Regulation of the epithelial Na+ channel by
extracellular acidification
Mouhamed S.
Awayda,
Michael J.
Boudreaux,
Roxanne L.
Reger, and
L. Lee
Hamm
Departments of Medicine and of Physiology, Tulane University School
of Medicine, New Orleans, Louisiana 70112
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ABSTRACT |
The effect of extracellular acidification was
tested on the native epithelial Na+ channel (ENaC) in A6
epithelia and on the cloned ENaC expressed in Xenopus
oocytes. Channel activity was determined utilizing blocker-induced
fluctuation analysis in A6 epithelia and dual electrode voltage clamp
in oocytes. In A6 cells, a decrease of extracellular pH
(pHo) from 7.4 to 6.4 caused a slow stimulation of the
amiloride-sensitive short-circuit current (INa)
by 68.4 ± 11% (n = 9) at 60 min. This increase
of INa was attributed to an increase of open
channel and total channel (NT) densities. Similar changes were observed with pHo 5.4. The effects of
pHo were blocked by buffering intracellular
Ca2+ with 5 µM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In
oocytes, pHo 6.4 elicited a small transient increase of the
slope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min)
followed by a decrease to 83.7 ± 11.7% of control at 60 min (n = 6). Thus small decreases of pHo
stimulate the native ENaC by increasing NT but
do not appreciably affect ENaC expressed in Xenopus oocytes.
These effects are distinct from those observed with decreasing
intracellular pH with permeant buffers that are known to inhibit ENaC.
epithelial sodium channel; A6 epithelia; Xenopus
oocytes; noise analysis; channel density
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INTRODUCTION |
THE NA+
channel in the apical membrane of many native electrically tight
Na+-absorbing epithelia is subjected to an environment with
a highly dynamic extracellular pH (pHo). It is well
established that large decreases (e.g., pH 2) of luminal pH
(pHo) decrease Na+ absorption and that this
inhibition is mediated via changes of intracellular pH
(pHi), leading to inhibition of the apical Na+
channel (6-10, 16, 19, 20, 22, 25). Indeed, Palmer and Frindt (20) found that channel activity and presumably
open probability (Po) is inhibited by
pHi in excised membrane patches. Zeiske et al.
(25) have also recently reported that open channel density
(No) of the Na+ channel found in A6
epithelia is inhibited by decreasing pHi.
Leaf et al. (16) have an unexplainable finding that small
decreases of pHo (down to 5.5) causes a stimulation, rather
than inhibition, of Na+ transport in the toad bladder. This
stimulation was observed in the absence of detectable effects on
pHi and was clearly distinct from the inhibitory effects
observed with intracellular acidification. The mechanism for this
stimulation, and whether such observation is applicable to other
Na+-absorbing epithelia, remains undetermined.
The regulation of the cloned epithelial Na+ channel (ENaC)
by pH has been recently investigated by Chalfant et al.
(4). These authors found that decreases of pHi
cause a rapid (within minutes) inhibition of ENaC expressed in
Xenopus oocytes through effects on channel activity. Similar
changes of pHo were without appreciable short-term (<10
min) effects on the channel. Thus it appears that the cloned ENaC has
the capability to rapidly (<5 min) respond to changes of
pHi and that these effects may represent a direct
interaction with H+, because an inhibition of
Po was found for ENaC incorporated into planar
lipid bilayers. Moreover, channel activity was relatively unaffected by
short-term (<10 min) deceases of pHo.
We have recently observed that a small decrease of pHo from
7.4 to 6.4 causes stimulation of the short-circuit current
(Isc) in the Xenopus kidney cell line
A6. This stimulation was not a direct effect of the pH change in that
the increase of Isc was not immediate. Moreover,
this effect was similar in its time course to that observed by Leaf et
al. (16) in toad bladder. Because the apical
Na+ channel (ENaC) is rate limiting to transepithelial
transport, this stimulation is likely mediated via effects on the
native ENaC. To determine the single channel basis of this stimulation, we utilized blocker-induced transepithelial fluctuation analysis. Similar experiments were also carried out on the cloned ENaC expressed in Xenopus oocytes to determine if prolonged extracellular
acidification also stimulates this channel in this system.
