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Ca²⁺-Activated Cl^– Channels: A Newly Emerging Anion Transport Family

Catherine M. Fuller and Dale J. Benos

C. M. Fuller and D. J. Benos are in the Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294.

	Abstract

 
A new family of Cl^– channels widely expressed in epithelia has been identified. These proteins are associated with Ca²⁺-sensitive conductive Cl^– transport when heterologously expressed. This family may underlie the Ca²⁺-mediated Cl^– conductance responsible for rescue of the cystic fibrosis knockout mouse from significant airway disease.

	Introduction

Top Introduction Mechanism of epithelial Cl-... A conundrum: the CF... Cloning of a new... A new family of... CaCCs: multifunctional proteins? Is the CaCC a... Is the CaCC a... References

 
Despite over 40 years of investigation, the exact mechanisms regulating the conductive movement of Cl^– across the apical membrane of secretory epithelia are still largely unknown. Although many of the components of the system (Na⁺ pump, cotransporter, K⁺ channel) have been described and their contribution to secretion is fairly well understood, a detailed picture of the Cl^– exit pathway at the apical membrane has, in many systems, proved refractory to identification. The problem lies in the sheer number of separate Cl^– conductance pathways that can contribute to secretion and the multiple opportunities that these conduits provide for different and sometimes contradictory modes of regulation. Moreover, the lack of high-affinity inhibitors of Cl^– channels has prohibited rapid molecular identification of specific channel proteins. Thus this class of ion transport proteins comprise a formidable system for the physiologist to dissect; determining which conductances contribute to physiological Cl^– secretion in any particular tissue has been hard to decipher, in large measure because of a lack of molecular information about the constituent channels that are present.

However, with the molecular cloning of several anion channels, including the cystic fibrosis transmembrane conductance regulator (CFTR), the ClC (Cl^– channel) family of anion channels, and the ligand-gated anion channels (e.g., -aminobutyric acid and glycine receptor-operated channels), this void is gradually dissipating. It is our intent in this article to describe the novel characteristics of an additional new family of anion channels, the Ca²⁺-activated Cl^– channels (CaCCs), and to discuss why these particular ion channels are well suited as pharmacological targets for the treatment of cystic fibrosis (CF).

	Mechanism of epithelial Cl– secretion

Top Introduction Mechanism of epithelial Cl-... A conundrum: the CF... Cloning of a new... A new family of... CaCCs: multifunctional proteins? Is the CaCC a... Is the CaCC a... References

 
The ability to secrete Cl^– is of fundamental importance to the maintenance of epithelial fluid and solute transport in a secreting epithelium. In secretory epithelial cells, Cl^– is accumulated to a concentration greater than that predicted by the Nernst equation for the equilibrium distribution of ions. This is dependent on both the concerted action of the Na⁺-K⁺-ATPase, a K⁺ channel and a Na⁺-K⁺-2Cl^– cotransporter, and on the polarized nature of the epithelium itself. Following an increase in the permeability of the apical cell membrane to Cl^–, Cl^– exits the cell down its electrochemical gradient accompanied by Na⁺ and water. The resultant primary secretion is essentially isotonic NaCl. The predominant Cl^– exit pathway in the majority of secretory epithelia is the CFTR protein. The primary physiological pathway for the activation of this ion channel is a receptor-mediated increase in cAMP and the consequent activation of protein kinase A (PKA). In the majority of individuals with CF, this small (10 pS) Cl^– channel is improperly processed and degraded by the quality control machinery of the endoplasmic reticulum and is thus missing from the apical plasma membrane.

	A conundrum: the CF knockout mouse

Top Introduction Mechanism of epithelial Cl-... A conundrum: the CF... Cloning of a new... A new family of... CaCCs: multifunctional proteins? Is the CaCC a... Is the CaCC a... References

 
In humans, the major symptoms of CF are largely commensurate with a lack of fluid secretion, i.e., thick secretions in the airways, obstruction in the gastrointestinal tract and pancreatic ducts, and a high sweat Cl^– concentration due to the failure of Cl^– reabsorption. As indicated above, this is largely attributable to the lack of functional CFTR at the apical plasma membrane of secretory epithelial cells. However, most of the mouse models of CF do not faithfully replicate the pulmonary consequences of the phenotype. The animals are largely free of airway disease, instead succumbing to intestinal obstruction.

