1 Department of Molecular and Cellular Physiology and 2 Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0576
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ABSTRACT |
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Rabbit and human ClC-2G
Cl channels are voltage
sensitive and activated by protein kinase A and low extracellular pH.
The objective of the present study was to investigate the mechanism involved in acid activation of the ClC-2G
Cl
channel and to determine
which amino acid residues play a role in this acid activation. Channel
open probability
(Po) at ±80 mV holding potentials increased fourfold in a concentration-dependent manner with extracellular H+
concentration (that is, extracellular pH,
pHtrans), with an
apparent acidic dissociation constant of pH 4.95 ± 0.27. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide-catalyzed amidation of the channel with glycine methyl ester increased
Po threefold at
pHtrans 7.4, at which the channel
normally exhibits low
Po. With
extracellular pH reduction (protonation) or amidation, increased
Po was due to a
significant increase in open time constants and a significant decrease
in closed time constants of the channel gating, and this effect was
insensitive to applied voltage. With the use of site-directed
mutagenesis, the extracellular region EELE (amino acids
416-419) was identified as the pH sensor and amino acid Glu-419
was found to play the key or predominant role in activation of the
ClC-2G Cl
channel by
extracellular acid.
human and rabbit chloride channels; Xenopus laevis oocytes; cystic fibrosis; acid-activated chloride channel; protein kinase-regulated chloride channel; protein kinase A; gastric secretion; epithelial transport; chloride transport; water-soluble carbodiimide; amidation
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INTRODUCTION |
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THE CHLORIDE CHANNEL, ClC-2G, was cloned
from the rabbit stomach (16), human intestine (2), and human lung and
stomach (23) and has also been shown to be present in a variety of
rabbit tissues (7). It is voltage dependent and activated by protein kinase A (PKA; Ref. 16). In the stomach, it is involved in acid secretion and is present in the apical membrane of the gastric parietal
cell (3, 16), which is exposed to a highly acidic environment
(approximate pH < 1). In this context, acid stability of ClC-2G
Cl channel function is
essential. In addition, rabbit and human ClC-2G
Cl
channels are activated
by extracellular acid (low
pHtrans) in the range of pH of
the gastric secretions (3, 16, 23).
The ClC-2G Cl channel is a
member of the rapidly expanding ClC family of
Cl
channels, which includes
at least nine different mammalian members (11). The common features of
this channel family are that they are voltage gated and their predicted
transmembrane topology is similar (11, 12). The location of the pore of
the channel is not known and has been variably suggested to be near the
carboxy-terminal (9) or the amino-terminal (5) transmembrane region.
Activation of both the rabbit and the human ClC-2G
Cl channels by low
extracellular pH has recently been confirmed by Furukawa et al. (8) and
Schwiebert et al. (22), respectively, but the mechanism involved was
not investigated. In addition, rat ClC-2 (13), rat ClC-1 (20), and
human ClC-1 (4) have also recently been shown to be affected by changes
in extracellular pH. Several different mechanisms have been suggested
to mediate these effects.
The objective of the present study was to investigate the mechanism
involved in acid activation of the ClC-2G
Cl channel as well as the
molecular basis for this acid activation. The half-maximal effect
occurred at an extracellular pH of ~5.0, suggesting that the effect
on channel activity occurred through neutralization of the negative
charge of carboxy groups by protonation. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC)-catalyzed amidation of reactive carboxy groups with glycine methyl ester (GME) (18) was
used to test this hypothesis and to probe the topology and accessibility of the potential pH activation site on the extracellular surface of the channel. This treatment activated the channel. To
investigate the molecular basis for activation of the ClC-2G Cl
channel by low
extracellular pH, a negatively charged potential pH sensor region
between predicted transmembrane regions D8 and D9 was examined using
site-directed mutagenesis. Analysis of the effect of low extracellular
pH on the channel gating was carried out to investigate the mechanism
of acid activation of the ClC-2G Cl
channel.
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MATERIALS AND METHODS |
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Expression in Xenopus laevis oocytes and measurement of
Cl channel activity.