We report that stimulation of the Isc observed
by small apical acidification in polarized A6 epithelia is due to
increases of total channel density (NT). A small
decrease of the single channel current (iNa) was
also observed and is likely due to apical membrane depolarization. This
decrease of iNa slightly underestimated the
stimulation of the macroscopic Isc. The changes
of NT and Isc were
mediated via intracellular Ca2+-dependent mechanisms, since
pretreatment of A6 epithelia with an intracellular Ca2+
buffer [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA)] prevented this stimulation.
In contrast, decreasing pHo in ENaC-expressing oocytes
caused a small transient stimulation of the amiloride-sensitive
conductance followed by recovery to below control levels. Thus
additional Ca2+-dependent mechanisms may be present in
tight epithelia and may be responsible for the sustained stimulation of
NT with an acidic luminal environment. We
speculate that the increase of NT observed in A6
cells may serve as a protective mechanism whereby an epithelium subjected to large acid loads, which would normally inhibit
Na+ transport through changes of pHi, is more
capable of resuming its Na+ reabsorption after recovery
from this acidic environment.
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MATERIALS AND METHODS |
A6 epithelia.
Cells were obtained from American Type Culture Collection (ATCC,
Manassas, VA). Cells were cultured to confluency in
75-cm2 flasks and were subcultured on permeable polyester
membrane inserts (Transwell Clear; Costar). Cells were trypsinized, and
the equivalent of 1/360th of cells from a single flask were seeded on a
single insert (area 4.7 cm2). Cell polarity was assessed by
their ability to develop a transepithelial potential difference
(Voc) and appreciable transepithelial resistance (RT). With the use of the solutions described
below, monolayers exhibited Voc in the range of
40 to
60 mV and RT in the range of 7-12
k
· cm2 within 10-14 days after plating.
These values were stable for ~2 mo.
Cells were grown at 26°C in a humidified incubator containing ambient
air with 1.2% CO2. The culture media was similar to that
previously described (23) and of the following
composition: 26.2% L-15 Leibovitz, 26.2% Ham's F-12, 7.6% FBS,
1.5% L-glutamine (200 mM solution), 0.3%
penicillin/streptomycin (10,000 U/ml penicillin and 10 mg/ml
streptomycin), and 0.3% of a 7% sodium-bicarbonate solution. ddH2O
was added (~38%) to a final solution osmolarity of ~200 mosmol/l.
Media in both flasks and membrane inserts was changed two times weekly.
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 (2). Injected oocytes were incubated at 18°C for 1-3 days until recording. All recordings were
performed at 19-21°C.
Solutions and chemicals.
All solutions and chemicals were as previously described by Awayda and
Subramanyam (3). ND-96 (96 mM NaCl, 1.8 mM
CaCl2, 1 mM MgCl2, 2 mM KCl, and 5 mM HEPES) at
pH 7.4 was used for initial recording from both oocytes and A6
epithelia. HCl was used to alter pH in ND-96 to pH 6.4 or 5.4. Amiloride was a gift from Merck-Sharp & Dohme (Rahway, NJ). BAPTA-AM
was obtained from Molecular Probes (Eugene, OR). All other chemicals
were of the highest grade and were obtained from Sigma Chemical (St.
Louis, MO).
Dual electrode clamp.
Defoliculated Xenopus oocytes were injected with cRNAs for
rat
-,
-, and
-ENaC (rENaC). Injected oocytes were
cultured as previously described (3). Whole cell currents
were recorded in oocytes held at 0 mV and pulsed from
100 to +40 mV.
Slope conductance (gm) was summarized between
80 and
100 mV (2). By convention, 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.
Fluctuation analysis.
Membranes were placed in a modified Ussing chamber, and the
transepithelial voltage was clamped to 0 mV using a low-noise, direct
current-powered, four-electrode voltage clamp. Short 2-mV pulses were
used to measure the transepithelial resistance.
