Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0576
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ABSTRACT |
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ClC-2 Cl channels represent a potential target for
therapy in cystic fibrosis. Key questions regarding the feasibility of using ClC-2 as a therapeutic target are addressed in the present studies, including whether the channels are present in human lung epithelia and whether activators of the channel can be identified. Two
new mechanisms of activation of human recombinant ClC-2
Cl
channels expressed in HEK-293 cells were identified:
amidation with glycine methyl ester catalyzed by
1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) and treatment with
acid-activated omeprazole. ClC-2 mRNA was detected by RT-PCR. Channel
function was assessed by measuring Cl
currents by patch
clamp in the presence of a cAMP-dependent protein kinase (PKA)
inhibitor, myristoylated protein kinase inhibitor, to prevent
PKA-activated Cl
currents. Calu-3, A549, and BEAS-2B cell
lines derived from different human lung epithelia contained ClC-2 mRNA,
and Cl
currents were increased by amidation,
acid-activated omeprazole, and arachidonic acid. Similar results were
obtained with buccal cells from healthy individuals and cystic fibrosis
patients. The ClC-2 Cl
channel is thus a potential target
for therapy in cystic fibrosis.
lung chloride channels; lung epithelia; 1-ethyl-3(3-dimethylaminopropyl) carbodiimide; pH-activated ion channels; water-soluble carbodiimides; Calu-3; A549; BEAS-2B; buccal cells
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INTRODUCTION |
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MUTATIONS in the
cystic fibrosis transmembrane regulator (CFTR) lead to death from lung
disease. Activation of other Cl channels including ClC-2
in the respiratory epithelia is a potential treatment for cystic
fibrosis (4, 6, 7, 9, 35, 37). Two key questions
include whether ClC-2 Cl
channels are present in the
epithelia of the lung and whether activators of ClC-2 Cl
channels can be identified. The patch-clamp studies of recombinant and
native human ClC-2 presented address these questions.
A number of studies have appeared exploring the possibility that ClC-2
Cl channels might serve as a potential target for therapy
in cystic fibrosis. It has been shown that ClC-2 Cl
channel expression in epithelial cells from cystic fibrosis patients can correct the defect in Cl
transport in vitro
(35). Keratinocyte growth factor (KGF) has been shown to
cause CFTR-independent changes in lung morphogenesis in vivo and to
raise the levels of ClC-2 Cl
channel protein in mouse
lung explants in vitro (2). KGF appears to act through
inhibition of degradation of the ClC-2 channel protein, providing a
possible means of upregulation of the channel protein level using KGF.
In rat lung, reduction of transcription of ClC-2 channel protein occurs
at birth (27), and transcription factors that control the
level of mRNA have been identified (3, 27). Nevertheless,
ClC-2 mRNA has been shown to be present in adult human lung
(37).
Extremes of reduction in extracellular pH (8, 26, 35, 37, 38,
40) and low concentrations of arachidonic acid (40)
previously have been shown to activate ClC-2 Cl channels.
In planar lipid bilayer studies (38), amidation of the
ClC-2 Cl
channel with glycine methyl ester (GME)
catalyzed by the water-soluble carbodiimide
1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) was shown to
increase open probability of the channel. Omeprazole is an anti-ulcer
agent that, when activated by acid, forms a charged species that reacts
with cysteines on a variety of proteins (1, 21, 24, 28, 30, 33,
42). One or more of the cysteines in ClC-2 may be extracellular
and accessible to reaction with acid-activated omeprazole.
The effects of these treatments were studied by whole cell patch clamp using recombinant ClC-2 in HEK-293 cells, in three human lung cell lines derived from different epithelia of the lung, and in human buccal epithelial cells from healthy individuals and cystic fibrosis patients. In parallel, mRNA for ClC-2 and CFTR was detected using RT-PCR. The results suggest that human ClC-2 is present in human cells and cell lines and that it can be activated by a variety of treatments, including arachidonic acid and covalent modification by amidation and acid-activated omeprazole.
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METHODS |
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Transfected HEK-293 cells. HEK-293 cells that had been stably transfected with His- and T7-tagged human ClC-2 cDNA in the mammalian expression vector pcDNA3.1 with the use of Lipofectamine were prepared and maintained as previously described (40). Mock transfected cells were prepared with the same vector but expressed a different protein as previously described (40). Stably transfected cell lines were grown and kept frozen as stocks.