What might underlie the apparent escape from CF-related airway disease in the CF mouse? Work from several laboratories had reported that a Ca²⁺-sensitive Cl^– pathway remained intact in cells of human CF origin. However, these reports did not gain clinical significance until experiments in the CF knockout mouse demonstrated the upregulation of a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive Ca²⁺-activated Cl^–-dependent short-circuit current across the nasal epithelium of this CF mouse model (5). Other studies using antisense oligonucleotides or antibodies to create functional CFTR knockouts in a variety of cell types also demonstrated that a Ca²⁺-sensitive Cl^– secretory pathway remained. However, upregulation of a similar pathway in "intact" humans (as opposed to CF cells of human origin) either does not occur or is ineffective. The absence of this potential backup pathway in the face of absent or dysfunctional CFTR would help to account for the invariably fatal consequences of CF in the human population. However, two important questions remained to be answered: 1) what was the molecular nature of the CaCC conductance? and 2) why doesn't a CaCC substitute for defective CFTR in humans as seems to occur in the airway of the CF mouse?

	Cloning of a new Cl– channel: the bCaCC

Top Introduction Mechanism of epithelial Cl-... A conundrum: the CF... Cloning of a new... A new family of... CaCCs: multifunctional proteins? Is the CaCC a... Is the CaCC a... References

 
Although there was considerable, if circumstantial, evidence for a distinct CaCC at the apical membrane of several epithelial cells, evidence of a distinct polypeptide that subserved this function had, until recently, been lacking. The first distinct CaCC to be cloned was identified in the bovine airway. With the use of classic biochemical purification techniques, Ran and co-workers (12) isolated a protein from the bovine tracheal epithelium that migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 140 kDa under nonreduced conditions, with minor components at 90 kDa, 60–64 kDa, and 32–38 kDa. When incorporated into planar lipid bilayers, this protein had a single-channel conductance of ~25 pS under symmetrical ionic conditions, an ion selectivity of I > Cl, (reversed from that observed for both CFTR and ClC-2), and was inhibited by DIDS. The protein could also be activated by Ca²⁺, albeit at rather high (5–10 µM) levels that would be unlikely to be seen by the cell under normal physiological conditions. However, the channel could be activated and phosphorylated by multifunctional calmodulin-dependent kinase II (CaMK II) (in the presence of calmodulin and ATP) at much lower (0.5–1 µM) concentrations of Ca²⁺ that more closely fell into the physiological range (Table 1 and Fig. 1). These results suggest that this CaCC is primarily regulated via Ca²⁺-dependent kinases. In addition, this channel was insensitive to regulation by PKA, unlike the PKA sensitivity exhibited by both CFTR and the outwardly rectifying Cl^– channel (ORCC). In the presence of the reducing agent dithiothreitol (DTT), only polypeptides migrating at a relative molecular mass (M_r) of 32,000–38,000 were observed by SDS-PAGE, and the protein could no longer form a channel when incorporated into a lipid bilayer. With the use of a polyclonal antibody raised against this 32- to 38-kDa protein to screen a tracheal cDNA expression library, a cDNA that coded for a 903 amino acid protein was isolated (2). Kyte-Doolittle secondary structure analysis of the primary amino acid sequence of the cloned protein (called bCaCC) predicted the presence of at least four transmembrane domains, several consensus sites for N-linked glycosylation, both consistent with a membrane protein, and several sites predicted to be targets for phosphorylation by CaMK II and protein kinase C (PKC), consistent with a protein subject to regulation by Ca²⁺. However, the cDNA predicted only two consensus sites for phosphorylation by PKA, neither of which were predicted to be accessible from the cytoplasmic solution. When expressed in Xenopus oocytes and recorded by dual-electrode voltage clamp, expression of the cDNA was associated with the appearance of a Ca²⁺-sensitive outwardly rectifying Cl^– conductance that was sensitive to both DIDS and DTT and had an ion selectivity profile of I > Cl. In lipid bilayer experiments and in whole cell patch-clamp recording of transiently transfected COS-7 cells, the expressed protein had a linear current-voltage relationship and a conductance of ~25 pS. Channel activity was significantly increased in the presence of CaMK II (increased Ca²⁺, calmodulin, and ATP). Increased current following exposure of bCaCC-expressing oocytes to phorbol ester was also observed. Because this current was sensitive to chelerythrine chloride, an inhibitor of PKC, these findings additionally implicated this kinase in CaCC regulation. The cloned channel was also insensitive to block by niflumic acid, a drug that is an effective blocker of an endogenous Ca²⁺-activated Cl^– conductance of the oocyte.

