The calcium-dependent chloride conductance mediator pCLCA1

Matthew E. Loewen1, Sherif E. Gabriel2, and George W. Forsyth1

1 Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatchewan, Canada S7N 5B4; and 2 Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27599-7020


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The regulatory behavior, inhibitor sensitivity, and properties of the whole cell chloride conductance observed in cells expressing the cDNA coding for a chloride conductance mediator isoform of the CLCA gene family, pCLCA1, have been studied. Common C-kinase consensus phosphorylation sites between pCLCA1 and the closely related human isoform hCLCA1 are consistent with a role for calcium in channel activation. Both channels are activated rapidly on exposure to the calcium ionophore ionomycin. Direct involvement of calcium in the activation of pCLCA1 was supported by the finding that treatment with the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM reduced the rate of chloride efflux from NIH/3T3 cells expressing the pCLCA1 channel. No combination of A-kinase activators used was effective in activating chloride efflux via this channel despite the presence of a unique strong A-kinase consensus site in pCLCA1. Notable differences of pCLCA1 from the reported properties of CLCA family members include the failure of phorbol 12-myristate 13-acetate to activate chloride efflux in cells expressing pCLCA1 and a lack of inhibition of chloride efflux from these cells after treatment with DIDS or dithiothreitol. However, selected inhibitors of anionic conductance inhibited pCLCA1-dependent anion efflux. The electrogenic nature of the ionomycin-dependent efflux of chloride from cells expressing pCLCA1 was confirmed by detection of outwardly rectifying chloride current and inhibition of this current by chloride conductance inhibitors in a whole cell patch-clamp study.

calcium activation; CLCA; ionomycin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THERE ARE AT LEAST three major types of chloride channels with the potential to act as gating molecules controlling the electrogenic release of chloride from the apical membrane of secretory epithelial tissues. The cystic fibrosis transmembrane regulator (CFTR) protein, some members of the related isoforms of the ClC protein family, and the CLCA family of calcium-dependent chloride channels are all expressed in the apical membrane of epithelial cells lining the surface of secretory tissues. There is experimental evidence supporting the roles of members of each of these channel families in transepithelial electrolyte and fluid transport (1, 8, 21). Cystic fibrosis disease arises from mutations in the CFTR protein that directly or indirectly reduce chloride transport by the CFTR molecule (2, 24, 26). ClC-2 is present in the apical membrane of intestinal epithelial cells and can contribute to chloride currents in these cells (18, 21). Isoforms bCLCA1, mCLCA1, and hCLCA2 are expressed in trachea and lung epithelium, and hCLCA1 expression was reported in the human gut, where it is postulated to participate with CFTR in regulated chloride transport (3, 8, 10, 13).

Our laboratory has reported the cloning of a porcine (pCLCA1) isoform of the CLCA gene family (11). Nucleotide sequence data show that the pCLCA1 channel is most similar to the hCLCA1 isoform of that family, with 78% identity in the predicted amino acid sequence. Predicted amino acid sequence data also suggest a common transmembrane topology for pCLCA1 and hCLCA1. Monobasic proteolytic cleavage sites likely to be involved in posttranslational processing of hCLCA1 are common to pCLCA1.

Heterologous expression of hCLCA1 and hCLCA2 as well as mCLCA1, mCLCA2, and bCLCA1 in HEK-293 cells and Xenopus oocytes is accompanied by the appearance of a calcium-sensitive anion conductance (13, 14, 27). The calcium-dependent anion conductance is reported to be sensitive to inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and by the reducing agent dithiothreitol (DTT). Weak consensus C-kinase phosphorylation sites in a predicted cytosolic loop at S535, T580, and T593 and the strong site at T600 are common to the predicted amino acid sequences of hCLCA1 and pCLCA1. CLCA channel activation by A-kinase has not been reported. The strong -RRSS587- A-kinase consensus site in the predicted cytosolic loop containing four conserved C-kinase phosphorylation sites is unique to predicted pCLCA1 amino acid sequence.

As with the b-1 and h-1 CLCA isoforms, pCLCA1 is expressed in tracheal and small intestinal epithelium, with highest levels in association with the secretory cell types in the tracheal submucosal glands and the small intestinal crypts of Lieberkuhn (11, 25). In addition to the expression sites for pCLCA1, two other lines of evidence relate to a potential role for this protein in epithelial electrolyte and fluid secretion. The initial evidence came from the monoclonal antibody used in expression cloning of the pCLCA1 gene. Incubation of this monoclonal antibody with apical membrane vesicles prepared from porcine ileum inhibited up to 95% of electrogenic chloride uptake by these vesicles (25). Expression of the pCLCA1 gene in permanently transfected 3T3 mouse fibroblasts introduced a calcium-dependent chloride efflux from the fibroblasts expressing pCLCA1 (11).

This report outlines the sensitivity of the pCLCA1 protein to second messenger activators and to some inhibitors of anion conductance, as well as some properties of the anion currents observed in whole cell patch-clamp measurements on 3T3 cells expressing pCLCA1.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Tissue culture media, G418 antibiotic, and Superscript II reverse transcriptase were purchased from GIBCO Life Technologies. Taq and Pfu DNA polymerases were obtained from Stratagene, dNTPs from Pharmacia, and restriction enzymes from New England Biolabs. Molecular biology grade chemicals and anion transport inhibitors including DIDS, glibenclamide, 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), alpha -phenylcinnamate (alpha -PC), and diphenylamine carboxylate (DPC) were obtained from Sigma-Aldrich. 36Cl was purchased from New England Nuclear. Oligonucleotides were synthesized at the Core DNA Services Laboratory, University of Calgary (Calgary, Canada).

