1 Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755; and 2 Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35233
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ABSTRACT |
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Protons regulate
electrogenic sodium absorption in a variety of epithelia, including the
cortical collecting duct, frog skin, and urinary bladder. Recently,
three subunits (,
,
) coding for the epithelial sodium channel
(ENaC) were cloned. However, it is not known whether pH regulates
Na+ channels directly by
interacting with one of the three ENaC subunits or indirectly by
interacting with a regulatory protein. As a first step to identifying
the molecular mechanisms of proton-mediated regulation of apical
membrane Na+ permeability in
epithelia, we examined the effect of pH on the biophysical properties
of ENaC. To this end, we expressed various combinations of
-,
-,
and
-subunits of ENaC in Xenopus
oocytes and studied ENaC currents by the two-electrode voltage-clamp
and patch-clamp techniques. In addition, the effect of pH on the
-ENaC subunit was examined in planar lipid bilayers. We report that
,
,
-ENaC currents were regulated by changes in intracellular pH
(pHi) but not by changes in
extracellular pH (pHo).
Acidification reduced and alkalization increased channel activity by a
voltage-independent mechanism. Moreover, a reduction of
pHi reduced single-channel open
probability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited
,
-ENaC,
,
-ENaC, and
-ENaC currents. We conclude that
pHi but not
pHo regulates ENaC and that the
-ENaC subunit is regulated directly by
pHi.
cortical collecting duct; amiloride; patch clamp; hydrogen ion; Xenopus oocyte
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INTRODUCTION |
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SODIUM ABSORPTION across a variety of epithelia, including the renal cortical collecting duct (CCD), frog skin, and urinary bladder, plays a vital role in maintaining Na+ and fluid homeostasis (14). Na+ absorption across these epithelia is a two-step, electrogenic process involving diffusion across the mucosal membrane through amiloride-sensitive, 5-pS, Na+-selective channels and active transport across the basolateral membrane via the Na+-K+-ATPase (14). The rate-limiting step in this process is Na+ entry across the mucosal membrane (14). Na+ channel activity is regulated by a variety of hormones as well as the intra- and extracellular milieu, and thus regulation of Na+ channel activity is an important component of Na+ and fluid homeostasis (14). Protons have long been recognized to influence amiloride-sensitive Na+ absorption in epithelia (12, 13, 16, 21, 25, 27, 28, 40). Acidification of the cytoplasm of frog skin (16, 40) and toad bladder (28) reduces Na+ absorption and the Na+ permeability of the mucosal membrane. Patch-clamp experiments have demonstrated that the activity of Na+ channels in the apical membrane of principal cells in the CCD is inhibited by acidification of intracellular pH (pHi) (30). In contrast, a reduction in pHi has been reported to stimulate Na+ transport in toad bladder (12, 21).
Recent studies indicate that the epithelial
Na+ channel (ENaC), which is
composed of three subunits, ,
, and
, with molecular masses of
70-80 kDa, constitutes the major
Na+ entry pathway in the mucosal
membrane of epithelial cells that absorb
Na+ by an electrogenic mechanism
(5, 6, 14, 24). Expression of the
-,
-, and
-subunits of ENaC
in Xenopus oocytes is sufficient for
the expression of Na+ channels
with biophysical properties similar to those expressed in epithelia
that exhibit pH-sensitive Na+
absorption (6, 14). At least six proteins may associate with and
regulate Na+ channels in renal
epithelial cells, including a 300-kDa protein that is phosphorylated by
protein kinase A (PKA), 95- and 70-kDa proteins, 150- and 55-kDa
proteins that are phosphorylated by protein kinase C, and a 40-kDa
protein that may be the heterotrimeric G protein subunit
G
i-3 (1, 3, 35). It is not
known whether pH regulates Na+
channels directly by interacting with one of the three ENaC subunits or
indirectly by interacting with a regulatory or associated protein. Accordingly, because the effect of pH on ENaC subunits has not been
reported, we examined the role of pH in regulating various combinations
of ENaC subunits expressed in Xenopus
oocytes and on
-ENaC in planar lipid bilayers. We report that
,
,
-ENaC currents are regulated by changes in
pHi but not by changes in the
extracellular pH (pHo).