View larger version (15K): [in this window] [in a new window]   FIGURE 1. Schematic illustrating effect of calmodulin-dependent kinase II (CaMK II) and D-myo inositol (3,4,5,6)-tetrakisphosphate (IP4) on open probability of the bovine Ca2+-activated Cl– channels (bCaCC) as a function of Ca2+ concentration ([Ca2+]). Recordings were made from membrane vesicles derived from Xenopus oocytes injected with the cRNA for the bCaCC and incorporated into planar lipid bilayers. Various ambient [Ca2+] were achieved using EGTA buffers. Experiments performed in the presence of CaMK II (0.4 mg/ml) also included calmodulin (1 mM) and Mg-ATP (100 mM). Some experiments also included 20 nM IP4, as indicated on the figure. Modified from Fig. 4 in Ref. 10.

	A new family of Cl– channels

Top Introduction Mechanism of epithelial Cl-... A conundrum: the CF... Cloning of a new... A new family of... CaCCs: multifunctional proteins? Is the CaCC a... Is the CaCC a... References

 
Expression and biochemical characteristics.
Until very recently, the bCaCC was the only cloned protein shown to act as a CaCC. Because mRNA for this isoform was not detected in a variety of other bovine tissues as determined by reverse transcription-polymerase chain reaction (RT-PCR) analysis, it was thought that the channel might be unique to the tracheal epithelium. However, this view has had to be revised as several new members of what is now apparently a large and intriguing family of related proteins has recently emerged. The first new family member identified, lung endothelial cell adhesion molecule 1 (Lu-ECAM-1, bCLCA2), was cloned from bovine aortic endothelial cells (BAEC) raised on a pulmonary matrix (3). This polypeptide is 91% homologous to the bCaCC at the amino acid level and is indeed post-translationally processed to two major forms closely associated at the membrane (Tables 2 and 3). Lu-ECAM-1 exhibits a highly restricted pattern of expression, being found in BAEC, spleen, and lung. Immunohistochemical analysis with a polyclonal antibody raised against the 90-kDa fragment of Lu-ECAM-1 revealed staining at the apical membrane of the bovine trachea and in vesicles in the bronchus (3). Given the homology between bCaCC and Lu-ECAM, staining in the trachea probably represents cross-reactivity with the bCaCC, whereas staining in the bronchus may represent a contribution of both proteins.

There is good evidence that this new family of proteins undergoes post-translational processing. Lu-ECAM-1 is processed from a 120- to 130-kDa precursor to two proteins that migrate at 90 kDa and 32–38 kDa on SDS-PAGE. Both hCaCC-1 and -2 and mCaCC are thought to be subject to post-translational cleavage from larger precursors. Glycosylation site scanning of hCaCC-2 has shown that the larger 86-kDa fragment has three transmembrane regions, whereas the smaller cleaved 34-kDa portion contains two transmembrane domains (9). Because this detailed topology mapping has not yet been carried out for the other members of the family, it may be that the assignment of four transmembrane domains to the larger portion of the molecule, which was based on hydrophobicity plotting, was premature.