Production of cell lines. NIH/3T3 fibroblasts were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS) and glutamine (2 mM) (complete DMEM medium). Optimum settings for plasmid transfection by electroporation used voltage and capacitance conditions sufficient to kill ~20% of freshly suspended recipient cells within 48 h of receiving a single electrical discharge. NIH/3T3 cells (8 × 106 cells) were mixed with 100 µg of pcDNA3 plasmid containing pCLCA1 cDNA or pcDNA3 vector without insert and then subjected to a 250-V discharge at 250 µF in a 4-mm gap electroporation cuvette. After electroporation, cells were plated at 2 × 105 cells per well in 24-well plates. G418 (2.5 mg/ml) was added to wells 24 h later to select for successful transfectants. Cells were switched to maintenance medium (500 µg/ml G418 in complete DMEM) 7 days posttransfection.

Total RNA was isolated by extraction in guanidiniumthiocyanate-phenol (Trizol; GIBCO) and used as a template in a reverse transcriptase PCR reaction. Total RNA (5 µg) from transfected cells was used as a template in a reverse transcriptase reaction containing dNTPs, 200 units of Superscript reverse transcriptase, and 1 pmol of primer (5'-GAGAAAGCTTGCGGCCGCTCGTGCAGAAAGTCTAAAATG-3') specific to a section of unique sequence in the 3' untranslated region of pCLCA1 and not found in any other CLCA isoform. The reverse transcriptase reaction product (1 µl) was used as template in a nested PCR reaction with sense (5'-GTGAACACGCCACGCAGAAG-3') and antisense (5'-GTCCAACCAGAATAGCTGTC-3') primers for 24 cycles at 94°C for 45 s, 52°C for 45 s, and 72°C for 1 min to produce a 518-base pair product. PCR products were separated by electrophoresis in 0.1% agarose gels in Tris-borate-EDTA buffer and exposed to ethidium bromide, and the fluorescence of the ethidium bromide-DNA complex was recorded on a GelDoc visualizing system (Bio-Rad).

Chloride efflux measurements. Stable pCLCA1 transfectants of mouse 3T3 fibroblasts were grown in DMEM supplemented with 2 mM glutamine, 10% fetal calf serum (FCS), and G418 (500 µg/ml). Confluent 35-mm plates of these cells were loaded with 36Cl by removing growth medium and incubating with loading buffer containing 4 mM KCl, 2 mM MgCl2, 1 mM KH2PO4, 1 mM CaCl2, 5 mM glucose, 10 mM HEPES, pH 7.5, and 140 mM NaCl plus 2 µCi/ml 36Cl for 2 h. Extracellular 36Cl was removed by rapidly washing cells five times with 1 ml of efflux buffer (loading buffer without 36Cl). The 36Cl content of the last wash was reported as the time 0 efflux value. The rate of release of chloride from the cells was determined by repetitively adding 1 ml of efflux medium and removing the medium 2 min later. The chloride efflux values reported at each 2-min interval represent the amount of 36Cl (measured by liquid scintillation counting) released from the cells during the preceding 2 min. Cells were removed from the plates at the end of the efflux by addition of 1.0 mM EDTA and mechanical agitation for the determination of total protein (Bio-Rad protein assay).

Potential activators of pCLCA1 chloride conductance were added to the efflux buffer after the end of the last wash. When ionomycin (10 µM), phorbol 12-myristate 13-acetate (PMA), or 8-(4-chlorophenylthio)-AMP (CPT-cAMP; 0.5 mM), 3-isobutyl-1-methylxanthine (IBMX; 2.0 mM), and forskolin (10 µM) were added to the efflux buffer, they were also present in the buffer used for each successive 2-min efflux period.

Potential inhibitors of cell signaling including the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) and the calcium calmodulin kinase II (CaMKII) inhibitor 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)- N-methylbenzylamine (KN-93) were added to uptake medium during the 2-h 36Cl loading period. DTT and potential chloride transport inhibitors with lipid-soluble anion properties presumed to interact with an anion channel in the conductance protein (DIDS, NPPB, etc.) were added to the wash solution during the five cell washes preceding efflux measurements and were also in the buffer during the subsequent timed 36Cl efflux.

Whole cell voltage-clamp studies. The pipette solution for intracellular dialysis contained 1 mM sodium pyruvate, 40 mM Tris · HCl, 90 mM D-gluconic acid lactone, 90 mM Tris base, 5 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), 1 mM EGTA, 2 mM MgCl2, 0.1 mM CaCl2, 1 mM MgATP, and 0.1 mM Na2GTP (pH 7.4). Ca2+ activity was buffered to ~40 nM (1.0 mM EGTA and 0.1 mM CaCl2). The routine bath solution contained 150 mM NaCl, 2 mM MgCl2, 1 mM CaCl2, 5 mM TES, and 30 mM sucrose (adjusted to pH 7.4 with Tris). The low-chloride solution contained 40 mM NaCl, 2 mM MgCl2, 1 mM CaCl2, 5 mM TES, and 250 mM sucrose. Single-cell, rapid solution changes were applied by using a gravity-fed Perfusion Fast-Step SF-77B perfusion system (Warner Instrument, Hamden, CT). Ionomycin (10 µM) was added as a single-cell bath solution change. Patch-clamp electrodes were pulled and fire-polished from borosilicate glass capillaries (outer diameter 1.5 mm; inner diameter 1.17 mm) with an inner filament (Harvard Apparatus, Edenbridge, UK) on a DMZ universal puller (Dagan, Minneapolis, MN). Patch pipettes had a tip resistance of 3-5 MOmega with these solutions. Whole cell currents were acquired with an Axopatch-1D patch-clamp amplifier (Axon Instruments, Foster City, CA) at 500 Hz, filtered at 100 Hz with Clampex 8, and analyzed with Clampfit 8 (Axon Instruments). The standard voltage-clamp protocol had a holding potential of 0 mV with voltage pulses applied for 600 ms from -80 to +100 mV in 20-mV increments. After a (>1 GOmega ) seal was obtained, capacitance compensation was carried out before whole cell access. Subsequent to whole cell access, all cells were dialyzed for 2 min before recording. Only those cells that had a membrane resistance ~100 times that of the access resistance before ionomycin activation were used. Current differences were normalized by the whole cell capacitance recorded from an integrated 10-mV hyperpolarizing pulse. All membrane potentials were corrected at the time of analysis for the measured junction potentials of pipette to bath solution, -4.8 ± 0.2 mV in bath solutions containing 150 mM Cl- and 0.8 ± 0.2 mV in bath solutions containing 40 mM Cl-.