Noise or fluctuation analysis was carried out as previously described
by Helman et al. (13). After Isc
was allowed to stabilize, noise analysis was conducted using the
uncharged amiloride analog 6-chloro-3,5-diamino-2-pyrazinecarboxamide
(CDPC). CDPC was pulsed into the apical side of the Ussing chamber at
concentrations of 20 and 80 µM or at 15 and 40 µM. Current noise
was filtered at Nyquist frequency (~1,900 Hz). The filtered signal
was amplified and stored digitally. Signals were Fourier transformed to
yield the power-density spectra.
pH changes were made to the apical side of the tissue while the pH of
the basolateral side was held constant. All solutions used a
HEPES-based buffer and were therefore not expected to affect pHi. When used, BAPTA-AM was added to both sides of the
tissue at a final concentration of 5 µM in 0.05% DMSO (tissue
culture grade).
pHi.
These measurements used the pH-sensitive dye
2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF). BCECF was purchased from Molecular Probes in its
membrane-permeable BCECF-AM form. BCECF-AM was dissolved in DMSO on the
day of experiments and was used at a final concentration of 10 µM in
ND-96. Cells were allowed at least 60 min to uptake the dye.
Fluorescence studies were carried out on an inverted Nikon Diaphot-TMD
microscope as previously described (14). Fluorescence ratio was measured using the Nikon/PTI Photoscan II system, with excitation at 490 and 440 nm and emission at 530 nm. Background fluorescence was measured in BCECF-free cells and was <10% of the
fluorescence observed in BCECF-loaded cells. All experimental data were
corrected for background fluorescence.
pHi experiments were also carried out on polarized A6
monolayers grown on the same filters used for the noise analysis
experiments to allow us to correlate these two measurements. Cells were
studied in special chambers designed to allow pHi
measurements in cultured polarized cells and offered separate access to
the apical and basolateral compartments in addition to a means of rapid
solution exchange in each of these compartments (17).
Because the cells were grown on transparent membrane supports, it was
expected that the filter material would not obstruct the
pHi measurements. However, to circumvent any potential
problems, the filter and accompanying cells were inverted in the
chamber so that the apical membrane and the cells directly faced the
excitation source.
Statistical analysis was carried out using paired Student's
t-test where appropriate. Significance was determined at the
95% confidence level (P < 0.05).
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RESULTS |
Effects of decreasing pHo on the macroscopic properties
of the native ENaC.
Confluent A6 cells were short circuited according to conventional
methods in symmetrical ND-96 at pH 7.4. In these cells, Isc is essentially all attributed to
Na+ transport through the apical native ENaC and is blocked
by 10 µM amiloride. To noninvasively calculate the single channel
properties, we used blocker-induced fluctuation analysis. This protocol
is similar to that used by Helman et al. (13) and allows
the assessment of the time course of changes of the single channel
parameters. To utilize these methods, cells were continuously perfused
in Ringer solution containing a low blocker concentration (20 or 15 µM CDPC) and were periodically (every 10 min) pulsed for ~3 min
with solution containing a higher blocker concentration (80 or 40 µM
CDPC). This protocol is shown in Fig. 1
along with a continuous recording of the Isc and
the effect of a decrease of apical pHo. It is evident from
the examples in Fig. 1 that decreasing pHo to 6.4 or 5.4 caused a gradual stimulation of the Isc and presumably the apical Na+ channels.

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Fig. 1.
Noise analysis protocol and representative effect of extracellular
acidification on Na+ transport in A6 monolayers. Continuous
recording of the short-circuit current (Isc) at
a holding potential of 0 mV. A 2-s pulse was applied at fixed intervals
to determine monolayer resistance. This pulse was removed during
acquisition of the power density spectra. A: effect of pH
6.4 on the Isc. B: effect of pH 5.4 on the Isc. In these particular examples, a
6-chloro-3,5-diamino-2-pyrazinecarboxamide (CDPC) dose response
(20-100 µM) was carried out at the beginning and end of the
experiment. To carry out noise analysis during the control and
experimental periods, tissues were incubated in 20 µM CDPC and pulsed
for ~3 min with 80 µM CDPC (see text for details). Both pH 6.4 and
5.4 caused a gradual stimulation of the Isc.
[CDPC], CDPC concentration.