Cell culture. A549 and BEAS-2B cells were grown in DMEM (4.5 g/l glucose, 4 mM L-glutamine) containing 8% fetal bovine serum (FBS) and supplemented with 2 mM L-glutamine. Calu-3 cells were grown in DMEM/F-12 medium with 15% FBS in collagen-coated flasks as previously described (18, 36).
ClC-2, CFTR, and -actin
mRNA detected by RT-PCR.
mRNA was prepared from cultured cells (Calu-3, A549, and BEAS-2B) and
fresh buccal scrapings (pooled from 4-5 individuals) using
Oligotex Direct mRNA columns (Qiagen). The mRNAs were then treated with
DNase I (RNase free). First-strand cDNA was synthesized using the
SuperScript preamplification system. cDNA was amplified with the use of
Ex-Taq polymerase and sequence-specific primers for ClC-2, CFTR, and
-actin. The primers used were, for ClC-2, 5'-ggcactgcacaggaccaaga-3'
(sense) and 5'-cttgccagggagattcggac-3' (antisense); for CFTR,
5'-ggacagttgttggcggttgctgg-3' (sense) and 5'-cgcttctgtatctatattcatcataggccacacc-3' (antisense); and for
-actin, 5'-gtccctgtatgcctctggtc-3' (sense) and
5'-tcgtactcctgcttgctgat-3' (antisense), producing cDNA fragments of
468, 210, and 669 bp for ClC-2, CFTR, and
-actin, respectively. PCR
conditions consisted of denaturing for 45 s at 94°C; annealing
for 45 s at 53-55°C for ClC-2, 61°C for CFTR, and 57°C
for
-actin; and elongating for 2 min at 72°C. cDNAs were amplified
for 30-40 cycles for ClC-2, 40 cycles for CFTR, and 30-40
cycles for
-actin. Amplified cDNA products were separated on 2%
(ClC-2 and CFTR) or 1% (
-actin) agarose gels containing ethidium
bromide. Three negative controls containing no cDNA in the PCR
reaction, no mRNA in the RT reaction, and no reverse transcriptase in
the RT reaction as well as one positive control containing cDNA for
ClC-2, CFTR, and
-actin in the PCR reaction were always performed
with each amplification. Amplified cDNAs for ClC-2 and CFTR
were confirmed by sequence analysis.
Measurement of whole cell Cl
currents.
Currents were elicited by voltage-clamp pulses (1,500-ms duration)
between +40 and
140 mV in 20-mV increments from a beginning holding
potential of
30 mV as previously described (40).
Currents were measured 50-100 ms after start of the pulse. The
external solution was normal Tyrode solution containing (in mM) 135 NaCl, 1.8 CaCl2, 1 MgCl2, 5.4 KCl, 10 glucose,
and 10 HEPES, pH 7.35 or as indicated. The pipette solution was (in mM)
130 CsCl, 1 MgCl2, 5 EGTA, and 10 HEPES, pH 7.35. In some
cases, where indicated, 1 mM ATP-Mg2+ (pH 7.4) was also
present in the pipette. When indicated, solutions also contained 0.8 µM myristoylated protein kinase inhibitor (mPKI). Amidation was
carried out with 1 mM EDC followed by 10 mM GME as previously described
(38). Omeprazole was dissolved in dimethyl sulfoxide
(DMSO) and diluted threefold into pH 4.0 citric acid for 15 min to
activate it. The acid-activated omeprazole solution (40 mM omeprazole)
was then added at a final concentration of 100 µM to cells. Freshly
prepared arachidonic acid in DMSO was diluted into the bath solution,
resulting in a final concentration of 1% DMSO. The free-acid form of
arachidonic acid shipped under inert gas was used. All precautions
recommended by the manufacturer were taken to prevent oxidation of
arachidonic acid, including the use of solutions in organic solvent,
storage of stock solutions in organic solvent at
20°C in sealed
containers, and protection from light. All measurements with compounds
in DMSO were compared with controls containing 1% DMSO alone. Pipettes
were prepared from borosilicate glass and pulled by a two-stage
Narashige puller to give 1- to 1.5-M
resistance. Data were acquired
with an Axopatch CV-4 headstage with a Digidata 1200 digitizer and an
Axopatch 1D amplifier. Data were analyzed with pCLAMP 6.04 (Axon
Instruments, Foster City, CA), Lotus 123 (Microsoft), and Origin
(Microcal) software. Statistical significance of the difference between
two means was determined with the Student's t-test with
n representing the number of cells.