Acidification reduced and alkalization increased channel activity by a
voltage-independent mechanism. Moreover, a reduction of
pHi reduced single-channel open
probability (Po), reduced
single-channel open time, and increased single-channel closed time
without altering single-channel conductance. A reduction in
pHi also inhibited
,
-ENaC,
,
-ENaC, and
-ENaC currents. We conclude that
pHi is an important regulator of
ENaC activity and that the
-ENaC subunit is regulated directly by
pHi.
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METHODS AND MATERIALS |
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Isolation of Xenopus oocytes and injection of cRNA. Well-documented methods for oocyte isolation and cRNA injection were employed (9). Briefly, ovarian lobes were surgically removed from anesthetized frogs (Xenopus laevis) and stored in Ca2+-free OR-2 solution (in mM: 82.5 NaCl, 2 KCl, 1 MgCl2, 5 HEPES, with pH 7.5 adjusted with NaOH). Oocytes were isolated and defolliculated using a combination of enzymatic treatment for 2 h (2 mg/ml type 1A collagenase in OR-2; Sigma Chemical, St. Louis, MO) and manual dissection. Defolliculated stage V and VI oocytes were transferred to L-15 medium modified for use with amphibian cells and supplemented with gentamicin sulfate (25 mg/ml). Oocytes recovered overnight at 19°C before injection of cRNA.
Pipettes for microinjecting cRNA were prepared from borosilicate glass using a horizontal puller, followed by beveling of the tip. To examine the effect of pH on ratcRNA preparation.
Plasmids containing cDNA encoding the -,
-, and
-subunits of
rENaC cloned into the pSport vector were a generous gift of Dr. Bernard
C. Rossier (Lausanne, Switzerland) (5, 6). The vector was linearized
with Not I and was
used as a template for cRNA synthesis using a kit containing T7 RNA
polymerase, ribonucleotides, and a 7-methylguanosine cap analog
(mMessage mMachine, Ambion, Austin, TX) following the manufacturer's instructions.
Two-electrode voltage clamp.
For two-microelectrode voltage-clamping experiments, oocytes were
bathed in either a HEPES- or sodium acetate-buffered solution. The
HEPES-buffered solution contained (in mM) 110 NaCl, 2 KCl, 0.2 CaCl2, 1.0 MgCl2, and 5 HEPES, with pH
adjusted to values ranging from 6.4 to 7.4 using either NaOH or HCl, as
appropriate. The sodium acetate-buffered solution contained (in mM) 50 NaCl, 60 sodium acetate, 2 KCl, 0.2 CaCl2, and 1.0 MgCl2, with pH titrated to values
ranging from 6.4 to 7.4 using NaOH or HCl as appropriate. All
experiments were performed at 22-24°C. When filled with 3 M
KCl, the microelectrodes had an electrical resistance of 1-3 M.
A voltage-clamp amplifier (OC-725, Warner Instrument) was used to
measure whole cell current. The bath was grounded via a pair of Ag-AgCl
wires immersed in reservoirs filled with 3 M KCl and connected to the
bath via glass tubes filled with 4% agar in 3 M KCl. Whole cell
currents were passed through a low-pass, four-pole Bessel filter with a
cutoff frequency of 100 Hz, digitized at a sampling rate of 2 kHz, and
stored on the hard disk of a DOS-based computer for subsequent analysis
using pCLAMP software (version 6.03, Axon Instruments, Foster City, CA).
Single-channel current measurements.
