current at
100 mV,
gNa, and
PNa averaged 48.1 ± 2.2 meq,
2,583 ± 204 nA, 27.6 ± 2.3 µS, and
0.446 ± 0.038 × 10
6 cm/s
(n = 27), respectively.
PNa was
calculated from the amiloride-sensitive I-V relationship as described in
MATERIALS AND METHODS (also see below).
A representative effect of injecting 0.5 M
Na2SO4
on the whole cell current is shown in Fig.
1. Within 2 min, an inhibition of current was observed and
gNa decreased
from 45.2 to 37.1 µS (Fig. 1, A and
B). This rapid inhibition was
followed by a secondary inhibition that caused a decrease of the
gNa to 21.7 µS
at 30 min (Fig. 1C). The remaining
amiloride-insensitive current is shown in Fig.
1D and exhibited a conductance of 1.0 µS.

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Fig. 1.
Whole cell currents
(Im) in an
epithelial Na+ channel
(ENaC)-expressing oocyte and changes of
Im after a rapid
increase of intracellular Na+
concentration
([Na+]i).
A:
Im in an oocyte
preincubated in 81 mM solution [0.5× L-15 (Sigma)], and
recorded in high Na+ (100 mM
[Na+]o).
B: rapid inhibition is observed within
2 min of injection of 27.6 nl of 0.5 M
Na2SO4.
(C) Im were
further inhibited 30 min after increase of
[Na+]i.
The amiloride (10 µM)-insensitive
Im are shown in
D. Note the shift in reversal
potential after increase of
[Na+]i.
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The effect of 0.5 M
Na2SO4
injection on the amiloride-sensitive
I-V relationship is shown in Fig.
2. The current data were summarized from
the average of five points at the end of each voltage episode. The
solid line represents the fit to the Goldman equation as described in
MATERIALS AND METHODS. As expected
with Goldman-type rectification (13), the
I-V relationship is inwardly rectified
under conditions of higher
[Na+]o
than
[Na+]i.
The injection of
Na2SO4
increased
[Na+]i
from 52 to 88 meq and decreased
PNa from 0.69 × 10
6 to 0.47 × 10
6 cm/s (Fig. 2,
A and
B). The
[Na+]i
at 30 min was essentially the same as that at 2 min; however PNa decreased to
0.30 × 10
6 cm/s (Fig.
2C). Consistent with Goldman-type
rectification, the increase of
[Na+]i
beyond
[Na+]o
was accompanied by a change of the I-V
relationship from one that exhibited inward rectification to one that
exhibited outward rectification.

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Fig. 2.
Amiloride-sensitive current-voltage
(I-V) relationship for an
ENaC-expressing oocyte, and changes of this relationship after rapid
increase of
[Na+]i.
Amiloride-sensitive currents were obtained after subtraction of
amiloride-insensitive components obtained at either beginning or end of
experiment (no appreciable differences in these amiloride-insensitive
currents were detected). The I-V
values were fit to the Goldman equation as described in
MATERIALS AND METHODS.
A:
[Na+]i
and membrane Na+ permeability
(PNa) were 52 meq and 0.69 × 10 6
cm/s, respectively, under control high-Na+ solution-bathed
conditions. Injection of 27.6 nl of 0.5 M
Na2SO4
decreased PNa to
0.47 × 10 6 cm/s
within 2 min (B) and to 0.30 × 10 6 cm/s at 30 min
(C). These changes of
PNa were
accompanied by an increase of
[Na+]i
from 52 to 88 meq at 2 min and to 90 mM at 30 min. Data were fit in
voltage range of 100 to 20 mV.