Functional characteristics.
Despite uncertainties over the exact number of transmembrane domains, the high degree of structural similarity among all of these polypeptides strongly argues for a commonality of function. To date, hCaCC-1 and -2 and mCaCC-1 have been expressed either in Xenopus oocytes and/or in HEK 293 cells. Expression of each isoform in Xenopus oocytes was associated with an increase in Ca²⁺-sensitive Cl^– current as assessed by dual-electrode voltage clamp. This current could be induced by ionomycin and was inhibited by DIDS and DTT, compounds that were previously demonstrated to be effective blockers of the bCaCC in oocytes. However, niflumic acid, which was used in earlier studies to inhibit the endogenous CaCC of the oocyte and that in our hands had no effect on the bCaCC, markedly inhibited both hCaCC1 and mCaCC. This observation prompted the use of the HEK 293 cell as a basis for the heterologous expression of these new CaCC isoforms. When expressed in the HEK cell system, both hCaCC-1 and -2 and mCaCC-1 were associated with increases in Ca²⁺-sensitive current as determined under whole cell patch-clamp conditions. Cells were patched with low Ca²⁺ (~25 nM) in the pipette and superfused with 2–4 µM ionomycin in the presence of a bath Ca²⁺ concentration of 1 mM. This treatment resulted in an immediate increase in an outwardly rectified current. In other experiments, whole cell recording was carried out in the presence of 2 mM Ca²⁺ in the pipette; on achieving the whole cell configuration, the current was immediately activated. The current was nearly completely inhibited by DIDS (300 µM), niflumic acid (100 µM), and DTT (2 mM). In one series of experiments with hCaCC-2, the current was also inhibited by tamoxifen (10 µM) (9), a compound also widely used as an inhibitor of anion channels. With the use of the cell-attached patch recording configuration, single channels from HEK cells expressing hCaCC-1 have also been recorded. The slope conductance of the channel was estimated at 13.4 pS, and the current-voltage curve exhibited a reversal potential of approximately –45 mV. The current across the patch was also increased in the presence of 2 µM ionomycin in the bath. These observations are consistent with these proteins being the initial members of a family of Ca²⁺-activated Cl^– channels. However, it is also consistent with these proteins subserving a potential role as regulators of an endogenous, otherwise cryptic channel. In the absence of functional mutations, the role of these proteins in epithelial Cl^– secretion still needs to be established.

	CaCCs: multifunctional proteins?

Top Introduction Mechanism of epithelial Cl-... A conundrum: the CF... Cloning of a new... A new family of... CaCCs: multifunctional proteins? Is the CaCC a... Is the CaCC a... References

 
One highly intriguing aspect of this new family of anion conductance proteins is exhibited by Lu-ECAM-1. This protein functions as a bona fide endothelial cell adhesion molecule; antibodies to the protein prevent the attachment of metastatic melanoma cells to BAEC cells grown on a lung matrix (3). Whether or not Lu-ECAM-1 functions simultaneously as a channel protein and an adhesion molecule or whether the two roles are mutually exclusive is not known at present. However, the ability of ion channels to subserve multiple roles is not unique; for example, both the ß-subunit of the voltage-gated brain Na⁺ channel and one of the family of Na⁺ channels of Caenorhabditis elegans (unc-105) have been postulated to have roles in cell adhesion. Cl^– channels have also been proposed to be multifunctional; the best example of this is undoubtedly CFTR, which regulates at least three other ion channels (the epithelial Na⁺ channel ENaC, the renal outer medulla K⁺ channel ROMK1, and an ORCC) and has been proposed to regulate several other transporters and channels. Furthermore, diphenylamine carboxylic acid (DPC), which is often used as a blocker of Cl^– channels, including CFTR, was reported to block adherence of endothelial cells to an artificial substrate. CFTR has itself been reported to act as an attachment factor for bacteria, including Pseudomonas aeruginosa. Whether or not this new class of proteins represents sticky ion channels or leaky glues remains to be determined.