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The time course of the effects of decreasing pHo is
summarized in Fig. 2. These data
summarize the changes of the amiloride-sensitive currents
(INa). Extracellular acidification with
HEPES-based buffer caused a gradual stimulation of the
INa that reached a plateau within 30-40
min. To determine whether similar effects are observed for the cloned
channel, we carried out experiments in the Xenopus oocytes
expression system.

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Fig. 2.
Time course of the stimulation of amiloride-sensitive
Na+ transport with extracellular acidification.
A: pH 6.4 caused a sustained but gradual stimulation of the
amiloride-sensitive short-circuit current (INa)
that reached a relative plateau within ~40 min. B: similar
effects were observed with pH 5.4, except that the stimulation of
INa was slightly faster. All data are summarized
in the presence of the lower [CDPC]; n = 9 and
n = 6 experiments for pH 6.4 and 5.4, respectively.
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Effects of decreasing pHo on the macroscopic properties
of the cloned channel.
Figure 3 is a representative example of
the whole cell currents in an ENaC-expressing oocyte and their block by
10 µM amiloride. Amiloride causes a decrease of the whole cell
currents to levels not different from those observed in control
water-injected oocytes. The whole cell currents between
100 and
80
mV were used to calculate the inward gm. As
evident from Fig. 3, the majority of the gm is
amiloride sensitive and is attributed to ENaC.

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Fig. 3.
Representative whole cell currents in an epithelial Na+
channel (ENaC)-expressing oocyte and its block by amiloride.
A: currents in an ENaC-expressing oocyte bathed in control
Ringer solution. B: currents in the same oocyte after the
addition of 10 µM amiloride. C: INa
calculated as A B. The slope conductance
(gm) was calculated from the currents at 100
and 80 mV.
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The effects of a small decrease of pHo with HEPES-buffered
solution on the gm are summarized in Fig.
4. Within the resolution of the first
measurement (30 s), pHo of 6.4 caused an increase of
gm. This increase reached a peak value at 2 min
and then steadily declined to values below control. Thus the response
to decreasing pHo was different between oocytes and A6
cells. The origin of these differences is unclear but may be due to one
or a combination of differences in the expression system (oocytes vs.
epithelial cells) or differences in the channel itself (cloned vs.
native).

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Fig. 4.
Time course of the effects of extracellular acidification
on the cloned ENaC expressed in oocytes. Data are summarized as
gm and are normalized in each oocyte to the
value of gm immediately before the
pHo change. Decreasing pHo caused a transient
stimulation of gm. This stimulation reached a
peak value of ~12% at 2 min and was followed by inhibition to below
control values (n = 6). E/C, mean ratio of the paired
control and experimental groups.
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It is possible that the initial stimulation of the cloned ENaC is
similar to that observed from the native channel in A6 cells. It is
clear, however, that the cloned ENaC expressed in oocytes did not
exhibit a sustained stimulation. To further investigate the mechanisms
of the sustained stimulation observed in A6 cells, we used
blocker-induced noise analysis.
Effects of decreasing pHo on the single channel
properties of the native channel.
In the absence of blockers, the spontaneous rates of ENaC transition
are too slow for the resolution of noise analysis. This is indeed
consistent with single channel patch-clamp data that indicate open and
close times on the order of seconds [see Garty and Palmer
(9)]. These slow kinetics are manifested by the absence
of a spontaneous Lorentzian function in the power density spectrum
(Fig. 5A). To resolve channel
properties, a blocker is used to interact with the channel and speed up
its rates of opening and closing. CDPC is used as it is uncharged and
results in small inhibition of macroscopic currents. The on and off
rates for CDPC are also sufficiently fast enough and allow for better
resolution of the blocker-induced Lorentzian (Fig. 5B). The
corner frequency of the Lorentzian function exhibits a linear
relationship with CDPC concentration, as expected from a first-order
reaction (Fig. 5C). The corner frequencies, macroscopic
currents, and power plateaus are used to calculate the channel
properties as described by Helman et al. (13). The effects
of pHo 6.4 and 5.4 on the channel properties are summarized
below.

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Fig. 5.
Noise or fluctuation analysis of the native ENaC in A6 epithelia.