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Summarized in Table 1 are the
effects of intracellular Na+
injection on ENaC. Within 2 min of injecting 0.5 M
Na2SO4,
[Na+]i
was elevated from 52.1 ± 2.6 to 98.9 ± 4.5 meq
(n = 9). This increase of
[Na+]i
was accompanied by a 29.5 ± 3.8% decrease of
gNa and a 34.8 ± 3.1% decrease of
PNa. The
[Na+]i
at 30 min remained elevated at 97.8 ± 6.5 meq and was not different from the value observed at 2 min. Despite the absence of further increases of
[Na+]i,
gNa and
PNa decreased by
67.3 and 66.9% of control, respectively, at 30 min. This
indicated the presence of an additional slower effect of elevating
[Na+]i
on ENaC. Two inhibitory phases were also observed with the injection of
27.6 nl of 0.25 M
Na2SO4.
However, as expected this injection resulted in smaller increases of
[Na+]i
and was accompanied by smaller initial and secondary decreases of
gNa and
PNa (see Table
1).
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Table 1.
Changes of [Na+]i and ENaC activity
subsequent to intracellular Na2SO4
injection in oocytes in 100 mM [Na+]o
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To determine the dynamic effects of increasing
[Na+]i
on ENaC, the time course of changes of
gNa with
Na+ injection was examined. As
shown in Fig. 3, the response of
gNa was biphasic
and consisted of a rapid inhibition by ~31.9% within 3 min, followed
by a slower secondary inhibition by ~69.6% at 30 min. The initial
rapid inhibition was nearly complete within 2-5 min, and the
second inhibitory phase began thereafter. The effects of injecting 27.6 nl of 0.25 M
Na2SO4
were qualitatively similar, and
gNa decreased by
a smaller amount at both 3 and 30 min. It should be noted that ENaC is
not directly sensitive to mechanical perturbations in oocytes, and
therefore these effects cannot be attributed to the injected volume.
This volume (27.6 nl) is well below that which was found to cause
mechanical membrane disruption, and, additionally, the injection of
isotonic KCl at volumes <180 nl does not cause time-dependent changes
of ENaC conductance (3).

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Fig. 3.
Time courses of inhibition of slope conductance
(gNa) by intracellular
Na+. Data are summarized as
changes of gNa
(calculated between 100 and 80 mV) and are normalized to
value of gNa
immediately before injection of
Na2SO4.
Two concentrations of
Na2SO4
were injected: 0.5 M ( ; n = 9) and
0.25 M ( ; n = 5). In both cases,
rapid inhibition, which reached a relative plateau within ~2-5
min, was observed. This was followed by a secondary and much slower
inhibitory phase, which continued to 30 min. All data points with
exception of 10- and 20-s points in 0.5 M group and 0.5-min point in
0.25 M group were significantly different from control. E/C,
experimental value divided by control value;
[Na+]o,
extracellular Na+ concentration.
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The changes of currents observed in these experiments are specifically
due to changes of the
gNa because there
were no effects of injecting 0.5 M
Na2SO4
in ENaC-expressing oocytes treated with a saturating concentration of
amiloride (10 µM) or in control oocytes (see Fig.
4). Thus this injection
procedure does not cause any appreciable changes of endogenous or
amiloride-insensitive currents. Moreover, the injection of isotonic
K2SO4
(70 mM) did not affect ENaC's conductance (Fig. 4), and
gNa was 101.1 ± 1.5 and 99.1 ± 2.8% of control
(n = 3) at 3 and 30 min,
respectively. The injection of isotonic
K2SO4
made hypertonic with sucrose (70 mM
K2SO4-1.2
M sucrose) was also without effect, and
gNa was 103.3 ± 2.0 and 103.2 ± 4.8% of control
(n = 6) at 3 and 30 min, respectively (Fig. 4). Thus these data rule out any nonspecific-injection- or
time-dependent changes of
gNa.

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Fig. 4.