Statistical methods. Two-way repeated-measures ANOVA was used to compare treatment over time or voltage effects. The Fisher least significant difference (LSD) method for pairwise multiple comparisons was used as a post-ANOVA test to determine treatment effects at individual points. Reversal potential (Erev) values were compared using Student's t-test. The SigmaStat statistical software package was used to perform all these comparisons.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Untransfected NIH/3T3 fibroblasts are killed by continuous exposure to complete DMEM medium containing 500 µg/ml of G418. The expression of pCLCA1 mRNA in transfected cells resistant to G418 was verified by reverse transcriptase PCR. Amplification of a 518-base pair product from reverse transcribing 5 µg of total RNA from cells transfected with the pCLCA1-expressing construct (Fig. 1) indicates that the construct was intact and that the transfected cells would be expected to synthesize the pCLCA1 protein.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1.   Identification of pCLCA1 mRNA expression in transfected NIH/3T3 fibroblasts. Reverse transcriptase PCR reaction was carried out with total RNA isolated from 2,500 cells. Reverse transcriptase was primed with an antisense primer complimentary to a pCLCA1-specific 3' untranslated sequence. The reverse transcriptase reaction product (1 µl) was used as template for a PCR reaction with nested primers designed to amplify a 518-base pair product. Flanking standard lanes contain a 100-base pair ladder. Lane 1, PCR no-template control; lane 2, NIH/3T3 mRNA RT template; lane 3, pcDNA3-transfected NIH/3T3 mRNA RT template; lane 4, pCLCA1-transfected NIH/3T3 mRNA RT template; lane 5, pCLCA1 cDNA control.

The pCLCA1 mRNA was cloned from a porcine intestinal cDNA library (11). Apical chloride channels from crypt cells in the intestinal mucosa are activated in situ in response to treatment with cholera toxin, with the heat-labile and heat-stable enterotoxins produced by enteropathogenic strains of Escherichia coli. The cyclic nucleotide phosphodiesterase inhibitors theophylline and IBMX are rapid and potent activators of apical chloride conductance. Various calcium ionophores including A-23187 and ricinoleic acid are also recognized as in situ activators of apical chloride channel conductance and intestinal secretion. Exogenous expression of the pCLCA1 protein in mouse 3T3 fibroblasts provides a model that can be used to test for the sensitivity of this presumptive anion channel to activation by various second messenger substances.

The presence of a strong A-kinase consensus site in pCLCA1 raises the possibility of activation of this channel by cAMP. Addition of CPT-cAMP, forskolin, and IBMX to transfected 3T3 cells expressing pCLCA1 mRNA did not affect the rate of release of 36Cl from cells loaded with this isotope (Fig. 2; ±pCLCA1 × time, P = 0.482). This lack of response to A-kinase activation was also observed in control-transfected 3T3 cells. The conditions used in this study should be sufficient for A-kinase activation in most test systems, but significantly higher concentrations of IBMX (5-10 mM) are routinely used for in situ activation of secretion without any CPT-cAMP or forskolin. Exposure of pCLCA1-transfected 3T3 cells to 5 mM IBMX for 6 or 10 min before extracellular 36Cl was washed off failed to produce a significant efflux response to the cyclic nucleotide phosphodiesterase inhibitor (data not shown).


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of pCLCA1 transfection on chloride release by NIH/3T3 fibroblasts treated with activators of protein kinase A (PKA). Medium on confluent cells in 3.5-cm dishes was replaced with loading buffer containing 4 mM KCl, 2 mM MgCl2, 1 mM KH2PO4, 1 mM CaCl2, 5 mM glucose, 10 mM HEPES, pH 7.5, and 140 mM NaCl plus 2 µCi/ml 36Cl. After 2 h, cells were washed rapidly 5 times with 1 ml of efflux buffer (loading buffer without 36Cl). The 36Cl content of the last wash is reported as the time 0 efflux value. 36Cl release was measured by changing the efflux buffer at 2-min intervals. 8-(4-Chlorophenylthio)-AMP (CPT-cAMP; 0.5 mM), 3-isobutyl-1-methylxanthine (IBMX; 2.0 mM), and forskolin (10 µM) were added to the efflux buffer at time 0 and at each subsequent buffer replacement interval to facilitate activation of PKA. , 3T3 cells (control) transfected with pcDNA3; open circle , 3T3 cells transfected with pcDNA3 containing pCLCA1. Values are means ± SE (n = 6).

There is a strong in situ secretory response to calcium ionophores including ricinoleic acid, bile acids, the A-subunit of cholera toxin, and A-23187 in porcine small intestine (19, 20). Addition of ionomycin to pCLCA1-transfected 3T3 cells increased the rate of 36Cl efflux from these cells. The ionomycin-dependent stimulation of 36Cl efflux was not observed in control-transfected cells containing only the pcDNA3 vector (Fig. 3; ±pCLCA1 × time, P < 0.001). The calcium dependence of this ionomycin effect was investigated by adding the calcium chelator BAPTA-AM to the loading medium during the 2-h loading with 36Cl. Normal loading buffer was modified for trials with BAPTA-AM by omitting 1.0 mM CaCl2. There was a significant decrease in the rate of 36Cl efflux from cells expressing pCLCA1 after treatment with BAPTA. (Fig. 4; ±BAPTA × time, P < 0.001).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of ionomycin addition on chloride efflux from pCLCA1-transfected NIH/3T3 fibroblasts. See legend to Fig. 2 for efflux conditions. , pCLCA1-transfected 3T3 cells with 10 µM ionomycin added at time 0; open circle , pCLCA1-transfected 3T3 cells without ionomycin; down-triangle, pcDNA3.1 (control)-transfected 3T3 cells with 10 µM ionomycin added at time 0; black-down-triangle , pcDNA3.1 (control)-transfected 3T3 cells without 10 µM ionomycin. * Significant ionomycin effects on Cl- efflux in pCLCA1-transfected cells. ^ Significant effect of pCLCA1 transfection on ionomycin-dependent Cl- efflux. Values are means ± SE (n = 6).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of intracellular calcium chelation by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) on chloride efflux from pCLCA1-transfected 3T3 cells. BAPTA-AM (50 µM) was added to the 36Cl-containing loading buffer 90 min before the effect of ionomycin addition was measured on chloride release. , Ionomycin (10 µM) added at time 0; open circle , cells pretreated with BAPTA and then with ionomycin (10 µM) added at time 0; black-down-triangle , control, untreated cells. * Significant BAPTA effects on ionomycin-dependent Cl- efflux. Values are means ± SE (n = 6).