Rabbit ClC-2G Cl
channel
cRNA was made from the wild-type and mutant cDNAs using the mMessage
mMachine cRNA kit (Ambion). Xenopus
laevis oocytes were prepared and injected with cRNA as described by Malinowska et al. (16). One day after isolation, oocytes
were injected with 10 ng cRNA/50 nl
H2O per oocyte. After 4-5
days at room temperature, oocyte plasma membrane vesicles expressing
the recombinant Cl
channel
protein were isolated for electrophysiological studies as previously
described (16). The equipment and procedures for vesicle incorporation
into the bilayer and single-channel recording were essentially the same
as previously described by Cuppoletti et al. (3). Routinely, 100-s
channel recordings were obtained at set holding potentials and filtered
at 500 Hz. Identification of events and determination of dwell times
and amplitudes as well as curve fitting were carried out using the
pCLAMP program (version 5.5). Solutions for measuring
Cl
channel activity in the
bilayer contained 800 mM tetraethylammonium (TEA) chloride, 10 mM EGTA
(pH 7.4), 2 mM MgCl2, and 1 mM ATP and were buffered with either 10 mM TEA-HEPES (pH 7.4) or TEA citrate
(pH 3.0) as indicated. In titration experiments, concentrated citric
acid was added to the solutions on the
trans side of the bilayer in
concentrations sufficient to give the indicated pH, as determined in
separate measurements of pH. PKA catalytic subunit (50 U/ml) was added
to the cis side of the bilayer, which
corresponds to the cytosolic side of the channel. The electrical
reference side of the bilayer was the
trans side, which corresponds to the extracellular side of the channel. In most cases, two
channels were incorporated into the bilayer. However, occasionally a
single channel or three channels were observed. When two or more
open-state current levels were observed, which were even multiples of
the lowest open-state current level, they were interpreted as
reflecting the activity of two or more (as appropriate) channels
present in the bilayer. All open probability
(Po) values
reported have been corrected for the number of channels present.
Single-channel Po
was calculated using the relationship
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Site-directed mutagenesis. Mutants of the rabbit ClC-2G cDNA were prepared using the Transformer site-directed mutagenesis kit (Clontech), and the sequences of resulting mutant cDNAs were fully verified. Single independent clones of each mutant were functionally characterized. The following mutants of region E416-E417-L418-E419 (EELE) were prepared: E416Q-E417Q (QQLE), E419Q (EELQ), E419G (EELG), and E416Q-E417Q-E419G (QQLG). In addition, mutant V349E was prepared.
Materials. Palmitoyl-oleoyl-phosphatidylethanolamine and palmitoyl-oleoyl-phosphatidylserine (Avanti Polar Lipids) were dissolved in n-decane. GME and PKA catalytic subunit were from Sigma. DRIREF electrodes were obtained from World Precision Instruments (Sarasota, FL). EDC was obtained from Pierce. Primers for sequencing, mutagenesis, and selection were synthesized at the Univ. of Cincinnati College of Medicine DNA Core Facility.
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RESULTS |
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Effect of pHtrans reduction on
rabbit ClC-2G Cl channel
Po and gating.
Rabbit (3, 16) and human (22, 23) ClC-2G
Cl
channels are activated
by low pHtrans. The effect of
pHtrans 3.0 on rabbit ClC-2G
Cl
channel activity is
shown in Fig.
1A. A
channel recording at
80 mV is shown. Two channels
are present in the bilayer membrane as shown in the recordings
(left) and amplitude histograms
(right). At
pHtrans 7.4, both channels were
evident, and, when pHtrans was
reduced to 3.0, activity of both channels increased. The concentration
dependence of the increase in channel Po (normalized
with respect to the number of channels, see MATERIALS AND METHODS) in response to changes in extracellular
acidification was then measured. The results are summarized in Fig.
1B. At
80 mV holding potential,
Po significantly
(P < 0.001) increased from 0.22 ± 0.02 (n = 25, N = 10) to 0.36 ± 0.04 (n = 14, N = 6) when pHtrans was decreased from 7.4 to
6.0. Between pHtrans 6.0 and 4.5, there was no significant change in channel
Po. However, lowering pHtrans further from pH
4.5 to 3.0 resulted in an additional significant
(P < 0.001) increase in
Po to 0.79 ± 0.07 (n = 6, N = 3). Similar results were obtained
at +80 mV holding potential (Fig.