Lack of nonspecific effects of
Na2SO4
injection on ENaC currents. Time courses of changes of whole cell
gNa after
injection of
Na2SO4,
isotonic
K2SO4,
or hypertonic sucrose are shown. Data are summarized as changes of
gNa from control
or time 0 point. ENaC-expressing
oocytes treated with 10 µM amiloride were not affected by injection
of
Na2SO4
( ; n = 5), indicating that
amiloride-insensitive current does not appreciably change during
experiment. Control oocytes were also insensitive to
Na2SO4
injection ( ; n = 10), indicating
lack of effect on background currents. Injection of isotonic
K2SO4
( ; n = 3) was without effect on
ENaC currents, and so was injection of isotonic
K2SO4
made hypertonic with sucrose ( ; n = 6).
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The time course of changes of
[Na+]i
in
high-[Na+]i
oocytes is summarized in Fig.
5. As shown, the
[Na+]i
in oocytes injected with 0.5 M
Na2SO4
began changing within 10 s and continued to increase to a maximal value
in 2-5 min. These values were essentially stable between 5 and 30 min. A small, statistically insignificant decrease of
[Na+]i
between the 5- and 30-min time points
(P > 0.185) was observed. Similar
results for the time course of changes of
[Na+]i
in oocytes injected with 0.25 M
Na2SO4
were observed.

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Fig. 5.
Time courses of changes of
[Na+]i
in oocytes injected with 0.5 M ( ) and 0.25 M ( )
Na2SO4.
Data are summarized as changes of
[Na+]i
from control values immediately before injection of
Na2SO4.
All data were calculated from Goldman equation as described in
MATERIALS AND METHODS, by using same
oocytes used for Fig. 3. With both concentrations of
Na2SO4,
a rapid increase of
[Na+]i,
which reached a relative plateau within ~5 min, is observed. Values
were unchanged up to 30 min (n = 8).
See Fig. 3 legend for more detail.
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As seen from Figs. 3 and 5, the time course of the changes of
[Na+]i
and that of changes of
gNa correlate
well within the first 5 min when the rapid inhibition is observed. This
inhibition was followed by a secondary, slower inhibition that
continued to 30 min in the absence of changes of
[Na+]i.
This secondary decrease and the initial inhibition exhibited a
concentration dependence because they were larger in oocytes injected
with 0.5 M
Na2SO4.
These results provide strong evidence for immediate rapid and delayed
long-term concentration-dependent inhibitions of ENaC by intracellular
Na+.
The finding that injection of 0.5 M
Na2SO4
causes larger inhibition than injection of 0.25 M
Na2SO4
during both the rapid and slow inhibitory phases is consistent with a
dose dependency between the
[Na+]i
and ENaC activity. Thus it is expected that smaller increases of
[Na+]i
may also produce inhibition. To better understand this relationship, the conductance and permeability data obtained in the first 3 min were
plotted against the changes of
[Na+]i.
As shown in Fig.
6A, an
inverse linear relationship between the changes of
gNa and the
changes of
[Na+]i
is observed. The regression line exhibited a slope of
5.56 and a
y-intercept of 1.016, indicating no
self-inhibition of the gNa in the
absence of changes of
[Na+]i.
This is similar to the relationship observed when plotting the average
change of
[Na+]i
and the corresponding percent change of
gNa within the
first 5 min of injecting 0.5 or 0.25 M
Na2SO4
(Fig. 6B;
y-intercept of 1.025 and slope of
6.27). Moreover, as shown in Fig.
6C, an inverse relationship for the
PNa was also
observed, indicating decreased permeability with increased
[Na+]i.
This relationship exhibited a slope of
7.36 and also indicated no feedback inhibition in the absence of changes of
[Na+]i
because the y-intercept was 1.034. Thus these data provide evidence for a dose dependence between
gNa and
[Na+]i
during the initial rapid phase and indicate that small changes of
[Na+]i
could result in rapid inhibition of ENaC. It is important to point out
that it is not necessary to observe large changes of ENaC activity in
native tissues such as the CCT for this phenomenon to be
an important regulator of Na+
transport, because a change of just a few percentage points in ENaC
activity can have significant effects on body
Na+ homeostasis.

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Fig. 6.