	Is the CaCC a candidate for the Cl– conductance upregulated in the CF knockout mouse?

Top Introduction Mechanism of epithelial Cl-... A conundrum: the CF... Cloning of a new... A new family of... CaCCs: multifunctional proteins? Is the CaCC a... Is the CaCC a... References

 
Given the experimental evidence that an ionomycin-sensitive, Ca²⁺-dependent Cl^– secretion is upregulated in the nasal epithelia of CF mice, it is tempting to speculate that the protein underlying these observations is a member of this newly defined ion channel family. Other studies that used UTP (an agonist whose effect is mediated by Ca²⁺) to activate secretion have reported that both nasal and rectal potential differences in long-living subgroups of CF mice are reduced by DIDS. However, the specificity of DIDS to block a CaCC in these studies has been questioned, because this compound may also inhibit the metabotropic P₂Y₂ (UTP) receptor. Other candidates that have been proposed to fulfill the role of alternate Cl^– channel in CF, including ClC-2, are less satisfactory alternatives either because 1) they are downregulated in the airways at birth (ClC-2), 2) rely on the presence of CFTR or voltage for activation (ClC-2, ORCC), or 3) are inhibited by Ca²⁺-mediated agonists such as phorbol ester (PKC) and even Ca²⁺ itself (ClC-3).

The cloned CaCC would therefore appear to be the best candidate for the upregulated Cl^– conductance in CF mouse tissues. Since a Ca²⁺-mediated Cl^– conductance pathway is also preserved in CF cells of human origin, why does the CaCC not effectively substitute for CFTR in humans? One possibility is that the human CaCC is not expressed either in appropriate cell types or in the appropriate cellular location. It has been suggested that if a CaCC does exist, its most likely location will be at the basolateral membrane. Preliminary evidence in the rat parietal cell suggests that a CaCC is indeed present at the basolateral membrane of this tissue. However, protein(s) immunoreactive with antibodies raised against either the 90-kDa fragment of Lu-ECAM-1 or the 32- to 38-kDa fragment of bCaCC is found at the apical membrane of bovine tracheal epithelium and at the apical membranes of bovine tracheal submucosal glands, consistent with prevailing electrophysiological evidence garnered from the upper airways and CF cells of human origin.

If the CaCC is likely to present in the correct location, why then can it not substitute for CFTR? The answer to this riddle may lie in some findings concerning the effects of a by-product of the inositol trisphosphate (IP₃)/phospholipase C (PLC)/Ca²⁺ signaling cascade, D-(3,4,5,6)-inositol tetrakisphosphate (IP₄). Observations from two independent laboratories have shown that this compound is an effective inhibitor of both Ca²⁺-mediated Cl^– secretion and Ca²⁺-activated Cl^– current in T84 cells (14, 15). Studies of the bCaCC incorporated into planar lipid bilayers further demonstrated that this family member is exquisitely sensitive to 20 nM IP₄ in the presence of submicromolar levels of Ca²⁺ (10).

The interaction of IP₄ with the bCaCC is complex and highly dependent on the prevailing concentration of Ca²⁺. In Ca²⁺-free solutions (<1 nM free Ca²⁺ concentration), the open probability (P_o) of the bCaCC in the presence of 20 nM IP₄ was on the order of 0.4. However, in the presence of CaMK II, channel P_o began to increase in direct proportion to the Ca²⁺ concentration until near-maximal P_o (0.9) was observed at a Ca²⁺ concentration of ~30 nM. Because Ca²⁺ concentration was increased further, however, the channel began to shut down until, at a Ca²⁺ concentration of ~300 nM, P_o was on the order of 0.36. At 1 µM free Ca²⁺ concentration, channel P_o was negligible (Fig. 1 and Table 1). A similar biphasic behavior was observed in the absence of CaMK II, with the exception that the peak P_o was in the range of 0.6–0.7 at 300 nM Ca²⁺ concentration.