1B).
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Neutralization by amidation of the negative charge of carboxy groups
facing the extracellular side of the ClC-2G
Cl channel.
If neutralization of the negative charge of carboxy groups by
protonation (see Fig. 1) is responsible for the change in
Cl
channel activity,
amidation with GME catalyzed by the water-soluble EDC, both added to
the trans side of the bilayer, would result in a similar
neutralization of charge and might therefore activate the channel.
Channel activation by amidation will only occur if the target amino
acids are carboxy groups (and not histidines) and are easily accessible
to the bulky water-soluble compounds GME and EDC (18). Figure
3 shows the effect of amidation on ClC-2G
Cl
channel activity. A
channel recording at pHtrans 7.4 and
80 mV holding potential is shown in Fig.
3A. Two channels were present in the
bilayer membrane, and these displayed minimal activity as illustrated
by the corresponding amplitude histogram. Addition of 1 mM EDC alone to
the solution in the trans compartment of the bilayer had no effect, but, when 10 mM GME was subsequently added to the trans side, channel
activity increased, with a further increase when
pHtrans was reduced to 3.0. The
data are summarized in Fig. 3B. As
found with protonation by lowering
pHtrans, amidation with EDC and
GME at pHtrans 7.4 and at
80 mV holding potential resulted in a significant increase
(P < 0.01) in channel
Po from 0.16 ± 0.02 (n = 40, N = 19) to 0.55 ± 0.05 (n = 19, N = 13) (Fig.
3B). The
Po of the
amidated channel was ~75% of the value obtained by reduction of the
pHtrans of the untreated channel
to pH 3.0. When pHtrans was lowered to 3.0 in the presence of EDC and GME,
Po further
increased significantly (P < 0.001)
to 0.89 ± 0.04 (n = 16, N = 7), which was not significantly
different from the
Po at
pHtrans 3.0 in the absence of EDC
and GME. Similar results were obtained at +80 mV holding potential. EDC
alone (Fig. 3) and GME alone (data not shown) had no effect on channel
Po, ruling out
the possibility of intramolecular cross-linking or noncovalent binding
as responsible for the effect. Analyses of the open and closed times of
channel gating (Table 2) showed that
increased channel
Po with EDC and GME at pHtrans 7.4 was due to
significantly increased open time constants
(
1 and
2) and significantly
decreased closed time constants
(
1 and
2) at ±80 mV. However, no
additional change in the time constants occurred due to lowered
pHtrans following amidation at
pHtrans 7.4 at ±80 mV, except for a
significant but minor effect on the closed time constants at
80
mV (P < 0.02;
P < 0.01, respectively, for
1 and
2). These findings suggest
that neutralization of one or more negatively charged carboxy groups at
the extracellular surface of the channel protein is involved in acid
activation of the ClC-2G Cl
channel and that histidines are not involved in the activation.
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Identification of the pH sensor region of the rabbit ClC-2G
Cl channel.
A potential pH sensor region was identified in the loop between
transmembrane domains D8 and D9 of the channel. This region has been
shown to be extracellular through glycosylation studies (14, 21) and
contains a negatively charged region with three glutamic acid residues
in close proximity: E416-E417-L418-E419 (EELE). The effects of
replacing these negatively charged residues with neutral, isosteric
glutamines by site-directed mutagenesis were investigated. QQLE and
EELQ ClC-2G Cl
channel
mutants were expressed, and single-channel recordings were obtained
throughout the voltage range from
80 mV to +80 mV. Figure
4 shows channel recordings and amplitude histograms at
80 mV holding potential obtained using these two mutant
channels. In a comparison of mutant channel activity at
pHtrans 3.0 and that at
pHtrans 7.4, at
80 mV, the
QQLE mutant channel retained acid activation. In contrast, the EELQ
mutant channel lost its acid activation. The summarized data are shown in Fig. 5A. At
80 mV, the QQLE mutant channel retained significant (P < 0.001) (~6-fold) acid
activation. Although the QQLE mutant channel exhibited significantly
lower Po at both
pHtrans 7.4 (P < 0.001) and 3.0 (P < 0.02) compared with the
wild-type channel, its
Po at
pHtrans 3.0 was nevertheless
~75% of the Po
of the wild-type channel at
pHtrans 3.0. This value was
similar to that of the amidated wild-type channel at pHtrans 7.4. In contrast, the
EELQ mutant channel did not exhibit acid activation and
Po values at both
pHtrans 7.4 and 3.0 at
80
mV were significantly (P < 0.001)
decreased compared with the wild-type channel. Similar results were
obtained at +80 mV holding potential (Fig.