Relationship between changes of
[Na+]i
in oocytes in high
[Na+]o
(100 mM) and observed self-inhibition-induced changes of
gNa and
PNa. Data are
obtained from first 3 min after injection of
Na2SO4
from experiments summarized in legend for Fig. 3.
A: inverse relationship between
changes of
[Na+]i
and gNa.
B: similar relationship between mean
changes of
[Na+]i
and gNa in
oocytes injected with 0.5 or 0.25 M
Na2SO4.
C: inverse relationship for changes of
[Na+]i
and PNa. Solid
line, linear regression fit. See text for more detail.
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Effects of increasing the intracellular concentration of poorly
permeant ions.
To determine whether the above intrinsic regulatory processes are
selective for ions that permeate ENaC, oocytes were injected with 27.6 nl of 0.5 M
K2SO4
or N-methyl-D-glucamine (NMDG) sulfate. As
expected from a membrane that predominantly contains
Na+ channels that are also highly
selective for Na+ over
K+ and
NMDG+, there was little effect of
this procedure on the membrane reversal potential (data not shown). The
time courses of the effect on gNa in these two
groups of oocytes are shown in Fig. 7.
Injection of either solution caused a rapid inhibition of
gNa similar to that observed with the injection of
Na2SO4.
However, this inhibition was clearly much smaller than that observed
with the injection of 0.5 M
Na2SO4.
Moreover, the gNa
was essentially constant after 5 min, and the primary response was not
followed by any significant secondary response. Thus
injection of Na+ causes additional
inhibition that is not observed with injection of other salts.

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Fig. 7.
Time courses of effects of increasing intracellular concentration of
impermeant ions on
gNa. Injection of
0.5 M
K2SO4
(n = 8), 0.5 M NMDG sulfate
(n = 8), or 0.75 M NMDG gluconate
(n = 4) caused a small initial
inhibition similar in time course to that observed with injection of
Na2SO4.
In all 3 groups, secondary inhibition at 30 min was either slightly
smaller or statistically not different from that at 5 min. Data from
oocytes injected with 0.5 M
Na2SO4
are same as those summarized in Fig. 3 and are shown for comparison
purposes.
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Injection of solution containing NMDG gluconate resulted in an
inhibition similar to that observed with NMDG sulfate,
indicating the lack of any appreciable effects of the injected anion on
ENaC activity. In combination with the observation that injection of hypertonic sucrose does not cause inhibition (see above), these results
indicate a small but significant effect of increasing the intracellular
concentration of impermeant ions. The reason for this finding is
unknown but may be attributed to a crowding effect whereby these ions
shield the Na+ from interacting
with ENaC.
 |
DISCUSSION |
The Xenopus oocyte expression system
was utilized for its ability to reproduce the electrophysiological
properties of the epithelial Na+
channel to study the regulation of ENaC by
[Na+]i.
In this system the changes of
[Na+]i
are separated from the effects on
Vm as oocytes are
voltage clamped. A holding voltage of 0 mV was used, and this voltage is close to that observed across the apical membranes of many open-circuited Na+-transporting
epithelia. In this system the
[Na+]i
could also be changed by direct injection of solutions in the absence
of changes of
[Na+]o.
I report that rapid increases of
[Na+]i
by direct intracellular injection of
Na2SO4
caused a biphasic inhibition of ENaC consisting of rapid and slow
phases. The rapid phase was completed within 2-3 min. During this
phase the gNa changed almost instantaneously with changes of
[Na+]i.
This observation is consistent with a direct interaction of intracellular Na+ with ENaC. The
second phase was slower in its time course and continued to 30 min
after the increase of
[Na+]i.
This phase is consistent with an indirect mechanism because the
gNa was found to
decrease in the absence of additional changes of
[Na+]i.
Relationship to physiological levels of
[Na+]i.