If extrapolated to the situation in the intact epithelial cell, these findings suggest that, in response to repeated cholinergic or -adrenergic stimulation, i.e., at a time when Cl^– secretion is required, and under conditions when IP₄ has accumulated (14), the CaCC may have such a low P_o as to be irrelevant for functional Cl^– secretion, compared with CFTR and the ORCC. However, in the case of a CF epithelial cell, three potential Cl^– exit pathways (CFTR, ORCC, and CaCC) would be inactive, contributing at a fundamental level to the deleterious consequences of the disease (Fig. 2). Why then in isolated human CF cells can a Ca²⁺-regulated Cl^– secretory pathway be demonstrated? The answer to this question probably rests in the way in which the experiments are done. Electrophysiological experiments to determine the presence of a Ca²⁺-stimulated Cl^– conductance in isolated CF cells are frequently done using an ionophore to circumvent the receptor. In other cases, only a single application of an agonist such as carbamylcholine is used. In each of these experimental maneuvers, little or insufficient IP₄ would be expected to accumulate to effectively block the CaCC.

View larger version (14K): [in this window] [in a new window]   FIGURE 2. Alternate pathways for Cl– secretion in epithelial cells. Secretory epithelia are subject to regulation by the autonomic nervous system, principally norepinephrine (NEpi) and acetylcholine (ACh) via adrenergic and muscarinic receptors, respectively. Activation of cystic fibrosis transmembrane conductance regulator (CFTR) chiefly involves both a cAMP-dependent phosphorylation step and ATP binding to the nucleotide-binding folds. In addition, CFTR plus protein kinase A (PKA)/ATP can regulate an additional outwardly rectifying Cl– channel (ORCC) that is only active in the presence of CFTR. An alternate pathway, involving Ca2+ as a second messenger and activated by acetylcholine or norepinephrine, is also available. In this case, Ca2+ released from the endoplasmic reticulum may be able to activate a channel directly, although it seems more likely that it would act to potentiate the actions of protein kinase C (PKC) or multifunctional CaMK II. Consistent with this dual pathway for regulating Cl– secretion, evidence exists for two populations of K+ channels that are sensitive to either cAMP or Ca2+-mediated signals, although the latter channel is by far the best characterized. Other modifications of the system could include Ca2+-coupled purinergic and cGMP-coupled receptors located on the apical membrane and an inhibitory role for a side product of the inositol trisphosphate (IP3) cascade, IP4, as indicated by the dashed line.

	Is the CaCC a candidate for pharmacological intervention in CF?

Top Introduction Mechanism of epithelial Cl-... A conundrum: the CF... Cloning of a new... A new family of... CaCCs: multifunctional proteins? Is the CaCC a... Is the CaCC a... References

In summary, a new family of proteins whose function is consistent with their proposed role as epithelial Cl^– channels has recently been identified. These proteins may underlie the Ca²⁺-dependent Cl^– secretion observed in some epithelial tissues, but resolution of their function will require a greater understanding of these new potential channels, in particular in terms of their precise cellular location, tissue distribution, and regulatory characteristics. These channels may be appropriate targets for therapeutic intervention in CF. However, mechanisms designed to increase Ca²⁺ in the cell are unlikely to be successful; not only can Ca²⁺ have deleterious consequences on cell function, but the bCaCC at least is in fact not very sensitive to Ca²⁺ in the physiological range in the absence of CaMK II. It will likely be more appropriate to focus on the IP₄ inhibitory pathway or on drugs that could maintain channel P_o in the presence of IP₄ and CaMK II.

	Acknowledgments

 
We would like to acknowledge the work of all of those laboratories whose studies, for reasons of space limitations, were not cited directly.

Work in our laboratories is funded by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-53090 and DK-53480.

	References

Top Introduction Mechanism of epithelial Cl-... A conundrum: the CF... Cloning of a new... A new family of... CaCCs: multifunctional proteins? Is the CaCC a... Is the CaCC a... References

 

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