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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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),
-phenylcinnamate (
-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 M
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 G
) 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
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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.
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RESULTS |
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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.
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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).
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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).
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The activation of whole cell currents in Xenopus oocytes
expressing a truncated form of the bCLCA1 channel by 107
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).
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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).
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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).
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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. -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).
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-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
-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
-PC in the efflux buffer
(Fig. 11; ±
-PC in the presence of
ionomycin × time, P < 0.001).
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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).
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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.
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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, -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,
-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.
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DISCUSSION |
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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
-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 -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).
-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.
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ACKNOWLEDGEMENTS |
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Darlene Hall provided excellent technical assistance.
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FOOTNOTES |
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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.
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REFERENCES |
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Anderson, MP,
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