The spontaneous intracellular Na+
activity in the bulk cytoplasm in many
Na+-transporting epithelia is in
the range of 10-30 meq (21, 26, 30). Thus the baseline
[Na+]i
encountered in the present study may represent a supraphysiological concentration. Moreover, the largest increase of
[Na+]i
in this study (~47 meq) may also represent a supraphysiological increase. However, it is important to point out that inhibition was
also observed in the group injected with 0.25 M
Na2SO4,
in which
[Na+]i
increased by <20 meq. Moreover, the inverse relationships observed in
Fig. 6 indicate that smaller changes of
[Na+]i
will likely result in small but significant changes of
gNa or
PNa. This was
also observed for both groups injected with
Na2SO4 at time points shorter than 3 min, in which the
[Na+]i
was increased by small amounts but was nevertheless accompanied by
significant changes of
gNa.
It is important to recognize that the activities measured by
intracellular microelectrodes represent those found in the bulk cytoplasm and are not those present in the subapical membrane space at
the inner mouth of the channel, whereas those calculated from reversal
potentials are more representative of the activities in the submembrane
space and may not reflect the bulk cytoplasmic concentrations. It is
also important to consider that although microelectrode studies report
a low bulk
[Na+]i,
these values are dependent on the activity of transporters in both the
apical and basolateral membranes. For example, an [Na+]i
of 14 meq in frog skin is reported to increase to 66 meq after inhibition of the basolateral pump by ouabain (29).
An additional caution against a direct comparison between the
activities in the present study and those obtained from cytoplasmic measurements in epithelia is the presence of a standing voltage across
the apical membranes
(Va) of these
ENaC-containing epithelia. This
Va is dependent
not only on the apical membrane permeability to
Na+ but also on whether the
epithelium is studied in the open circuit or short circuit modes and in
high- or
low-[Na+]o
Ringer solution. In the short circuit mode, taking account of the
example of frog skin epithelia, the apical membrane is clamped to a Va
in the range of its intracellular voltage of
60 to
80 mV
(8). In combination with an
[Na+]o
of ~110 meq, it is clear that the equilibrium activity at the inner
mouth of the channel could exceed 1,100 meq! Because the membrane is
not at equilibrium, as evident from the presence of a short circuit
current, the
[Na+]i
at the subapical space is not 1,100 meq; however, it is also unlikely
to be 10 meq. A similar situation applies in the open circuit mode, in
which Va is in
the range of 0 to
20 mV (5, 30, 31) and is very close to the
Thevenin equilibrium potential. At an
[Na+]o
of 110 meq, the subapical
[Na+]i
near the inner mouth of the channel is expected to be ~50 meq or higher.
Possible origin of these inhibitory phases.
The initial inhibitory phase is a rapid process and is consistent with
a direct effect of
[Na+]i
on ENaC. Indeed the onset of this response (within 10-20 s) is in
the range of the delay expected if one assumed a free-diffusion rate of
~50 µM/s. The effects of injecting second messenger proteins such
as protein kinase A or PKC into oocytes expressing ENaC or cystic
fibrosis transmembrane conductance regulator are delayed by 1-2
min and are also prolonged, such that a plateau is not observed until
30-60 min after injection (M. S. Awayda, unpublished observation).
Moreover, at any one instant during the initial response the changes of
reversal potential, and presumably the increases of
[Na+]i,
correlate well with the decrease of
gNa. Although the
involvement of a rapid second messenger system cannot be ruled out, the
observed changes during this phase are consistent with a direct effect of Na+ on ENaC (see Figs. 3, 5,
and 6).
On the other hand, the observed secondary inhibition is unlikely to be
directly caused by Na+, because
changes of gNa
and PNa are
observed during this phase in the absence of further changes of
[Na+]i.
Thus this phase may be consistent with one that involves a second
messenger system such as PKC (10, 20) or Nedd4 (4). It should be
emphasized that Na+-dependent
feedback regulation of ENaC by ubiquitination and that by PKC are not
necessarily mutually exclusive because Nedd4, which interacts with
ENaC, also possesses Ca2+ and
phospholipid binding sites in addition to its ubiquitin ligase site
(25) and is likely activated by
Ca2+ and phospholipids, similar to PKC.