The activation of whole cell currents in Xenopus oocytes expressing a truncated form of the bCLCA1 channel by 10-7 M PMA was cited as evidence for channel activation by protein kinase C (PKC) (15). The conservation of four C-kinase phosphorylation acceptor sites between hCLCA1 and pCLCA1 in the predicted cytoplasmic loop between TM3 and TM4 domains implies an importance of these sites in control of channel activity. However, there was only an insignificant effect of 1 × 10-7 M PMA on 36Cl efflux from 3T3 cells transfected with pCLCA1 (Fig. 5; ±pCLCA1 in presence of PMA × time, P = 0.106).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of phorbol 12-myristate 13-acetate (PMA) on chloride efflux from control and pCLCA1-transfected NIH/3T3 fibroblasts. Cells were loaded and chloride efflux carried out as described in Fig. 2. PMA (100 nM) was added to efflux buffer at time 0. open circle , 3T3 cells transfected with pCLCA1; , 3T3 cells transfected with pcDNA3.1 vector with no insert in polylinker region. Values are means ± SE (n = 8).

There are also conserved CaMKII phosphorylation sites in hCLCA1 and pCLCA1. The CaMKII inhibitor KN-93 (29) was added to pCLCA1-transfected 3T3 cells to test for any effects of this reportedly specific inhibitor on the ionomycin-dependent increase in 36Cl efflux rate from these cells. Although the KN-93 inhibitor is reported to have an inhibition constant of 0.37 µM, there was no inhibitory effect on 36Cl efflux when pCLCA1-transfected 3T3 cells were exposed to 20 µM KN-93 for 1 h before 36Cl efflux was stimulated by addition of ionomycin (Fig. 6). Statistical analysis of ±KN-93 in the presence of ionomycin × time gave a significant ionomycin-KN-93 interaction (P = 0.03). Post-ANOVA analysis indicated KN-93 effects approaching significance (e.g., for 2-min time, P = 0.061).


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 6.   Inhibition of ionomycin-dependent chloride efflux from pCLCA1-transfected 3T3 cells by calcium-calmodulin kinase inhibitor KN-93. Loading and efflux conditions were as described for Fig. 2. Ionomycin (10 µM) was added at time 0. open circle , Ionomycin alone; black-down-triangle , ionomycin plus 20 µM KN-93; , pCLCA1 control without ionomycin. Values are means ± SE (n = 6).

In addition to being able to distinguish chloride transporters by the second messenger signal pathways involved in their activation, it would be helpful to distinguish channels or to be able to rule out particular channels as contributors to an endogenous chloride conductance through the use of specific ion channel inhibitors. DTT was reported to inhibit chloride currents that could be due to the native form of bCLCA1 (4) as well as the other isoforms that have been expressed for functional study. We found that the reducing agent DTT did not inhibit the efflux of 36Cl from 3T3 fibroblasts expressing pCLCA1 (data not shown).

DIDS is recognized as an inhibitor of whole cell and single-channel chloride currents mediated by CLCA proteins. Addition of DIDS to 3T3 cells transfected with pCLCA1 had no effect on the rate of ionomycin-dependent release from these cells. The 36Cl efflux response in the presence of 500 µM DIDS (Fig. 7; ±DIDS in the presence of ionomycin × time, P = 0.71) was similar to the lack of effect observed at a concentration of 100 µM DIDS (not shown).


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on ionomycin-dependent chloride efflux from pCLCA1-transfected 3T3 cells. Loading and efflux conditions were as described for Fig. 3. Ionomycin (10 µM) was added to all cells at time 0. , Ionomycin alone, with no DIDS added; open circle , DIDS (500 µM) present in efflux buffer during the 5 washes and for the first 8 min of the efflux, with ionomycin added at time 0. Values are means ± SE (n = 6).

Glibenclamide has been reported to be a relatively specific inhibitor of the chloride transport activity of CFTR. Addition of glibenclamide to the cell wash solutions and to the efflux medium at a concentration of 100 µM reduced the initial rate of ionomycin-dependent 36Cl efflux from pCLCA1-transfected 3T3 cells (Fig. 8; ±glibenclamide in the presence of ionomycin × time, P = 0.044). Ionomycin-dependent 36Cl efflux from pCLCA1-transfected 3T3 cells was inhibited by other anion channel blockers that fall in the broad category of lipid-soluble anions. alpha -PC, DPC, and NPPB all inhibited 36Cl efflux from loaded cells when used at concentrations employed by others for this purpose. Walsh et al. (31) have reported a Ki value of 130 µM for the inhibition of CFTR-mediated chloride conductance by NPPB. Preliminary investigations into the apparent affinity of DPC and NPPB for inhibition of 36Cl efflux indicated that both of these inhibitors were effective at concentrations as low as 10 µM for DPC (Fig. 9; ±10 µM DPC, P = 0.012) and 50 µM for NPPB (Fig. 10; ±50 µM NPPB, P < 0.001). Hence, it appears that distinctive inhibitor affinities could be investigated as a tool for discriminating between possibly competing chloride conductance proteins until a thorough search for specific inhibitors can be completed (17).