5B). Corresponding open and closed
time constants (
1 and
2) at ±80 mV for the
mutant channels are shown in Table
3. With the QQLE
mutant channel, low pHtrans resulted in significantly increased (P < 0.001) open time constants and significantly decreased
(P < 0.01;
P < 0.02) closed time constants, as
observed with the wild-type channel. With the EELQ mutant channel, low
pHtrans had no effect on open
time constants, whereas a significant but minor effect on closed time
constants was obtained at
80 mV
(P < 0.05;
P < 0.02 for
1 and
2, respectively), resulting in
the channel being virtually closed. The conductance of these two mutant
channels remained similar to that of the wild-type channel (Table 1).
These results strongly indicate that E419 is of predominant importance
to acid activation of the ClC-2G Cl
channel, mediating
~75% of the maximal acid activation effect. E419 provides negative
control over channel gating, which is relieved by neutralization.
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DISCUSSION |
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The ClC-2G Cl channel is
voltage and PKA activated (16, 23). ClC-2G
Cl
channels are
significantly activated by low extracellular pH without affecting the
voltage dependence of activation (3, 8, 16, 23). This was shown in
studies using native parietal cell apical membranes (3) and using
recombinant rabbit (16) and human (23) ClC-2G
Cl
channels. The goal of
the present study was to investigate the mechanism of acid activation
of the ClC-2G Cl
channel
and to localize the region involved in this activation at the molecular
level.
The concentration-dependent response to change of
pHtrans covered the range from pH
7.4 to pH 3.0. Extremes of low pH are physiologically relevant for the
ClC-2G Cl channel in the
context of the gastric parietal cell, where it is likely to be involved
in gastric acid secretion. Reduction of pH on the extracellular surface
of the channel from pH 7.4 to pH 3.0 induced a fourfold increase in
ClC-2G Cl
channel
Po to a value
approaching 0.80. Increased channel
Po was due to
significantly increased open time constants and significantly decreased
closed time constants, which indicates that lowering the
pHtrans affects the channel
gating mechanism.
Channel Po increased in a concentration-dependent manner with increasing H+ concentration. Channel Po was half of its maximal value at pHtrans 4.95. This value was interpreted to be the apparent pKa of an amino acid involved in pH sensor function. Aspartic acid, glutamic acid, and histidine residues are all likely candidates for this role, with respective pKa values of 3.85, 4.25, and 6.0 for each amino acid in free solution (25). Although the value obtained is closest to the value for glutamic acid, the pKa of amino acids is known to be influenced by the environment. The hydrophobicity of the environment of the amino acid, as well as the presence of other charged amino acids in its vicinity, could alter its pKa. In addition, the nature of the solution bathing the amino acid could have an effect on the pKa, depending on how closely the microenvironment around the residue reflects that of the bulk solution.
It was therefore necessary to determine whether the amino acid
responsible for acid activation was exposed to the extracellular surface of the channel and to distinguish between carboxylic amino acids and histidine. If the increased
Po resulted from
protonation and thus neutralization of one or several charged amino
acids, then neutralization by amidation should lead to the same effect, namely, an increase in
Po. Amidation by
EDC occurs under very mild conditions and GME forms a neutral adduct.