Intracellular vs. extracellular
Na+.
The present experiments were designed to increase
[Na+]i.
Although small changes of
[Na+]o
due to Na+ exit through ENaCs
cannot be completely ruled out, there are two observations that favor
the conclusion that these changes of
gNa are the
result of changes of
[Na+]i
rather than
[Na+]o.
First, the time course of the changes of
gNa mimics that
of changes of
[Na+]i
during the initial rapid phase. These changes occur within seconds,
thereby eliminating the contribution of
Na+ exit. Second, the reversal
potentials after the injection of Na+ increase and reach a plateau
during this initial phase and then remain constant through the
remainder of this phase and throughout the entire secondary phase (see
Fig. 5).
Similarly, it is also unlikely that
Na+ leak through ENaC is causing
the secondary slower inhibition because the
[Na+]i
remained essentially constant during this period, indicating little or
no Na+ loss. It should be noted
that, because the intracellular volume is much smaller than the
extracellular volume, any Na+ loss
resulting in appreciable change of
[Na+]o
would be accompanied by a much larger and exaggerated decrease of
[Na+]i.
Na+ vs. other
ions.
The lack of effect of injecting 70 mM
K2SO4-1.2
M sucrose indicates that both of these inhibitory processes are
independent of intracellular osmolarity. Additionally, the observation
that the injection of various anions or cations other than
Na+ results in a much smaller
nonspecific initial inhibition with no appreciable secondary inhibition
indicates that Na+ selectively
causes these two inhibitory processes.
It is not known whether the initial small inhibition observed with
injection of
K2SO4,
NMDG sulfate, or NMDG gluconate is due to the injection of cations or
anions or a combination of both. However, it is clear that the effect
of a general increase of intracellular ionic strength is distinct from
that of a specific increase of
[Na+]i
for both the initial and delayed phases of inhibition. If the effect of
increased ionic strength is attributed to cations, one can speculate
that these cations may possess a finite but smaller affinity to a
Na+ binding site on ENaC.
Alternatively, if this effect is due to a general increase of ionic
strength, one can speculate that this may be due to a crowding effect
whereby these ions shield the Na+
from interacting with ENaC.
Conclusions.
[Na+]i
was found to regulate ENaC activity in a dose-dependent manner. This
regulation can be divided into rapid and slow phases. These two phases
would be expected to control tonic and phasic channel activities,
respectively. These two modes of regulation are likely to correspond to
a direct and indirect effect of
[Na+]i
on ENaC and are not observed with the injection of various other
cations or anions.
 |
ACKNOWLEDGEMENTS |
I thank Dr. Bernard Rossier (University of Lausanne, Lausanne,
Switzerland) for the gift of rat ENaC subunits and Dr. Lawrence Palmer
(Cornell University) and Roxanne Reger for reading the manuscript.
 |
FOOTNOTES |
This work was supported by a Grant-In-Aid from the Louisiana American
Heart Association and by a Louisiana Education Quality Support Fund
grant from the Louisiana Board of Regents.
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: M. S. Awayda,
Dept. of Medicine, SL 35, Tulane Univ. School of Medicine, New Orleans,
LA 70112 (E-mail: mawayda{at}mailhost.tcs.tulane.edu).
Received 2 March 1999; accepted in final form 8 April 1999.
 |
REFERENCES |
1.
Abramcheck, F. J.,
W. Van Driessche,
and
S. I. Helman.
Autoregulation of apical membrane Na+ permeability of tight epithelia. Noise analysis with amiloride and CGS 4270.
J. Gen. Physiol.
85:
555-582,
1985[Abstract].
2.
Awayda, M. S.,
I. I. Ismailov,
B. K. Berdiev,
C. M. Fuller,
and
D. J. Benos.
Protein kinase regulation of a cloned epithelial Na+-channel.
J. Gen. Physiol.
108:
49-65,
1996[Abstract].