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 8.   Inhibition of ionomycin-dependent chloride efflux from pCLCA1-transfected 3T3 cells by glibenclamide. Loading and efflux conditions were as described for Fig. 2. Ionomycin (10 µM) was added to all cells at time 0. , Ionomycin alone, with no glibenclamide added; open circle , glibenclamide (100 µM) present in efflux buffer during the 5 washes and for the first 8 min of the efflux, with ionomycin added at time 0. * Significant effect of glibenclamide on ionomycin-dependent Cl- efflux. Values are means ± SE (n = 6).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 9.   Inhibition of ionomycin-dependent chloride efflux from pCLCA1-transfected 3T3 cells by diphenylamine carboxylate (DPC). Loading and efflux conditions were as described for Fig. 2. Ionomycin (10 µM) was added to all cells at time 0. , Ionomycin alone, with no DPC added; open circle , ionomycin plus 10 µM DPC; black-down-triangle , ionomycin plus 50 µM DPC; down-triangle, ionomycin plus 200 µM DPC. * Significant inhibition of Cl- efflux by 200 µM DPC. ^ Significant inhibition of Cl- efflux by 50 µM DPC. -Significant inhibition of Cl- efflux by 10 µM DPC. Values are means ± SE (n = 6).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 10.   Inhibition of ionomycin-dependent chloride efflux from pCLCA1-transfected 3T3 cells by 5-nitro-2-(3-phenylpropylamino) benzoate (NPPB). Loading and efflux conditions were as described for Fig. 2. Ionomycin (10 µM) was added to all cells at time 0. , Ionomycin alone, with no NPPB added; open circle , ionomycin plus 2 µM NPPB; black-down-triangle , ionomycin plus 10 µM NPPB; down-triangle, ionomycin plus 50 µM NPPB. * Significant effect of 50 µM NPPB on ionomycin-dependent Cl- efflux. Values are means ± SE (n = 6).

alpha -PC has been reported to inhibit conductive chloride uptake by ileal mucosal brush border vesicles (5). The anion conductance in this vesicle system was inhibited by the monoclonal antibody that was used to clone pCLCA1 from a porcine small intestinal cDNA library. These circumstances would lead to the prediction that alpha -PC may be an inhibitor of expressed pCLCA1 activity. The rate of ionomycin-dependent chloride efflux from 3T3 cells transfected with pCLCA1 was significantly reduced by the inclusion of alpha -PC in the efflux buffer (Fig. 11; ±alpha -PC in the presence of ionomycin × time, P < 0.001).


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 11.   Inhibition of ionomycin-dependent chloride efflux from pCLCA1-transfected 3T3 cells by alpha -phenylcinnamate (alpha -PC). Loading and efflux conditions were as described for Fig. 2. Ionomycin (10 µM) was added to cells at time 0. open circle , Ionomycin alone, with no alpha -PC added; , ionomycin plus 100 µM alpha -PC. * Significant effect of 100 µM alpha  -PC on ionomycin-dependent Cl- efflux. Values are means ± SE (n = 6).

pCLCA1 contributes to an outwardly rectifying calcium-activated chloride current when expressed in the NIH/3T3 cell line. Transfection of NIH/3T3 cells with pCLCA1 significantly increased an outwardly rectifying calcium-activated chloride current above the levels present in cells transfected with pcDNA3 vector (Fig. 12). The current density in a high-chloride bath solution (156 mM Cl-) was significantly higher than in controls at 60, 80, and 100 mV in the pCLCA1-transfected cell line (±pCLCA1 vs. voltage, P < 0.001, n = 8). The Erev in the pCLCA1-transfected cell line (-10.26 ± 2.8 mV, n = 8 ) was significantly more negative than the Erev measured in the vector control cell line (-0.738 ± 2.6 mV, n = 8, P = 0.027).


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 12.   Effect of ionomycin on whole cell chloride currents in NIH/3T3 fibroblasts transfected with the pcDNA3.1 vector or with the vector containing pCLCA1 cDNA. Current (bottom) vs. 20-mV increments in transmembrane potential (top) are shown for control cells transfected with vector alone (A) and for cells transfected with pcDNA3 containing pCLCA1 cDNA (B). open circle , 10 µm ionomycin added to cells at t = 0; , no ionomycin added. P < 0.001 for voltage × gene interaction (2-way repeated-measures ANOVA). * Voltages at which current is significantly higher in cells transfected with pCLCA1 (post-ANOVA multiple comparison analysis). At 42 mM internal Cl- and 156 mM external Cl-, the mean Erev values were -0.74 ± 2.6 mV for control-transfected cells and -10.3 ± 2.8 mV for pCLCA1-transfected cells (P = 0.027). Representative current-voltage tracings are given for each cell line. Values are means ± SE (n = 8).

The current density in a low-chloride bath solution (46 mM Cl-) was significantly higher than in controls at -80, 60, 80, and 100 mV in the pCLCA1-transfected cell line (±pCLCA1 vs. voltage, P < 0.001, n = 8 ). The Erev in the pCLCA1-transfected cell line shifted significantly from that seen in the high-chloride bath solution (-0.725 ± 2.9 mV, n = 8, P = 0.036), fulfilling one criterion of a chloride current (Fig. 13). However, the Erev did not shift on transfer of the pcDNA3, control-transfected cell line to the bath solution containing 46 mM Cl- (-0.763 ± 2.1 mV, n = 8). No difference was found between the Erev of the two cell lines at low chloride concentrations.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 13.   Whole cell current-voltage relationship in control- and pCLCA1-transfected NIH/3T3 fibroblasts with symmetrical chloride concentrations. Erev values were -0.76 ± 2.1 mV for controls and -0.73 ± 3.0 mV for pCLCA1-transfected cells (P = 0.99) with pipette and extracellular chloride concentrations at 44 and 46 mM, respectively. open circle , control-transfected cells; , pCLCA1-transfected cells. P < 0.001 for voltage × gene interaction (2-way repeated-measures ANOVA). * Voltages at which current is significantly higher in cells transfected with pCLCA1 (post-ANOVA multiple comparison analysis). Values are means ± SE (n = 8).