A: background power density spectrum in the absence of a
blocker. Consistent with the known kinetics of ENaC, there are no
observed spontaneous Lorentzian functions. B: addition of a
blocker, such as CDPC, induces the appearance of a Lorentzian function.
This function can be described by power plateau and a corner frequency.
C: as expected from a first-order reaction, the corner
frequency is linearly related to blocker concentration, and this
relationship can be used to calculate the blocker on and off rates. See
text for more details.
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Figure 6 shows the effects of decreasing
pHo on the blocker equilibrium constant
(KB). This constant is calculated from the ratio
of the blocker off and on rates and is therefore independent of
Po, which can affect the apparent equilibrium
constant calculated from the half-maximal block of the macroscopic
currents. CDPC is electroneutral at pH 7.4 and down to approximately pH
4, and therefore its net charge is unaffected by a change of pH from 7.4 to 5.4. In this respect, it becomes of additional advantage to use
CDPC in the current experiments since any pH-related changes of
KB are not due to simple changes of the net
charge on this blocker. As evident from Fig. 6,
KB is unaffected by changes of pHo,
and thus pHo does not affect the interaction between CDPC with the externally accessible portion of ENaC. Because amiloride, the
parent molecule for CDPC, behaves as a plug (18) that
protrudes ~25% of the way into the mouth of the channel
(24), the lack of effects on KB may
also indicate that the outer portion of the channel that interacts with
amiloride and CDPC is not modified by these changes of pHo.
This, however, does not rule out pHo-induced modification
of an accessory protein or of different extracellular regions of the
channel that do not interact with CDPC.

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Fig. 6.
Lack of effects of extracellular acidification on the blocker
equilibrium constant (KB).
KB was insensitive to pHo 6.4 (A) and 5.4 (B); n = 9 and 6 for
pHo 6.4 and 5.4, respectively.
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To determine the mechanisms of stimulation of the
INa, we calculated the single channel properties
and channel density. As shown in Fig. 7,
the stimulation observed with pHo 6.4 is predominantly due
to a stimulation of No. These changes were
accompanied by a compensatory decrease of iNa.
These effects on iNa are likely due to a
decrease of the electrochemical gradient across the channel rather than
a change of the single channel conductance (see
DISCUSSION).

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Fig. 7.
Time course of the effects of pHo 6.4 on the
single channel properties. A: pHo 6.4 caused a
small but significant decrease of the single channel current
(iNa). These changes appeared to be more rapid
than those of the INa. B: open
channel probability (No) was stimulated with a
similar time course to that observed for the changes of
INa. C: no effects were observed on
open probability (Po; n = 9).
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The mechanisms underlying the stimulation of
INa by pHo 5.4 were similar to those
observed above with pHo 6.4. As shown in Fig.
8, pHo 5.4 caused a
stimulation of No and a compensatory decrease of
iNa. Consistent with the slightly faster changes
of INa with pHo 5.4, these changes
of No appear to reach a relative plateau within
30 min. The relatively slow time course of these changes may indicate
the presence of channel trafficking events resulting from the
involvement of second messenger cascades. The two most prominent second
messenger cascades that affect Na+ transport involve cAMP
and Ca2+. We focused our attention on the Ca2+
pathway because of our ongoing interest in ENaC regulation by Ca2+ and/or protein kinase C.

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Fig. 8.
Time course of the effects of pHo 5.4 on the
single channel properties. A: pHo 5.4 also
caused a small but significant decrease of iNa.
The time course of the changes of iNa was
clearly more rapid than that of the changes of
INa. B: No was
stimulated with a similar time course to that observed for the changes
of INa. C: no effects were observed
on Po (n = 6). Both
No and iNa exhibited
similar but more rapid changes compared with pHo 6.4 (see
Fig. 7).
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Role of intracellular Ca2+
concentration in the observed stimulation.