3.
Awayda, M. S.,
and
M. Subramanyam.
Regulation of the epithelial Na+ channel by membrane tension.
J. Gen. Physiol.
112:
97-111,
1998[Abstract/Free Full Text].
4.
Dinudom, A.,
K. F. Harvey,
P. Komwatana,
J. A. Young,
S. Kumar,
and
D. I. Cook.
Nedd4 mediates control of an epithelial Na+ channel in salivary duct cells by cytosolic Na+.
Proc. Natl. Acad. Sci. USA
95:
7169-7173,
1998[Abstract/Free Full Text].
5.
Drewnowska, K.,
E. J. Cragoe, Jr.,
and
T. U. Biber.
pH in principal cells of frog skin (Rana pipiens): dependence on extracellular Na+.
Am. J. Physiol.
255 (Renal Fluid Electrolyte Physiol. 24):
F930-F935,
1988[Abstract/Free Full Text].
6.
Els, W. J.,
and
K.-Y. Chou.
Sodium-dependent regulation of epithelial sodium channel densities in frog skin; a role for the cytoskeleton.
J. Physiol. (Lond.)
462:
447-464,
1993[Abstract].
7.
Els, W. J.,
and
S. I. Helman.
Activation of epithelial Na channels by hormonal and autoregulatory mechanisms of action.
J. Gen. Physiol.
98:
1197-1220,
1991[Abstract].
8.
Fisher, R. S.,
D. Erlij,
and
S. I. Helman.
Intracellular voltage of isolated epithelia of frog skin.
J. Gen. Physiol.
76:
447-453,
1980[Abstract].
9.
Fuchs, W.,
E. H. Larsen,
and
B. Lindemann.
Current-voltage curve of sodium channels and concentration dependence of sodium permeability in frog skin.
J. Physiol. (Lond.)
267:
137-166,
1979[Medline].
10.
Frindt, G.,
L. G. Palmer,
and
E. E. Windhager.
Feedback regulation of Na channels in rat CCT. IV. Mediation by activation of protein kinase C.
Am. J. Physiol.
270 (Renal Fluid Electrolyte Physiol. 39):
F371-F376,
1996[Abstract/Free Full Text].
11.
Frindt, G.,
R. B. Silver,
E. E. Windhager,
and
L. G. Palmer.
Feedback regulation of Na channels in rat CCT II. Effects of inhibition of Na entry.
Am. J. Physiol.
264 (Renal Fluid Electrolyte Physiol. 33):
F565-F574,
1993[Abstract/Free Full Text].
12.
Garty, H.,
and
L. G. Palmer.
Epithelial sodium channels: functions, structure, and regulation.
Physiol. Rev.
77:
359-396,
1997[Abstract/Free Full Text].
13.
Goldman, D. E.
Potential, impedance, and rectification in membranes.
J. Gen. Physiol.
27:
37-60,
1943[Free Full Text].
14.
Ishikawa, T.,
Y. Marunaka,
and
D. Rotin.
Electrophysiological characterization of the rat epithelial Na+ channel (rENaC) expressed in MDCK cells. Effects of Na+ and Ca2+.
J. Gen. Physiol.
111:
825-846,
1998[Abstract/Free Full Text].
15.
Kellenberger, S.,
I. Gautschi,
B. C. Rossier,
and
L. Schild.
Mutations causing Liddle syndrome reduce sodium-dependent downregulation of the epithelial sodium channel in the Xenopus oocyte expression system.
J. Clin. Invest.
101:
2741-2750,
1998[Abstract/Free Full Text].
16.
Komwatana, P.,
A. Dinudom,
J. A. Young,
and
D. I. Cook.
Cytosolic Na+ controls an epithelial Na+ channel via the Go guanine nucleotide-binding regulatory protein.
Proc. Natl. Acad. Sci. USA
93:
8107-8111,
1996[Abstract/Free Full Text].
17.