Both EDC and GME are hydrophilic and relatively bulky. If amidation
from the outer surface affects channel activity, this would imply
accessibility of the residue or residues to the solution bathing the
extracellular surface of the channel. Amidation with EDC and GME at
pHtrans 7.4 led to a threefold
increase in channel
Po that
approximated the
Po obtained with
the QQLE mutant channel at
pHtrans 3.0. This
Po is ~75% of
that obtained for the wild-type channel under the same conditions. When
pHtrans was reduced to pH 3.0, the Po of the
amidated ClC-2G channel increased to that of the untreated channel at
pHtrans 3.0. This additional increase in Po at
low pH could be due to acid-induced exposure of new amino acids to the
reagents after the initial modification of a single, highly reactive
amino acid and/or the known increase in the amidation reaction
rate at low pH (18) or protonation of other sites, which are not
initially accessible to the reagents. Noncovalent binding of either EDC
or GME could affect channel function, and, in the absence of GME,
formation of linkages between carboxy and amino groups of the protein
could occur. However, neither EDC alone (Fig. 3) nor GME alone (not
shown) affected ClC-2G Cl
channel Po or
open or closed time constants (Table 2). These findings suggest that
the pH sensor is likely to be a carboxylic acid and not histidine,
since histidine is not reactive with EDC (18).
Because these large water-soluble compounds were added only to the trans chamber (corresponding to the outside of the channel), the susceptible amino acid(s) must be exposed at the extracellular surface of the channel and to the medium. Amino acid charge neutralization, whether by protonation or by formation of a neutral product through amidation, affected the Po and the closed and open time constants. There are numerous carboxylic amino acid residues in the ClC-2G channel protein (16). Although the primary reaction catalyzed by EDC is with carboxylic amino acids under these mild conditions, the amidation results with EDC must also be interpreted within the framework of knowledge that EDC can react with amino acids other than carboxylic acids and that EDC can gain access to amino acids that may be partially buried within the structure of proteins (18).
All members of the ClC Cl
channel family have a similar predicted topology of 10-12
transmembrane domains with both amino and carboxy termini localized to
the cytoplasmic or intracellular side (21). With the assumption that
the pH sensor contains carboxylic amino acid residues facing the
extracellular environment of the channel, a region of the protein that
contains a cluster of negatively charged amino acids and is known to be
extracellular was selected for investigation as a possible pH sensor.
Although the entire topology has not yet been experimentally verified,
the loop between the D8 and D9 transmembrane domains has been shown by
glycosylation experiments and protease protection assays (14, 19, 21) to be extracellular. In the rabbit ClC-2G
Cl
channel, this loop
contains a cluster of negatively charged glutamic acid residues E416,
E417, and E419 (EELE). Therefore, to investigate the molecular basis of
acid activation of the rabbit ClC-2G
Cl
channel, these glutamic
acid residues were modified and channel function was studied. The
effects of pHtrans on channel Po, open and
closed time constants, and voltage dependence of activation were
measured.
The results with the EELQ and QQLE mutant channels suggest that E419
plays the key or predominant role in acid-activated gating of the
rabbit ClC-2G Cl channel.
The reduction of negative control of the EELG mutant channel, along
with loss of acid activation, demonstrates that the structure of the
region containing EELE as well as charge neutralization is important to
acid activation of channel gating. The voltage dependence of acid
activation of the QQLG mutant channel illustrates that acid activation
of the ClC-2G channel can occur through other mechanisms, perhaps
through changes in surface potential (10) that do not affect channel
gating.
Recently, it has been shown by whole cell current studies of rat ClC-2
and several other members of the ClC
Cl channel family, that
changes in extracellular pH result in increases in the
Cl
current. In rat (20) and
human ClC-1 (4), lowering extracellular pH from 7.0 to 5.5 increased
the Cl
current in the
hyperpolarized range of the membrane potential (~1.3- to 1.6-fold at
80 mV), whereas there was no effect in the depolarized range and
there was no effect on the time constants of the
Cl
currents. These authors
concluded that low extracellular pH affects the voltage dependence of
activation of these channels. In studies of rat ClC-2 (13), the time
constants of the Cl
currents were not analyzed. However, because acid activation did not
occur at positive membrane potentials, the authors concluded that low
extracellular pH affected the voltage-dependent activation of these
channels.