The effect of anionic current inhibitors on whole cell currents in pCLCA1-transfected and control cell lines was measured because the Erev deviated from the theoretical value for a pure chloride current. Whole cell currents were studied in the presence of inhibitory concentrations of DPC, alpha -PC, NPPB, and DIDS. The case for the mediation of a chloride conductance by pCLCA1 was supported by inhibition of the whole cell current by DPC, alpha -PC, and NPPB (Fig. 14); compared with controls, P values were 0.027, 0.009, and 0.011, respectively, for these inhibitors. There was no significant effect of inhibitors on whole cell currents from control pcDNA3-transfected cells.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 14.   Effect of inhibitors on whole cell patch-clamp currents at +100 mV in pCLCA1- and pcDNA3-transfected 3T3 cells. Open bars, 3T3 cells expressing pCLCA1; hatched bars, 3T3 control cells. Final inhibitor concentrations were 100 µM DPC, 100 µM alpha -PC, 50 µM NPPB, and 300 µM DIDS. * Significant difference from whole cell current measured in uninhibited pCLCA1 cells (1-way ANOVA with post-ANOVA Fisher LSD test). Values are means ± SE (n = 8).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The presence of calcium-dependent electrogenic chloride currents in fibroblasts transfected with pCLCA1 cDNA is an indication of the identity of a component of the secretory apparatus in porcine small intestine. There is a significant amount of evidence pointing to a signaling role for calcium in this tissue. Ricinoleate and the bile acid deoxycholate have calcium ionophore activity and calcium-dependent secretory activity when placed in the lumen of the porcine small intestine (19). Even the secretory effects of cholera toxin have been reported to operate in this tissue through calcium ionophore activity present in the A-subunit of the enterotoxin (20). This feature is particularly salient in the porcine small intestine where cholera toxin causes fluid secretion without detectable increases in cAMP concentration (6). Though there is very little evidence for calcium-dependent chloride conductance in mouse and human small intestine, these species have calcium-dependent chloride conductance in other important secretory tissues including the tracheal epithelium (9, 12). Upregulation of a calcium-dependent chloride conductance may occur as a compensatory response to a loss of CFTR function in the trachea (9). The failure to observe calcium-dependent chloride conductance in small intestine of cystic fibrosis patients (30) may also be a reflection of a regulatory interdependence of proteins involved in chloride conductance.

The absence of a chloride efflux response to cAMP in pCLCA1-transfected cells raises questions about the role of the strong A-kinase consensus site located on what appears to be an important cytosolic loop of this protein. More complex combinatorial experiments are required to investigate possible interactions between A- and C-kinase phosphorylation acceptor sites that could be involved in regulating the conductance activity associated with pCLCA1 transfections. The effects of simultaneous activation of two or more classes of protein kinase acceptor sites remain to be determined.

Conservation of C-kinase sites between hCLCA1 and pCLCA1 isoforms could be construed as evidence of a role for these acceptor sites in channel regulation. This proposed role is supported by evidence of activation of bCLCA1 conductance by PMA. However, the failure of treatment with PMA to increase the rate of chloride efflux from pCLCA1-transfected cells creates significant uncertainty about the role of C-kinase in the stimulation of this conductance by calcium. Activation of the epsilon -isoform of C-kinase by PMA or epidermal growth factor is reported to inhibit tissue short-circuit currents induced by forskolin or carbamyl choline (3, 28). Although a component of these inhibitory effects could also be operating via a basal potassium conductance, it is clear that there is no necessary connection between C-kinase activation and increased calcium-dependent chloride conductance. At least the evidence in this study of the reduced rate of ionomycin-dependent 36Cl efflux from cells treated with the intracellular calcium chelator BAPTA reinforces the assumption that Ca2+ is directly involved in the regulation of pCLCA1 activity.

The role of disulfide bonding in stabilizing CLCA channel structure and activity is somewhat controversial. Polypeptides arising from posttranslational cleavage of bCLCA1 may require disulfide bonding to retain conformation or aggregation properties necessary for function. However, the lack of an inhibitory DTT effect on chloride conductance activity of nonglycosylated forms of bCLCA1 is not completely consistent with a role for disulfide bonding in structural stabilization (8). The predicted amino acid sequence of pCLCA1 contains conserved posttranslational cleavage sites found in bCLCA1 and hCLCA1, but posttranslational processing has not been investigated for this protein. We conclude that there is no evidence for a requirement for disulfide bonding in pCLCA1 translation products in assays measuring the rate of 36Cl efflux from transfected 3T3 fibroblasts.

The chloride conductance of most endogenous calcium-activated chloride channels is inhibited by DIDS. The overall similarity of predicted amino acid sequence between hCLCA1 and pCLCA1 leads to a prediction of similar charge distribution and geometry for an anion pore involved in chloride transport. However, there are also significant differences in amino acid sequence that could affect the access of a bulky organic anion like DIDS to inhibitory sites. There is no information currently available concerning the three-dimensional structure of a possible anion pore in the CLCA proteins.

Both NPPB and DPC inhibited 36Cl efflux at relatively low concentrations. There was significant inhibition at 50 µM NPPB, consistent with reports of Ki values in the range of 22 µM for blockage of calcium-activated chloride currents in Xenopus oocytes (17, 32). Chloride conductance by CFTR is also reported to be inhibited by NPPB, but significantly higher concentrations of NPPB are required (Ki = 166 µM) (17, 31). Hence, this inhibitor should be useful in distinguishing between the chloride conductance of pCLCA1 and CFTR.