For patch-clamp experiments, the vitelline membrane was removed as
described (26), with minor modifications. Briefly, oocytes were placed
in a hypertonic solution containing (in mM) 220 N-methyl-D-glucamine (NMDG) aspartate, 1 MgCl2, 5 EGTA, and 5 HEPES, with
pH titrated to 7.4 with NMDG-OH. After several minutes, the vitelline
membrane was removed using a pair of fine forceps, and devitellinated
oocytes were transferred to the patch-clamp recording chamber (RC-24, Warner Instrument, Hamden, CT) mounted on the stage of an inverted microscope (Diaphot-TMD, Nikon, Tokyo, Japan). For cell-attached and
inside-out patch-clamp experiments, the pipette solution contained (in
mM) 110 NaCl, 1.0 CaCl2, and 5.0 HEPES, with pH titrated to 7.4 with NMDG-OH, and was supplemented with
0.1 mM GdCl3 to block endogenous,
stretch-activated cation channels (46). In preliminary experiments,
GdCl3 (0.1 mM) had no effect on
ENaC currents in oocytes (n = 4). For
inside-out patches, the cytoplasmic bath solution contained (in mM) 110 KCl, 2.5 EGTA, and 5.0 HEPES, with pH titrated to values ranging
between 8.0 and 6.4 using NMDG-OH or HCl. The solution bathing
the cytoplasmic surface of inside-out membrane patches was exchanged
rapidly by a perfusion pipette. Patch-clamp pipettes were pulled from
borosilicate glass (Corning no. 7052, Garner Glass, Claremont, CA)
using a horizontal puller (Sutter Instrument, San Rafael, CA). When
filled with the pipette solution, the electrodes had resistances
ranging from 1.5 to 5 M. Pipette currents were amplified, filtered
with a low-pass, four-pole Bessel filter with a cutoff frequency of 200 Hz, digitized at a sampling rate of 2 kHz, and stored on the hard disk
of a DOS-based computer for subsequent analysis using pCLAMP software version 6.03. Single-channel currents were measured as previously described in detail (22). All voltages refer to the cell interior referenced to the patch pipette. After a gigaseal patch was attained and the membrane patch was excised to form the inside-out
configuration, recordings were made for the generation of
I-V
plots and the determination of
NPo, the product
of the number of observed open channels
(N) and
Po.
NPo was
calculated as follows
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Planar lipid bilayers.
The effect of pHi on the
single-channel properties of -ENaC was studied in planar lipid
bilayers. Stage V-VI Xenopus oocytes were harvested and injected with 5 ng
-ENaC cRNA or 50 nl water as
described above. Membrane vesicles were prepared 48 h postinjection following the method of Pérez et al. (31). Thirty to forty oocytes in each group were washed and homogenized in a
high-K+ sucrose medium containing
the following protease inhibitors: aprotinin (1 µg/ml), leupeptin (1 µg/ml), pepstatin (1 µg/ml), phenylmethylsulfonyl fluoride (100 µM), and DNase I (2 µg/ml). Oocyte membranes were
isolated by discontinuous sucrose gradient density centrifugation and
resuspended in 300 mM sucrose, 100 mM KCl, and 5 mM MOPS (pH 6.8).
Membrane vesicles were separated into 50-µl fractions and stored at
80°C until use. Planar lipid bilayers were made from a
phospholipid solution containing a 1:1 mixture of diphytanoyl
phosphatidylethanolamine and diphytanoyl phosphatidylserine (in
n-octane; final phospholipid
concentration 25 mg/ml). Membrane vesicles were applied with a
fire-polished glass rod to one side
(trans) of a preformed bilayer
bathed with a symmetrical solution of 100 mM NaCl and 10 mM MOPS-Tris
(pH 7.4). Acquisition and analysis of single-channel
recordings were performed as described (20).
Statistical analysis. Differences between means were compared by ANOVA and the Bonferroni post hoc comparison test or the paired or unpaired Student's t-test, as appropriate. Statistical analyses were performed with the InStat statistical software package (Graphpad, San Diego, CA). Data are expressed as means ± SE. P < 0.05 is considered significant.
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RESULTS |
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Characterization of
,
,
-rENaC currents.
In water-injected oocytes, we did not observe any amiloride-sensitive
currents (Fig. 1), consistent
with previous observations (6). In contrast, injection of cRNAs
encoding the
-,
-, and
-subunits of rENaC into oocytes induced
the expression of amiloride-sensitive Na+ currents (Fig. 1). The
amiloride-sensitive
I-V
relationship had a positive reversal potential and a slight inward
rectification (Fig. 1G). To
calculate the ionic selectivity of the amiloride-sensitive currents,
ion substitution experiments were conducted in which Na+ was replaced on a equimolar
basis with Li+. When the bath
solution contained NaCl (110 mM), the reversal potential of the
amiloride-sensitive
I-V
plot was 6.5 ± 0.5 mV (n = 5).