The effect of extracellular acidification of rabbit ClC-2G has also
been studied by measurement of whole cell
Cl currents in
Xenopus oocytes (8). Lowering
extracellular pH from 7.6 to 3.6 resulted in an increase in the
Cl
current in both negative
and positive ranges of membrane potential. Similar findings were
obtained with whole cell current measurements of human ClC-2G
Cl
channels overexpressed
in IB3 cells (22), indicating that acid activation is independent of
voltage. Although the mechanism of activation was not investigated, the
results obtained with rabbit and human ClC-2G using whole cell current
measurements are in full agreement with those at the single-channel
level as shown in the present study but are in contrast to the findings
with rat ClC-2 and ClC-1.
In whole cell recordings, rat ClC-2 (13) and rabbit (8) and human (22)
ClC-2G Cl currents
displayed a slowly activating process and no deactivation, but gating
models were not suggested in these studies. ClC-0, which has both fast
and slow gating modes, has been described with a six-state Markovian
model with a minimum of six time constants (1, 15). For ClC-1, which
has slow, fast, and ultrafast transitions, a five-state model with six
time constants has been proposed (4). To date, none of the members of
the ClC Cl
channel family
has shown simple two-state gating (open-closed) with only two time
constants,
open and
closed. In the present study,
two channels were generally present, and the "closed" and "open" times may differ from open and closed times that might be
obtained with single-channel studies (see MATERIALS
AND METHODS) or after "correction" (17).
However, the open and closed times obtained were best fit by two
exponentials with time constants
1 (fast) and
2 (slow), suggesting that the
gating mechanism of the ClC-2G
Cl
channel could be
described by at least a three-state Markovian model with fast and slow
gating modes and a minimum of four time constants. Similar behavior at
the single-channel level has been described for the cardiac
Ca2+ release channel (26) and the
amiloride-sensitive Na+ channel
(6). A model for the ClC-2G
Cl
channel would require a
detailed analysis of the lifetime distribution of each state and
fitting by computer simulation, which is beyond the scope of the
present studies.
The region EELE in the extracellular loop between transmembrane domains
D8 and D9 is conserved in both rabbit and human ClC-2G Cl channels (16, 23),
whereas, in the rat ClC-2
Cl
channel, this region
contains the sequence EDLG. Different amino acid composition between
the rat ClC-2 and the human and rabbit ClC-2G
Cl
channels, especially in
position 419, may be responsible for the different mechanisms in
responses to extracellular pH changes.
In rat ClC-2, a point mutation of residue V352E led to a constitutively
open channel and loss of pH sensitivity (13). Channel function of the
corresponding V349E mutant of the rabbit ClC-2G channel was examined,
and significant differences were found. In direct contrast with the
findings with rat ClC-2 (13), the V349E mutant of the rabbit ClC-2G
Cl channel did not lead to
a constitutively open Cl
channel with loss of pH sensitivity. Instead, the rabbit V349E channel
mutant had not only a significant level of pH sensitivity but also had
a reduced channel
Po compared with
the wild type at pHtrans 7.4 and
3.0. In addition, this was the only mutant that showed altered
(reduced) conductance compared with the wild-type channel, suggesting
that the mutation may be affecting the folding of the protein in the
vicinity of the channel pore. These results may reflect the differences
in amino acid sequence between the ClC-2 and ClC-2G
Cl
channels and the
different methodologies used to study channel function.
In summary, the EELE419 sequence within the extracellular D8-D9 loop
region in the rabbit ClC-2G
Cl channel is involved in
activation of the channel by low
pHtrans. Analyses of the channel
open and closed time constants indicate that this region exerts
negative control of channel gating. Charge neutralization and the
structure of this region are both important to the control of channel
gating. Of these amino acids, E419 plays the major role.
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ACKNOWLEDGEMENTS |
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We thank Lisa M. Knapp for her help with the initial experiments performed and Dr. J. W. Hanrahan for helpful discussions regarding analysis of multichannel records.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-43816, DK-43377, and DK-58399 and the Cystic Fibrosis Foundation Award CUPPOL96PO to J. Cuppoletti and D. H. Malinowska.
Address for reprint requests: J. Cuppoletti, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, PO Box 670576, Cincinnati, OH 45267-0576.
Received 30 June 1997; accepted in final form 10 July 1998.
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