ClC-2 Clminus channels in human lung epithelia: activation by arachidonic acid, amidation, and acid-activated omeprazole

John Cuppoletti, Kirti P. Tewari, Ann M. Sherry, Elena Y. Kupert, and Danuta H. Malinowska

Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0576


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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 beta -actin, 5'-gtccctgtatgcctctggtc-3' (sense) and 5'-tcgtactcctgcttgctgat-3' (antisense), producing cDNA fragments of 468, 210, and 669 bp for ClC-2, CFTR, and beta -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 beta -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 beta -actin. Amplified cDNA products were separated on 2% (ClC-2 and CFTR) or 1% (beta -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 beta -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-MOmega 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:20cisDelta 5,8,11,14) (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).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Effects of arachidonic acid, amidation, and acid-activated omeprazole on whole cell Cl- currents in HEK-293 cells expressing human ClC-2 (hClC-2) Cl- channels. A: representative scans of Cl- currents of HEK cells expressing human ClC-2 before and after treatment with 1 µM arachidonic acid (+AA), amidation by 1 mM 1-ethyl-3(3-dimethylaminopropyl) carbodiimide plus 10 mM glycine methyl ester (GME) (+EDC), and 100 µM acid-activated omeprazole (+OME). B: representative scans of Cl- currents of nontransfected cells before and after treatment with arachidonic acid, amidation, and acid-activated omeprazole. C: current-voltage (I-V) curves for Cl- currents normalized to cell capacitance (pA/pF) before () and after treatment with arachidonic acid (), amidation (black-triangle), or acid-activated omeprazole (black-down-triangle ). Vm, membrane potential. D: summarized data for normalized slope conductance (nS/pF) for nontransfected, mock-transfected, and transfected cells before (-) and after (+) treatment with each of the three activators. In C and D, data are plotted as means ± SE; n = 3-10 cells for each condition with matched controls. #P < 0.01, *P < 0.001 with respect to control. No forskolin/IBMX or myristoylated protein kinase inhibitor (mPKI) was present.

As shown in Table 1, the presence of 1 mM ATP-Mg2+ in the patch pipette was without effect on the extent of activation by arachidonic acid, amidation, or acid-activated omeprazole. Therefore, these data were combined in Fig. 1D. Omission of ATP-Mg2+ also had no effect on the extent of activation by forskolin plus IBMX (Table 2), suggesting that sufficient ATP was being produced in the cell from bath glucose or remained in the cell during the course of the experiment to support PKA activation. mPKI was not present in HEK cell experiments. All subsequent experiments with Calu-3, A549, BEAS-2B, and buccal cells were carried out in the absence of forskolin/IBMX, with 0.8 µM mPKI present, and in the absence of ATP in the pipette to ensure that CFTR and ClC-2 were not activated by PKA.

                              
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Table 1.   Effect of ATP on ClC-2 channel activation


                              
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Table 2.   Effect of ATP on forskolin/IBMX stimulation of ClC-2

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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Fig. 2.   Presence of ClC-2 mRNA by RT-PCR and effects of arachidonic acid, amidation, and acid-activated omeprazole on Cl- currents in human airway Calu-3 cells. A: sequence-specific primers were used to amplify cDNA fragments of ClC-2 (468 bp), cystic fibrosis transmembrane regulator (CFTR; 210 bp), and beta -actin (669 bp) from Calu-3 mRNA pretreated with DNase. Lane 1, no cDNA in PCR reaction; lane 2, no mRNA in RT reaction; lane 3, no reverse transcriptase in RT reaction; lane 4, experimental mRNA; and lane 5, positive controls (cDNAs for ClC-2, CFTR, or beta -actin as indicated). RT reaction product (2 µl) was used for cDNA amplification, and 5 µl of the PCR reaction products were loaded on the gel. B: representative scans of Cl- currents recorded from Calu-3 cells before and after treatment with 1 µM arachidonic acid, amidation by 1 mM EDC plus 10 mM GME, and 100 µM acid-activated omeprazole. C: I-V curves for Cl- currents normalized to cell capacitance (pA/pF) before () and after treatment with arachidonic acid (), amidation (black-triangle), or acid-activated omeprazole (black-down-triangle ). D: summarized data for normalized slope conductance (nS/pF) for the data in B and C before (-) and after (+) treatment with each of the three activators. Data are plotted as means ± SE; n = 3 cells for each condition. #P < 0.01, **P < 0.02 with respect to control. In B-D, bath forskolin/IBMX and pipette ATP were absent, and 0.8 µM mPKI was present in the bath.