Replacing Na+ in the bath solution
with Li+ changed the reversal
potential of the amiloride-sensitive
I-V plot to +19.4 ± 1.2 mV (n = 5;
P < 0.0001). From the change in the
reversal potential and the Goldman-Hodgkin-Katz equation, we calculate
a
Li+-to-Na+
permeability ratio of 1.6. Thus, in agreement with previous studies in
oocytes,
,
,
-rENaC is more permeable to
Li+ than to
Na+ (6, 32, 43).
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Acidification inhibits
,
,
-rENaC currents.
To change pHi, oocytes were bathed
in sodium acetate solutions titrated to pH values from 7.4 to 6.4. H+-acetate
diffuses across plasma membranes and, once inside the cell, dissociates to H+ and
acetate
, thereby reducing
pHi (8, 39). The change in
pHi is proportional to the
pHo (8, 39). For example, reducing
pHo from 7.4 to 6.3 in an
acetate
-buffered bath
solution decreased pHi in
Xenopus oocytes from 6.8 to 6.1 (8).
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Voltage independence of pH-sensitive
,
,
-rENaC currents.
To determine whether the inhibition of ENaC by a reduction in
pHi is influenced by membrane
voltage, we examined the effects of voltage on ENaC currents
at pH 6.4. At positive voltages, the amiloride-sensitive currents
were small, and thus our analysis was restricted to
100 to 0 mV. The percent inhibition of the amiloride-sensitive ENaC current was similar at all voltages
and ranged from a low of 81.4 ± 2.7% to a high of 83.6 ± 5.3%
(n = 4). Thus the pH-induced
inhibition of
,
,
-rENaC is voltage independent. This result is
in agreement with studies in frog skin demonstrating that inhibition of
the apical membrane Na+
conductance is independent of voltage over the range from
100 mV
to
20 mV (16).
Effect of pHi on single-channel
,
,
-rENaC currents.
In the experiments described above, we did not measure
pHi in oocytes, and thus it was
not possible to describe the relationship between
pHi and ENaC activity. To
determine more accurately the relationship between
pHi and ENaC activity, we
conducted inside-out patch-clamp experiments in which we could control
the pH of the solution bathing the cytoplasmic side of the membrane
patch. In water-injected oocytes, we never observed ENaC-like channels
(i.e., amiloride-sensitive, ~5 pS,
Na+-selective channels); however,
ENaCs were readily identified in oocytes injected with
,
,
-ENaC
cRNAs. ENaCs were readily identified in cell-attached and inside-out
patches due to their low conductance (~5 pS) and slow gating kinetics
(e.g., open and closed times >1 s) (14). A representative experiment
demonstrating the effect of changing
pHi on ENaC activity is shown in
Fig. 6. Acidification of the solution
bathing the cytoplasmic surface of excised, inside-out membrane patches
from pH 7.4 to 6.4 decreased
NPo as fast as we could change the bath solution. Moreover, in every membrane patch studied, inhibition of channel activity was reversible when the pH was
returned to 7.4 (Fig. 7). Figure
8A
summarizes experiments examining the effects of
pHi over the range of 6.0-8.0
on channel NPo. A
reduction of pHi from 7.4 to 6.9 reduced NPo;
however, further reduction in pHi
to 6.4 and 6.0 had no additional effect on
NPo (Fig.
8A). In contrast, an increase in
pHi from pH 7.4 to 8.0 enhanced
NPo (Fig.
8A). These results demonstrate that
,
,
-rENaC is regulated by
pHi in the physiological range
(i.e., pH 7.0-7.4) (7, 29, 34).
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Effect of pH on different combinations of -,
-, and
-rENaC subunits.
To identify the subunit(s) of rENaC that are regulated by pH, we
examined the effects of reducing pH on various combinations of ENaC
subunits expressed in Xenopus oocytes.
As illustrated in Fig. 9, a reduction in
the pH of the sodium acetate-buffered bath solution from 7.4 to 6.4 reduced ENaC conductance in oocytes expressing
,
,
-ENaC,
,
-ENaC,
,
-ENaC, and
-ENaC. We did not examine the effect
of pH on
,
-rENaC because these subunits do not form
amiloride-sensitive Na+ channels
in oocytes (6). These studies demonstrate that the
-rENaC subunit is
regulated by pH and that
- and
-rENaC are not required for pH
regulation of the channel.