Kroll, B.,
S. Bremer,
B. Tummler,
G. Kottra,
and
E. Fromter.
Sodium dependence of the epithelial sodium conductance expressed in Xenopus laevis oocytes.
Pflügers Arch.
419:
101-107,
1991[Medline].
18.
Lide, D. R.
(Editor).
Handbook of Chemistry and Physics (72nd ed.). Boca Raton, FL: CRC, 1991, p. 5-99
19.
Lindemann, B.
Fluctuation analysis of sodium channels in epithelia.
Annu. Rev. Physiol.
46:
497-515,
1984[Medline].
20.
Ling, B. N.,
and
D. C. Eaton.
Effects of luminal Na+ on single Na+ channels in A6 cells, a regulatory role for protein kinase C.
Am. J. Physiol.
256 (Renal Fluid Electrolyte Physiol. 25):
F1094-F1103,
1989[Abstract/Free Full Text].
21.
Nagel, W.,
J. F. Garcia-Diaz,
and
W. M. Armstrong.
Intracellular ionic activities in frog skin.
J. Membr. Biol.
61:
127-134,
1981[Medline].
22.
Palmer, L. G.
The epithelial Na channel: inferences about the nature of the conducting pore.
Comments Mol. Cell. Biophys.
5:
259-283,
1991.
24.
Palmer, L. G.,
H. Sackin,
and
G. Frindt.
Regulation of Na+ channels by luminal Na+ in rat cortical collecting tubule.
J. Physiol. (Lond.)
509:
151-162,
1998[Abstract/Free Full Text].
25.
Plant, P. J.,
H. Yeger,
O. Staub,
P. Howard,
and
D. Rotin.
The C2 domain of the ubiquitin protein ligase Nedd4 mediates Ca2+-dependent plasma membrane localization.
J. Biol. Chem.
272:
32329-32336,
1997[Abstract/Free Full Text].
26.
Rick, R.,
A. Dorge,
E. Von Arnim,
and
K. Thurau.
Electron microprobe analysis of frog skin epithelium: evidence for a syncytial sodium transport compartment.
J. Membr. Biol.
39:
313-331,
1978[Medline].
27.
Rokaw, M. D.,
E. Sarac,
E. Lechman,
M. West,
J. P. Johnson,
and
M. L. Zeidel.
Chronic regulation of transepithelial Na+ transport by the rate of apical Na+ entry.
Am. J. Physiol.
270 (Cell Physiol. 39):
C600-C607,
1996[Abstract/Free Full Text].
28.
Silver, R. B.,
G. Frindt,
E. E. Windhager,
and
L. G. Palmer.
Feedback regulation of Na channels in rat CCT I. Effects of inhibition of Na pump.
Am. J. Physiol.
264 (Renal Fluid Electrolyte Physiol. 33):
F557-F564,
1993[Abstract/Free Full Text].
29.
Smets, I.,
W. Zeiske,
P. Steels,
and
W. Van Driessche.
Na+ dependence of single-channel current and channel density generate saturation of Na+ uptake in A6 cells.
Pflügers Arch.
435:
604-609,
1998[Medline].
30.
Stoddard, J. S.,
and
S. I. Helman.
Dependence of intracellular Na+ concentration on apical and basolateral membrane Na+ influx in frog skin.
Am. J. Physiol.
249 (Renal Fluid Electrolyte Physiol. 18):
F662-F671,
1985[Medline].
31.
Thurman, C. L.,
and
J. T. Higgins, Jr.
Norepinephrine stimulation of sodium transport in Necturus urinary bladder.
Biochim. Biophys. Acta
1022:
79-86,
1990[Medline].
32.
Turnheim, C.
Intrinsic regulation of apical sodium entry in epithelia.
Physiol. Rev.
71:
429-445,
1991[Abstract/Free Full Text].
33.
Van Driessche, W.,
and
B. Lindemann.
Concentration dependence of currents through single sodium-selective pores in frog skin.
Nature
282:
519-520,
1979[Medline].
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