Similar experiments were carried out on A549 cells derived from a human lung carcinoma and that have properties similar to human alveolar type II cells (22) and on BEAS-2B cells derived from virus-transformed human bronchial epithelial cells (31). As shown in Fig. 3A, these cells contained ClC-2 mRNA, but CFTR mRNA was not detected. Arachidonic acid, amidation, and acid-activated omeprazole increased Cl- currents significantly over the whole range of holding potentials in both A549 and BEAS-2B cells (Fig. 3, B and C) but to a lower level than that measured with Calu-3 cells (Fig. 2D).


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Fig. 3.   Presence of ClC-2 mRNA by RT-PCR and effects of arachidonic acid, amidation, and acid-activated omeprazole on Cl- currents in human airway A549 and BEAS-2B cells. A: sequence-specific primers were used to amplify cDNA fragments of ClC-2 (468 bp), CFTR (210 bp), and beta -actin (669 bp) from A549 and BEAS-2B mRNA pretreated with DNase. Lane 1, no cDNA in PCR reaction; lane 2, no mRNA in RT reaction; lane 3, no reverse transcriptase in RT reaction; lane 4, experimental mRNA; and lane 5, positive controls (cDNAs for ClC-2, CFTR, or beta -actin as indicated). RT reaction product (2 µl) was used for cDNA amplification, and 25 µl of the PCR reaction products were loaded on the gel. B: I-V curves for Cl- currents normalized to cell capacitance (pA/pF) in A549 cells and BEAS-2B cells before () and after treatment with 1 µM arachidonic acid (), amidation by 1 mM EDC plus 10 mM GME (black-triangle), or 100 µM acid-activated omeprazole (black-down-triangle ). C: summarized data for the normalized slope conductance (nS/pF) for the data in B before (-) and after (+) treatment with each of the three activators. Data are plotted as means ± SE; n = 3 cells for each condition. #P < 0.01, **P < 0.02, ##P < 0.05 with respect to control. In B and C, bath forskolin/IBMX and pipette ATP were absent, and 0.8 µM mPKI was present in the bath.

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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Fig. 4.   Presence of ClC-2 mRNA by RT-PCR and effects of arachidonic acid, amidation, and acid-activated omeprazole on buccal cells from normal individuals and cystic fibrosis patients. A: sequence-specific primers were used to amplify cDNA fragments of ClC-2 (468 bp), CFTR (210 bp), and beta -actin (669 bp) from human buccal epithelial cell mRNA obtained from normal subjects (healthy) and cystic fibrosis patients (CF). Lane 1, no cDNA in PCR reaction; lane 2, no mRNA in RT reaction; lane 3, no reverse transcriptase in RT reaction; lane 4, experimental mRNA; and lane 5, positive controls (cDNA for ClC-2, CFTR, or beta -actin as indicated). RT reaction product (2 µl) was used for cDNA amplification except for ClC-2, for which 4 µl of RT reaction product were used for both normal and CF samples; 20 µl of PCR reaction products were loaded on the gel. B: I-V curves for Cl- currents normalized to cell capacitance (pA/pF) in buccal cells from healthy individuals and cystic fibrosis patients before () and after treatment with 1 µM arachidonic acid (), amidation by 1 mM EDC plus 10 mM GME (black-triangle), or 100 µM acid-activated omeprazole (black-down-triangle ). C: summarized data for normalized slope conductance (nS/pF) from I-V curves of healthy individuals (normal) and cystic fibrosis patients before (-) and after (+) treatment with each of the three activators. Data are plotted as means ± SE; n = 4-6 cells for each condition with matched controls. #P < 0.01, **P < 0.02 with respect to control. In B and C, bath forskolin/IBMX and pipette ATP were absent, and 0.8 µM mPKI was present in the bath.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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