It is well known that large increases of the intracellular
Ca2+ concentration ([Ca2+]i)
inhibit ENaC (5, 7, 19, 20). However, Ca2+
is also involved in many second messenger-mediated signaling cascades, including those resulting in vesicular trafficking. To
determine the role of [Ca2+]i, A6 monolayers
were incubated with 5 µM BAPTA-AM, an intracellular Ca2+
chelator. This chelator is added in a membrane-permeable form that
allows it to enter the cell. Intracellular BAPTA is then deesterified,
which renders it membrane impermeant, and is trapped within the cell
where it binds free Ca2+ and buffers the changes of
[Ca2+]i. Figure
9 is a representative effect of BAPTA
followed by pHo 6.4 on the Isc.
Within minutes, the addition of BAPTA caused a marked decrease of
transport. These monolayers were challenged with pHo 6.4 1 h after the addition of BAPTA. In this example, there were no
changes of the Isc with pHo 6.4.

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Fig. 9.
Representative effect of extracellular acidification on
Na+ transport in A6 monolayers pretreated with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA). Addition of 5 µM BAPTA caused a gradual inhibition of the
Isc. Subsequent extracellular acidification ~1
h after the initial BAPTA treatment was without effect. Thus the
stimulation observed with pHo 6.4 is dependent on
[Ca2+]i. See Fig. 1 legend for additional
details.
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The effects of pHo 6.4 on the macroscopic and single
channel parameters in BAPTA-pretreated monolayers are summarized in
Fig. 10. BAPTA treatment (5 µM)
caused a decline of INa. This trend was not
altered after treatment with pHo 6.4, and
INa showed no appreciable evidence of
stimulation (Fig. 10A). Similarly, no significant effects of
pHo were observed on iNa,
No, and Po in
BAPTA-pretreated cells within the first 30 min. A significant increase
of Po was observed in measurements >30 min
after the pHo change. The reason for this increase is
unknown. It may be unrelated to the change of pHo but
related to prolonged intracellular Ca2+ depletion.
Nevertheless, the effects of pHo on
No appear to involve [Ca2+]i.

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Fig. 10.
BAPTA pretreatment eliminated the effects of extracellular
acidification on the macroscopic and single channel properties.
A: addition of BAPTA caused a gradual and continuous
decrease of INa. This trend was not appreciably
affected by pHo 6.4. B: no appreciable effects
were observed on iNa. C:
No exhibited a small transient tendency for an
increase after pHo 6.4. However, this effect was not
statistically significant. D: no effects were observed on
Po (n = 6).
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A major advantage of noise analysis is that this technique allows the
calculation of single channel parameters and NT.
This circumvents problems with variable channel gating, as observed with ENaC, and allows a more accurate estimate of
Po and NT. The effects on
NT of decreasing pHo and its block
by buffering [Ca2+]i are summarized in Fig.
11. This figure demonstrates that
pHo causes a greater than twofold increase of
NT via mechanisms that involve increases of
[Ca2+]i. As observed from Figs. 1-10,
the changes of NT with pHo 5.4 were more rapid than those observed with pHo 6.4.

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Fig. 11.
Time course of the stimulation of total channel density
(NT) by extracellular acidification.
Extracellular acidification caused a >2-fold increase of
NT. The magnitude of the changes was similar
between pHo 6.4 (circles, n = 9) and 5.4 (squares, n = 6); however, the effects of
pHo 5.4 were significantly faster. Pretreatment of cells
with BAPTA caused a decrease of NT (triangles,
n = 6) that was unaffected by subsequent acidification,
as NT continued to decrease to ~30% on
control within 60 min.
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Table 1 summarizes our findings with
extracellular acidification. The only significant changes observed at
both decreased pHo were those involving
iNa and NT. No
significant changes were observed in the BAPTA-pretreated cells. In
both cases, and in the absence of BAPTA, decreasing pHo
caused an ~60% increase of INa mediated via
an approximately twofold increase of NT and a small ~20% decrease of iNa.
Measurements of pHi.
It is highly possible that prolonged changes of pHo, even
in HEPES-buffered solutions, could lead to appreciable changes of pHi. To determine whether such a hypothesis is tenable in
the present experiments, we utilized fluorescence measurements of pHi in polarized A6 monolayers. As shown in the
representative example in Fig. 12,
decreasing apical pHo did not have any detectable changes
of the BCECF fluorescence ratio, indicating lack of effects on
pHi. On the other hand, an appreciable and reversible
effect could be observed with the permeable ion NH4. These
observations do not rule out small local changes of pHi but
indicate the lack of large changes of pHi.