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Effect of pHi on -rENaC
in planar lipid bilayers.
To determine more accurately the relationship between
pHi and
-rENaC activity, we
examined the regulation of
-rENaC by
pHi in channels reconstituted in
planar lipid bilayers (Fig. 10). A sequential reduction in pHi (i.e.,
cis bath solution) from 7.4 to 6.9 and
to 6.5 progressively decreased the
Po from 0.62 ± 0.06 to 0.29 ± 0.05 to 0.08 ± 0.04 (P < 0.05), respectively, without changing the single-channel conductance
(n = 3-9/pH). In contrast, a
similar reduction in pHo
(i.e., trans bath solution)
had no effect on
Po (Fig. 10).
Thus the
-rENaC subunit expressed in planar lipid bilayers, like
-rENaC expressed in oocytes, is inhibited by a reduction
in pHi but not by a reduction in
pHo.1
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DISCUSSION |
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The major new finding of this study is that ,
,
-ENaC currents
are regulated directly by changes in
pHi in the physiological range but
not by changes in pHo. Although it
is possible that other proteins in addition to
,
,
-ENaC may
regulate Na+ channels in
epithelial cells, these subunits are not required for
pHi regulation of
ENaC.2
Acidification reduced and alkalinization increased channel activity by
a voltage-independent mechanism. Moreover, a reduction of
pHi reduced
Po, reduced
single-channel open time, and increased single-channel closed time,
without altering single-channel conductance. A reduction in
pHi also inhibited
,
-ENaC,
,
-ENaC, and
-ENaC currents. Thus our data demonstrate that
-ENaC alone is sufficient for pHi regulation of rENaC.
Other members of the ENaC/FaNaC/DRASIC/degenerin superfamily are also regulated by pH and are, like ENaC, H+-gated, Na+-selective channels. The recently cloned acid-sensing ion channel (ASIC), MDEG1, and DRASIC are activated directly by a reduction in the pH of the extracellular solution but not the intracellular solution (23, 41, 42). Accordingly, H+ not only affects ENaC-mediated Na+ absorption but also influences mechanotransduction, neurotransmission, and nociception by regulating Na+ uptake via MDEG1, FaNaC, and ASIC, respectively (23, 41, 42). It is interesting to note that ENaC may mediate salt taste perception in taste cells on the surface of the tongue and that sour taste, perhaps via H+, alters the perception of salt (14). Thus H+, in addition to regulating ENaC-mediated transepithelial Na+ transport, may also play a role in modulating the perception of salt by ENaC.
The decrease in ENaC activity with acidification of the cytoplasmic solution is consistent with observations that a reduction in pHi inhibits Na+ absorption across frog skin (16, 40) and toad bladder (28). In addition, a reduction in pHi also reduces the Na+ permeability of the mucosal membrane of frog skin (16, 40), toad bladder (28), and principal cells in rabbit CCD (38). In contrast, a reduction in pH has been reported to stimulate Na+ transport in toad bladder (12, 21). Moreover, neither acidosis nor alkalosis affects Na+ absorption across rabbit CCD perfused in vitro (4, 38) or rat distal tubule perfused in vivo (37). These apparent differences in the reported effects of pH may be related to the fact that a number of parameters in addition to the Na+ permeability of the mucosal membrane influence transepithelial Na+ absorption, including the electrochemical gradient for Na+ across the mucosal membrane and the rate of Na+ efflux across the basolateral membrane via the Na+-K+-ATPase, all of which may be regulated by pHi (38, 45).
Changes in pHi between 8.0 and
7.0, but not pHo, influenced ENaC
gating. Several different molecular mechanisms have been proposed to
account for pH-dependent regulation of ion channels. First, protons may
bind in the channel pore and block the channel, as proposed for the
voltage-dependent proton block of the frog nerve
Na+ channel (44). However, because
inhibition of ENaC by H+ is
voltage independent, it is unlikely that
H+ inhibits the channel by this
mechanism. Second, protons may bind near the pore and influence ion
conduction through an electrostatic interaction. Channel subconductance
states caused by this type of regulation have been observed for the
Torpedo
Cl channel (15), the
olfactory cyclic nucleotide-gated cation channel (33), and the
ATP-sensitive cardiac inwardly rectifying K+ channel (11). This type of
inhibition is also unlikely for ENaC, because a reduction in
pHi neither affected the channel conductance nor promoted the appearance of subconductance states (Fig.