Human buccal cells. Buccal cells were harvested from healthy volunteers and cystic fibrosis patient volunteers with a sterile cytology brush. Buccal cell suspensions were centrifuged for 1 min at 1,000 g and cultured at room temperature for 1-24 h in MEM supplemented with penicillin and streptomycin. Buccal cells that settled on polylysine-coated dishes and that excluded trypan blue were used for patch-clamp studies. All procedures involving human volunteers were approved by the Institutional Review Boards of Children's Hospital (Cincinnati, OH) and the University of Cincinnati.
Materials.
HEK-293 cells and Calu-3 cells were obtained from American Type
Culture Collection (ATCC). Arachidonic acid 5,8,11,14-eicosatetraenoic acid 20.4 (C:20cis5,8,11,14) (free acid) was from Avanti Chemicals. Omeprazole, HEPES, DMSO, Tris, cAMP-dependent protein kinase (PKA), IBMX, GME, EGTA, and inorganic and organic salts were obtained from
Sigma Chemical. MEM, Lipofectamine, the Superscript preamplification system, and G418 were obtained GIBCO. pcDNA3.1 was from InVitrogen. mPKI was from Calbiochem. Ex-Taq polymerase was from Pan Vera. Oligotex
Direct mRNA columns were from Qiagen. Borosilicate glass (no. 7052) was
from Garner Glass. EDC was from Pierce Chemical. Cytosoft brushes were
from Medical Packaging Group (Camarillo, CA). A549 and BEAS-2B cells
were obtained from Dr. J. A. Whitsett (Children's Hospital,
Cincinnati, OH).
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RESULTS |
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Effect of arachidonic acid, amidation, and acid-activated
omeprazole on recombinant human ClC-2 Cl
channels expressed in HEK-293 cells.
Figure 1A shows representative
Cl
channel current traces of HEK-293 cells stably
transfected with the human recombinant ClC-2 Cl
channel
before and after addition of channel activators. Arachidonic acid
activation of human recombinant ClC-2 has been previously demonstrated
(40). In the present studies, arachidonic acid effects
were studied for comparison. EDC-catalyzed amidation and acid-activated
omeprazole significantly (P < 0.001) increased Cl
currents throughout the range of holding potentials to
levels similar to those measured with arachidonic acid (Fig. 1,
A, C, and D). These treatments had no
effect on Cl
currents measured in nontransfected HEK-293
cells (Fig. 1, B and D) or mock-transfected
HEK-293 cells (Fig. 1D).
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Effect of arachidonic acid, amidation, and acid-activated
omeprazole on ClC-2 Cl
currents measured in Calu-3, A549, and
BEAS-2B cells.
Calu-3 cells are derived from a human pulmonary adenocarcinoma. These
human airway cells have similarities to submucosal gland serous cells
(11, 18), are well known to be enriched in CFTR (17), and are used extensively in studies of CFTR
(18, 36). To investigate whether ClC-2 is present in
Calu-3 cells, we performed RT-PCR using Calu-3 mRNA. Figure
2A shows that ClC-2 and CFTR are present in Calu-3 cells (lane 4). All negative controls
showed no bands (lanes 1-3), and lane 5 was
the positive control. Cl
currents were then measured by
patch clamp of single Calu-3 cells. In Calu-3 cells, basal slope
conductance was significantly (P < 0.05) reduced from
0.227 ± 0.029 nS/pF before mPKI to 0.155 ± 0.015 nS/pF
(n = 9) after mPKI. In contrast, basal slope
conductance of transfected HEK 293 cells was not significantly
different before and after treatment with 0.8 µM mPKI [0.069 ± 0.008 and 0.061 ± 0.008 nS/pF (n = 13),
respectively]. In Calu-3 cells, arachidonic acid, amidation,
and acid-activated omeprazole greatly increased the Cl
currents throughout the range of holding potentials (Fig. 2, B-D).
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Effect of arachidonic acid, amidation, and acid-activated
omeprazole on Cl currents of human normal
and cystic fibrosis buccal cells.
Figure 4A shows that ClC-2
mRNA is present in the buccal epithelial cells of healthy humans and
cystic fibrosis patients. CFTR mRNA was not evident. Arachidonic acid,
amidation, and acid-activated omeprazole all significantly increased
Cl
currents in buccal cells from healthy individuals and
cystic fibrosis patients (Fig. 4, B and C).