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Fig. 12.
Representative example of the effects of apical
acidification on pHi. In all experiments, a control period
of ~20 min was established. The apical solution was then switched to
one with a pH of 6.4. Ammonium chloride (20 mM) was then added as a
positive control to verify that the measured fluorescence ratio
reflected pHi. This maneuver resulted in a reversible
change of pHi. Data are representative of 6 experiments.
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DISCUSSION |
We used A6 epithelia and the Xenopus oocyte expression
system to study the effects of extracellular acidification on ENaC. Consistent with a previous report by Leaf et al. (16) in
the toad bladder, extracellular acidification caused a stimulation of
Na+ transport in A6 epithelia. This response was sustained
over 60 min. In contrast, currents in oocytes expressing the cloned
ENaC were only slightly and transiently stimulated by extracellular acidification. The stimulation in A6 cells was reflected as a large
increase of NT and a small compensatory decrease
of iNa. These effects were dependent on
[Ca2+]i, as pretreatment with BAPTA prevented
the changes of INa, iNa, and NT.
Role of pHi.
It is established in a variety of preparations that the native channel
is inhibited by decreasing pHi (7-10, 16, 19,
20, 22). Moreover, recent evidence and our own unpublished
observations indicate that the channel in cultured A6 epithelia is also
inhibited by decreasing pHi (25). However,
aside from a report in toad bladder (16), pHo
is not thought to affect ENaC. Thus the present findings indicate that
this phenomenon is not restricted to toad bladder and may be present in
other Na+-absorbing epithelia. Our findings also provide
the single channel basis for this increase along with potential
mechanisms. The changes of pHo to 5.4 are well within the
range of those encountered in the urinary bladder and in the distal
nephron; thus, these findings represent an important physiological
effect of external H+.
We cannot rule out with absolute certainty that the observed
stimulation of No was due to small localized
changes of pHi, despite the fact that we could not detect
any changes of the BCECF fluorescence ratio and therefore bulk
pHi. However, three additional lines of indirect evidence
argue against this possibility. First, both pHo 6.4 and 5.4 were without effects on Po, which is shown to be
rapidly inhibited by small intracellular acidification (4, 19,
20). Second, a decrease of pHi is expected to
decrease No in A6 cells (25), which
is opposite to our observed effects with decreasing pHo.
Third, similar experiments in toad bladder found that a pHo
down to 5.4 was also without detectable effects on pHi
(16).
Effects in oocytes vs. A6 cells.
It is well established that many of the ENaC properties found in native
and cultured epithelia are well reproduced for the cloned channel
expressed in Xenopus oocytes. However, overexpression of the
three cloned ENaC subunits may result in the formation of a channel
that reproduces the basic native Na+ channel properties but
lacks the regulation conferred by association with other endogenous
proteins. One such example was proposed by Awayda et al.
(2) to explain the lack of regulation of the cloned ENaC
by protein kinase A in Xenopus oocytes. It is possible that
this may also be applicable to the observed differences in the response
to pHo. However, at the present time, we cannot distinguish whether the variance in the response to pHo is due to
differences between the native and cloned ENaC or oocytes and A6 cells.
In either case, a better understanding of the origins of these
differences will ultimately depend on elucidation of the mechanisms for
sensing pHo and/or the role of Ca2+.
Effects on NT (role of
Ca2+).
The observed effects of pHo on NT
were gradual, and appreciable changes were observed up to 30 min at
pHo 5.4 and 40 min at pHo 6.4. This time course
rules out an effect of external H+ on the channel, leading
to direct activation of electrically silent but membrane-resident
channels. However, it is possible that external H+
activates the channel via indirect effects on channel-related regulatory mechanisms. Decreasing pHi may protonate an
accessory or a regulatory protein, e.g., a kinase or a phosphatase,
that may be involved in channel trafficking. Alternatively, ENaC itself may be modified to alter its membrane residency to decrease its rates
of endocytosis. At present, we are not able to select a likely
mechanism among these; however, the finding that these changes were
dependent on [Ca2+]i may indicate the
potential involvement of a cell signaling cascade in the observed
increase of NT.