Inhibitory concentrations of DPC and alpha -PC may also permit discrimination between alternate chloride conductance proteins as mediators of a measured chloride conductance. Nishikawa et al. (22) reported that chloride influx into porcine tracheal submucosal glands was inhibited by 10-9 M DPC. Maximal inhibition of 36Cl efflux from pCLCA1-transfected 3T3 cells was obtained at 10 µM DPC in this study, although lower concentrations were not investigated. Much higher concentrations of DPC are necessary to inhibit CFTR (reported Ki values of 280 µM for internal application) (33). alpha -PC is a potential specific inhibitor of pCLCA1, but its effects on CFTR-mediated chloride conductance have not been reported.

Previous investigations of cloned pCLCA1 function were based on net in situ 36Cl efflux from cells grown as confluent monolayers. Whole cell chloride current measurements provide the information necessary to distinguish between anion exchange and electrogenic ion transport (11, 25). The increased current density in pCLCA1-transfected cell lines over control-transfected cells and the Nernstian shift of Erev on introduction to a low-chloride bath solution provide a rigorous demonstration of the electrogenic movement of chloride that cannot be supplied by 36Cl efflux. However, in a bath solution with high chloride concentration, the calculated Erev using the Nernst equation at 25°C (16) is -32.4 mV. This calculated value is significantly different from the measured Erev of -10.26 ± 2.8 mV. This deviation of the measured from the calculated Erev is assumed to be caused by an undefined endogenous calcium-activated current. The contaminating endogenous current in control-transfected cells had an Erev of -0.738 ± 2.6 mV, in violation of the Nernst equilibrium potential for chloride with a bath solution concentration of 156 mM and a pipette solution at 44 mM Cl-. The Erev of this current was unaffected upon a shift to low chloride concentration (46 mM) in the bath solution. The identity of the conductive ion affecting the Erev values for the channel is difficult to discern. The ionic compositions of bath and pipette solutions were set to permit direct identification of the ion producing the current from Erev values. However, the measured Erev was not consistent with a current attributable to any single ionic species in the bathing solution.

The activation of both cationic and anionic channels or a channel with a high permeability to both cations and anions could have produced the observed result. The calculated Erev for sodium is 128.7 mV in our high-chloride bath solution. A significant increase in sodium permeability would cause a positive shift from the expected chloride Erev. Cation permeability of anion channels is known, and anion channels with PNa/PCl ratios as high as 0.2 have been reported (7, 16, 23). In our case an endogenous chloride channel with some cation permeability could alter the Erev at high bath chloride concentration without shifting the Erev when both sodium and chloride concentrations were reduced.

The identity of the anionic current associated with pCLCA1 expression was confirmed by an electrogenic response distinctive from endogenous contaminating currents and by the reduction of ionomycin-induced currents to control levels by anionic conductance inhibitors. Additional confirmation was provided by the lack of effect of anion conductance inhibitors on the basal levels of contaminating current in cells transfected only with the expression vector. The nature of the contaminating current has not been pursued beyond its insignificant response to these inhibitors.

The pathophysiological significance of a calcium-activated chloride current in the airway epithelium is well established. Expression of pCLCA1 in tracheal epithelium has been documented (11). The current studies support the role of pCLCA1 as a chloride conductance mediator. The extent to which this protein contributes to the in vivo conductance in both the presence and absence of a functional CFTR molecule is an area for future investigation.


    ACKNOWLEDGEMENTS

Darlene Hall provided excellent technical assistance.


    FOOTNOTES

These investigations would not have been possible without the generous financial support provided by the Canadian Cystic Fibrosis Foundation.

Address for reprint requests and other correspondence: G. Forsyth, Veterinary Biomedical Sciences, Univ. of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5B4 (E-mail: george.forsyth{at}usask.ca).

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.

April 10, 2002;10.1152/ajpcell.00477.2001

Received 9 October 2001; accepted in final form 27 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anderson, MP, and Welsh MJ. Calcium and cAMP activate different chloride channels in the apical membrane of normal and cystic fibrosis epithelia. Proc Natl Acad Sci USA 88: 6003-6007, 1991[Abstract].

2.   Berschneider, HM, Knowles MR, Azizkhan RG, Boucher RC, Tobey NA, Orlando RC, and Powell DW. Altered intestinal chloride transport in cystic fibrosis. FASEB J 2: 2625-2629, 1988[Abstract/Free Full Text].

3.   Chow, JY, Uribe JM, and Barrett KE. A role for protein kinase Cepsilon in the inhibitory effect of epidermal growth factor on calcium-stimulated chloride secretion in human colonic epithelial cells. J Biol Chem 275: 21169-21176, 2000[Abstract/Free Full Text].

4.   Cunningham, SA, Awayda MS, Bubien JK, Iskander I, Ismailov Arrate P M, Berdiev BK, Benos DJ, and Fuller CM. Cloning of epithelial chloride channel from bovine tracheal. J Biol Chem 270: 31016-31026, 1995[Abstract/Free Full Text].

5.   Forsyth, GW, and Gabriel SE. Inhibiting conductive chloride uptake in membrane vesicles: specificity of alpha -phenylcinnamate. Biochim Biophys Acta 977: 19-25, 1989[ISI][Medline].

6.   Forsyth, GW, Hamilton DL, Goertz KE, and Johnson MR. Cholera toxin effects on fluid secretion, adenylate cyclase, and cyclic AMP in porcine small intestine. Infect Immun 21: 373-380, 1978[ISI][Medline].

7.   Franciolini, F, and Nonner W. Anion and cation permeability of a chloride channel in rat hippocampal neurons. J Gen Physiol 90: 453-478, 1987[Abstract].

8.   Fuller, CM, and Benos DJ. Electrophysiological characteristics of the Ca2+-activated Cl- channel family of anion transport proteins. Clin Exp Pharmacol Physiol 27: 906-910, 2000[ISI][Medline].