6). Third, changes in pH may regulate ENaC currents in part by altering
interfacial potentials (17). Finally, protonation of key amino acid(s)
may alter channel conformation and shift the equilibrium between the
open and closed state such that the closed state is favored. Although
our analysis of single-channel open and closed times favors this
mechanism, additional studies are required to determine how protons
regulate ENaC.
Alterations in pHi but not
pHo regulate ENaC, and thus it is
likely that amino acids in the amino and carboxy termini of -rENaC, which are thought to be cytoplasmic, are involved in pH regulation (36). A highly conserved histidine (His-94) in the amino terminus of
-rENaC is a likely candidate. In preliminary studies, we found that
mutation of His-94 eliminated ENaC currents in oocytes, thereby precluding an examination of the role of His-94 in the regulation by
protons. There are numerous other highly conserved candidate amino
acids in the amino and carboxy termini (5, 6, 14, 24). Additional
studies are required to address the role of these amino acids in pH
modulation of ENaC activity.
In summary, we report that ,
,
-ENaC is regulated directly by
changes in pHi in the
physiological range but not by changes in
pHo. Although expression of all
three subunits is required for maximum ENaC currents, expression of
-rENaC alone is sufficient for
pHi regulation of rENaC. Although
several proteins may associate with and regulate the
,
,
-ENaC
complex, it does not appear that these regulatory subunits are required
or necessary for pHi regulation.
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ACKNOWLEDGEMENTS |
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B. A. Stanton is grateful for the support and encouragement of Dr. Ralph E. Stanton, who published his first paper on pH regulation of cell function in 1923 (37a). We thank Flora Ciampolillo for valuable technical assistance.
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FOOTNOTES |
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These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-34533, DK-51067, and DK-07301 (to B. A. Stanton) and DK-37206 (to D. J. Benos) and a grant from the National Kidney Foundation (to M. L. Chalfant).
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.
1
The responses to changes in
pHi are somewhat different in
,
,
-ENaC expressed in oocytes (Fig. 8) vs.
-ENaC expressed
in planar lipid bilayers (Fig. 10). A reduction in
pHi below 7 has no additional
effect on
,
,
-ENaC (Fig. 8), whereas a reduction in
pHi below 7 decreases
-ENaC
activity (Fig. 10). This difference suggests, but does not prove, that
the
- and
-ENaC subunits may influence the
pHi sensitivity of the
-ENaC
subunit. It is also possible that the response to changes in
pHi may depend on the experimental
preparation (i.e., oocytes vs. bilayers). It is important to note,
however, that this subtle difference in the response to
pHi does not change our major
conclusion that changes in pHi
within the physiological range (i.e., 7.0 to 7.4), but not
pHo, modulate ENaC activity.
2 It is possible that the regulatory proteins associated with ENaC in epithelial cells are also expressed in Xenopus oocytes and present in lipid bilayers. Thus we cannot categorically exclude the possibility that pH regulates ENaC indirectly. However, indirect regulation by pHi is unlikely for two reasons. First, PKA does not activate ENaC in lipid bilayers or oocytes (2), most likely because the regulatory subunit that is phosphorylated by PKA is not present in bilayers or expressed in oocytes. Second, studies by Benos and colleagues (3) have shown that recombinant ENaC synthesized by rabbit reticulocytes and ENaC isolated from Xenopus oocytes have similar biophysical properties and are regulated in a similar fashion in lipid bilayers. Thus, because channels produced in vitro by rabbit reticulocytes are unlikely to be associated with regulatory proteins, the most parsimonious conclusion is that pHi directly regulates ENaC.
Address for reprint requests: B. A. Stanton, Dept. of Physiology, Dartmouth Medical School, 615 Remsen Bldg., Hanover, NH 03755-3836.
Received 12 August 1998; accepted in final form 6 November 1998.
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