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DISCUSSION |
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ClC-2 is a Cl channel that is found in epithelial
cells throughout the respiratory tract. Importantly, it was found to be present in Calu-3 cells, a cell line that has CFTR (17, 18, 36) and shows molecular and functional properties similar to serous cells of the submucosal glands (11). These glands
are thought to play an important role in the pathophysiology of cystic fibrosis (11). In the present study, ClC-2 was found to be
present not only in Calu-3 cells but also in A549, BEAS-2B, and human buccal epithelial cells. Buccal cell scrapings provide a convenient source of cells for studies of mRNA levels and Cl
channel
function in healthy individuals and cystic fibrosis patients.
Arachidonic acid activation effects have been previously shown for recombinant ClC-2 in HEK-293 cells (40), and amidation was shown to activate recombinant ClC-2 in single-channel studies (38). In a preliminary report, acid-treated omeprazole activated recombinant ClC-2 (10). In the present studies, these agents were shown to activate native ClC-2 channel activity in a variety of human lung epithelial cell lines and in human buccal epithelia. Acid-activated omeprazole also activated the channel in all of these cells.
It has been previously demonstrated that ClC-2 is activated by reduced
extracellular pH (8, 26, 35, 37, 38, 40). In the present
studies, it was not possible to use reduced extracellular pH, since pH
activation of recombinant ClC-2 does not occur when PKA is blocked by
mPKI (40). All experiments with airway cell lines and
buccal cells were carried out in the presence of mPKI to eliminate
Cl currents resulting from PKA activation of CFTR or
ClC-2. In addition, ClC-2 activation by low pH, per se, does not seem
to be a viable option for treatment of cystic fibrosis, since the
half-maximal pKa for activation is in the range
of pH 4.5 (38), a value that is not consistent with
maintaining tissue viability.
In contrast, both amidation and acid-activated omeprazole have the
potential to activate ClC-2 under mild conditions in situ and,
therefore, have promise for use in therapy. EDC, a water-soluble carbodiimide, has been used in a variety of biological contexts as a
catalyst for coupling amines with carboxyl groups of proteins. When
reacted with a neutral amine under very mild conditions, the modified
channel becomes active at neutral pH (38). Omeprazole has
been used as an anti-ulcer agent by virtue of reactivity of the
acid-activated form with free sulfhydryls in the
H+-K+-ATPase. It also reacts with a variety of
other proteins (1, 21, 24, 28, 30, 33, 42). Although
omeprazole requires activation by acid, once the active species is
formed, it can react at neutral pH (21, 24). Arachidonic
acid has been shown to inhibit CFTR (23), but arachidonic
acid and inhibitors of arachidonic acid metabolism such as nonsteroidal
anti-inflammatory agents activate ClC-2 Cl channels
(5, 40). It is not known whether other ClC
Cl
channels are affected by arachidonic acid or whether
arachidonic acid could serve as a therapeutic agent because of its
numerous other cellular effects.
ClC-2-mediated currents in HEK-293 cells are essentially linear and
show little rectification or time- and hyperpolarization-dependent voltage activation. The rat form of the channel, when expressed in
HEK-293 cells, showed time-dependent currents that were activated at
negative potentials and approached a steady state within 200-400 ms with a time constant of ~30 ms at 100 mV, for example
(29). This is much faster than when the same channel was
studied in Xenopus oocytes (25, 29). In
addition to differences in rat ClC-2 Cl
channel function
between expression systems, the magnitude of channel currents, the
amount of rectification, and the time dependence of rat ClC-2 currents
are affected by several other factors, including the amount of time
that elapses between achieving the whole cell mode and measurement of
currents (25, 29). Indeed, even with the rat ClC-2
Cl
channel expressed in Xenopus oocytes, a
short pulse protocol similar to that used in the present studies does
not significantly activate rat ClC-2 (25), whereas long
pulses give very large time- and hyperpolarization-dependent activation
and, consequently, large inward rectification (25). It has
been suggested that differences might arise from differences in the
state of phosphorylation or differences in assembly with other channels
(29). However, the human form of the channel, studied
here, did not show major inward rectification or time- and
hyperpolarization-dependent current increases. Others have found that
the current-voltage (I-V) responses showed only "slight
inward rectification" with human cells overexpressing human ClC-2
when measured at physiological pH (35).