Stimulation of Na+ transport in A6 epithelia by various
mechanisms has been linked to Ca2+ mobilization. Hayslett
and colleagues (11, 12) found that stimulation by
adenosine and by vasopressin is linked to Ca2+
mobilization, since this effect could be blocked by BAPTA pretreatment. These authors measured the equivalent Isc;
therefore, their methods did not allow for an assessment of the single
channel properties and channel density. Nevertheless, our data are
consistent with their observations and establish a role for channel
trafficking or channel activation by [Ca2+]i.
This may occur via a simple dependence of the cellular trafficking machinery on [Ca2+]i similar to that observed
in many exocytic fusion events. Alternatively, it may involve a more
complicated second messenger cascade. In any case, the potential roles
of Ca2+, as delineated by buffering with BAPTA, should be
distinguished from those observed with large and sometimes
pharmacological increases of Ca2+ with ionophores
that are known to inhibit ENaC.
Assuming that pHi is not altered, how do we envision an
increase of external H+ concentration causing an increase
of [Ca2+]i? There are no known
H+-sensing proteins in A6 cells. However, these cells are
thought to contain an external Ca2+ sensor
(15). This sensor is mildly Ca2+ selective in
that it can also respond to Mg2+ and other multivalent
cations (1, 21). Moreover, is it also known that this
sensor is coupled to intracellular Ca2+-signaling cascades
and to G protein-coupled cascades (1). It is unclear if
such a sensor is also affected by extracellular H+
concentration; however, such a process could account for the effects of
pHo on NT and the involvement of
[Ca2+]i.
A notable alternative hypothesis worth mentioning is that
pHo may not alter [Ca2+]i but may
affect the interaction of intracellular Ca2+ with ENaC. To
our knowledge, such a mechanism has not been described previously.
However, a relevant mechanism was described by Garty and colleagues
(7). Using toad bladder vesicles, these investigators found that pHi affects the interaction of the
Na+ channel with [Ca2+]i. In
these experiments, the ability of Ca2+ to inhibit
Na+ uptake was greatly reduced by decreasing
pHi from 7.4 to 7.0. In this case, it would be expected
that a decrease of pHi may relieve the inhibition of the
channel by Ca2+ and cause its stimulation. It is unclear if
a similar process occurs with changes of pHo, as this
requires that protonation of an externally accessible site on the
channel or associated protein leads to changes in the interaction with
internal Ca2+. The above hypotheses await further
experimental testing.
Potential physiological significance.
We demonstrated that extracellular acidification caused a more than
twofold increase of channel density. In a native
Na+-absorbing epithelium, such as the cortical collecting
duct, this could cause major changes in Na+ reabsorption.
If the present findings can be extended to native epithelia, it is
possible that this mechanism may prime the principal cells to increase
their capacity for Na+ transport and allow for a better
recovery after inhibition by a large acid load. A second hypothesis was
proposed by Leaf et al. (16) who made the original
observation of stimulation of Na+ transport by
pHo in the toad bladder. They remarked that many clinically
encountered conditions associated with excess plasma acidosis and
increased urinary H+ secretion are also accompanied by the
need for Na+ conservation. In this case, the stimulation of
Na+ transport by luminal acidity would constitute an
intrinsic mechanism that conserves Na+. This mechanism may
also serve to prevent excess Na+ loss through
Na+/H+ exchangers in the presence of increased
luminal H+.
pHo was found to activate Na+ transport in A6
epithelia. This activation was primarily due to an increase of
NT. The increase of NT
was [Ca2+]i dependent and was prevented by
buffering [Ca2+]i with BAPTA. This
stimulation may represent an intrinsic mechanism of channel regulation
leading to increased Na+ reabsorption. It is unclear if the
cloned channel behaves in a similar manner, since the currents in
oocytes expressing the cloned ENaC were only transiently stimulated by
pHo.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Willy Van Driessche (K.U. Leuven) for helpful
discussions regarding the pKa of CDPC.
 |
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 to M. S. Awayda.
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}tulane.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 January 2000; accepted in final form 6 July 2000.
 |
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