9.   Gabriel, SE, Makhlina M, Martsen E, Thomas EJ, Lethem MI, and Boucher RC. Permeabilization via the P2X7 purinoreceptor reveals a Ca2+-activated Cl- conductance in the apical membrane of murine tracheal epithelial cells. J Biol Chem 275: 35028-35033, 2000[Abstract/Free Full Text].

10.   Gandhi, R, Elble RC, Gruber AD, Schreur KD, Ji HL, Fuller CM, and Pauli BU. Molecular and functional characterization of a calcium-sensitive chloride channel from mouse lung. J Biol Chem 273: 32096-32101, 1998[Abstract/Free Full Text].

11.   Gaspar, KJ, Racette KJ, Gordon JR, Loewen ME, and Forsyth GW. Cloning a chloride conductance mediator from the apical membrane of porcine ileal enterocytes. Physiol Genomics 3: 101-111, 2000[Abstract/Free Full Text].

12.   Grubb, BR, and Gabriel SE. Intestinal physiology and pathology in gene-targeted mouse models of cystic fibrosis. Am J Physiol Gastrointest Liver Physiol 273: G258-G266, 1997[Abstract/Free Full Text].

13.   Gruber, AD, Elble RC, Ji HL, Schreur KD, Fuller CM, and Pauli BU. Genomic cloning, molecular characterization, and functional analysis of human CLCA1, the first human member of the family of Ca2+-activated Cl- channel proteins. Genomics 54: 200-214, 1998[ISI][Medline].

14.   Gruber, AD, Schreuer KD, Hong-Long J, Fuller CM, and Pauli BU. Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea and mammary gland. Am J Physiol Cell Physiol 276: C1261-C1270, 1999[Abstract/Free Full Text].

15.   Ji, HL, DuVall MD, Patton HK, Satterfield CL, Fuller CM, and Benos DJ. Functional expression of a truncated Ca2+-activated Cl- channel and activation by phorbol ester. Am J Physiol Cell Physiol 274: C455-C464, 1998[Abstract/Free Full Text].

16.   Hille, B. Ionic Channels of Excitable Membranes (3rd ed.). Sunderland, MA: Sinauer, 2001.

17.   Hipper, A, Mall M, Greger R, and Kunzelmann K. Mutations in the putative pore-forming domain of CFTR do not change anion selectivity of the cAMP activated Cl- conductance. FEBS Lett 374: 312-316, 1995[ISI][Medline].

18.   Loewen, ME, MacDonald DW, Gaspar KJ, and Forsyth GW. Isoform-specific exon skipping in a variant form of CLC-2. Biochim Biophys Acta 1493: 284-288, 2000[ISI][Medline].

19.   Maenz, DD, and Forsyth GW. Ricinoleate and deoxycholate are calcium ionophores in jejunal brush border vesicles. J Membr Biol 70: 125-133, 1982[ISI][Medline].

20.   Maenz, DD, Gabriel SE, and Forsyth GW. Calcium transport affinity, ion competition and cholera toxin effects on cytosolic Ca concentration. J Membr Biol 96: 243-249, 1987[ISI][Medline].

21.   Mohammad-Panah, R, Gyomorey K, Rommens J, Choudhury M, Li C, Wang Y, and Bear CE. ClC-2 contributes to native chloride secretion by a human intestinal cell line, Caco-2. J Biol Chem 276: 8306-8313, 2001[Abstract/Free Full Text].

22.   Nishikawa, K, Ishihara H, Ozawa K, and Tamura K. Chloride transport mechanism in swine tracheal submucosal gland cells. Respiration 62: 274-279, 995[ISI][Medline].

23.   Qu, Z, and Hartzell HC. Anion permeability in Ca2+-activated Cl- channels. J Gen Physiol 116: 825-844, 2000[Abstract/Free Full Text].

24.   Quinton, PM. Chloride impermeability in cystic fibrosis. Nature 301: 421-422, 1983[ISI][Medline].

25.   Racette, KJ, Gabriel SE, Gaspar KJ, and Forsyth GW. Monoclonal antibody against conductive chloride transport in pig ileal apical membrane vesicles. Am J Physiol Cell Physiol 271: C478-C485, 1996[Abstract/Free Full Text].

26.   Riordan, JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, Drumm ML, Iannuzzi MC, Collins FC, and Tsui LC. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066-1073, 1989[ISI][Medline].

27.   Romio, L, Musante L, Cinti R, Seri M, Moran O, Zegarra-Moran O, and Galietta LJV Characterization of murine gene homologous to the bovine CaCC chloride channel. Gene 228: 181-188, 1999[ISI][Medline].

28.   Song, JC, Hanson CM, Tsai V, Farokhzad OC, Lotz M, and Matthews JB. Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms. Am J Physiol Cell Physiol 281: C649-C661, 2001[Abstract/Free Full Text].

29.   Sumi, M, Kiuchi K, Ishikawa T, Ishii A, Hagiwara M, Nagatsu T, and Hidaka H. The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells. Biochem Biophys Res Commun 181: 968-975, 1991[ISI][Medline].

30.   Taylor, CJ, Baxter PS, Hardcastle J, and Hardcastle PT. Failure to induce secretion in jejunal biopsies from children with cystic fibrosis. Gut 29: 957-962, 1988[Abstract].

31.   Walsh, KB, Long KJ, and Shen X. Structural and ionic determinants of 5-nitro-2-(3-phenylpropylamino)benzoic acid block of the CFTR chloride channel. Br J Pharmacol 127: 369-376, 1999[Abstract/Free Full Text].

32.   Wu, G, and Hamill OP. NPPB block of Ca++-activated Cl- currents in Xenopus oocytes. Pflügers Arch 420: 227-229, 1992[ISI][Medline].

33.   Zhang, ZR, Zeltwanger S, and McCarty NA. Direct comparison of NPPB and DPC as probes of CFTR expressed in Xenopus oocytes. J Membr Biol 175: 35-52, 2000[ISI][Medline].


Am J Physiol Cell Physiol 283(2):C412-C421
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society