A variety of functions have been attributed to CFTR that are distinct
from intrinsic Cl channel activity
(34). These include regulation of epithelial Na+ channels, interactions with K+ channels,
involvement in the transport of other substances, and regulation of
other Cl
channels, including ClC Cl
channels (34). Defects in these functions may also play a
role in cystic fibrosis. Therefore, augmentation of epithelial
Cl
transport by activation of ClC-2 may not be effective
in cystic fibrosis if the Cl
transport defect is not
primary in the etiology of the disease.
It was important to rule out PKA-activated Cl currents in
the human lung cell lines and in buccal cells, whether due to ClC-2 or
CFTR. For this reason, the PKA inhibitor mPKI was included in the bath.
ATP was left out of the patch pipette to further prevent activation by
PKA. However, the omission of ATP was insufficient to prevent
activation by forskolin plus IBMX over the time course of our
experiments using recombinant ClC-2 in HEK-293 cells, presumably because of the presence of glucose in the media, the high affinity of
PKA for ATP (19, 41) or because of the short time course of our studies. Longer time courses (10-30 min) have been shown to
be required to induce rundown of cAMP-dependent processes (32, 39). The bath and pipette solutions were similar to the
solutions used for most studies of ClC Cl
channels
expressed in HEK-293 cells where neither ATP nor Ca2+ were
present in the pipette (12-16, 29). In one case,
Ca2+ alone was included in the pipette, and the authors
showed that ClC-2 was insensitive to Ca2+
(20). In one study of human ClC-2, the pipette solution
contained both ATP and Ca2+ (35), and these
authors showed similar I-V curves. In the absence of
statistically significant differences with and without ATP in
recombinant cells observed in the present studies, ATP was left out of
the pipette solutions in all studies of native human cells.
The molecular and physiological basis for basal Cl
currents in these cells was not identified in the present studies. A
part of the basal current in Calu-3 cells was sensitive to mPKI.
However, increases in Cl
currents that were elicited by
arachidonic acid, amidation, and acid-activated omeprazole are likely
to be due to ClC-2, since amidation and acid-activated omeprazole
increased Cl
currents with ClC-2 Cl
channels in planar lipid bilayers and since all three treatments increased Cl
currents in HEK-293 cells stably transfected
with human ClC-2 (10, 38, and 40; present study). Mock-transfected
cells were not affected by these treatments, suggesting that ClC-2 is
not a major component of HEK-293 Cl
currents. No other
Cl
channels are known to respond similarly to these
treatments, but further study with recombinant channels is required to
rule out the possibility that other channels may also be affected by these treatments.
The mechanism of action of amidation in increasing the activity of
ClC-2 has been previously studied (38) and appears to result from amidation of one or more carboxyl groups that are available
on the outer surface of the channel. Amidation removes tonic inhibition
at neutral pH (38). Omeprazole, when activated by low pH,
inhibits the gastric H+-K+-ATPase by covalent
modification of sulfhydryl groups in the enzyme (21, 24,
28) that are likely present at the outer surface of the enzyme.
Acid-activated omeprazole also affects other proteins including
carbonic anhydrase (30), other Cl channels
(33), and Helicobacter pylori urease
(42). In the stomach, ClC-2 is found in the same membrane
as the gastric H+-K+-ATPase (8)
and is also a potential target for omeprazole. As demonstrated here,
recombinant and native human ClC-2 channels are activated by
acid-activated omeprazole in a variety of human cells. The site(s) of
action of acid-activated omeprazole on ClC-2 is not known, although
cysteines on the outer surface of the channel represent likely targets.
ClC-2 channel mRNA was present in Calu-3, A549, and BEAS-2B cell lines and in buccal cells from healthy individuals and cystic fibrosis patients. Quantitative determination of the level of ClC-2 mRNA between the various cells was not carried out. CFTR mRNA was only detected in Calu-3 cells, as found by others (17), whereas ClC-2 mRNA was present in all cells tested. It is possible that treatments that activate ClC-2 in Calu-3 cells would also activate ClC-2 in serous glands. It is not yet known whether ClC-2 is in the apical membrane of the cells in serous glands. Future studies using polarized Calu-3 monolayers and glands will be required to address this question.
Together, these results suggest that ClC-2 Cl channels
can be activated in human airway epithelial cells and that this may provide a means of repairing the Cl
channel defect that
occurs in cystic fibrosis.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43816 and National Heart, Lung, and Blood Institute Grant HL-58399.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. Cuppoletti, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way ML 0576, Cincinnati, OH 45267-0576 (E-mail John.Cuppoletti{at}uc.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 26 April 2000; accepted in final form 26 January 2001.
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