HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Eggermont, J. Search for Related Content PubMed PubMed Citation Articles by Eggermont, J.

Calcium-activated Chloride Channels

(Un)known, (Un)loved?

Laboratory of Physiology, Katholieke Universiteit Leuven, Campus Gasthuisberg, Leuven, Belgium

Correspondence and requests for reprints should be addressed to Jan Eggermont, M.D., Ph.D., Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium. E-mail: jan.eggermont{at}med.kuleuven.ac.be

	ABSTRACT

TOP ABSTRACT THE CACC PHENOTYPE MOLECULAR IDENTITY OF CACC LACK OF SPECIFIC PHARMACOLOGIC... CACC AS A MOLECULAR... REFERENCES

 
Calcium-activated chloride channels (CaCCs) participate in many different physiologic processes such as transepithelial transport, excitability of neurons and muscle cells, and oocyte fertilization. Within the airways, they contribute to epithelial fluid secretion. This review focuses on three outstanding questions about CaCCs. First, although their biophysical fingerprint (anion selectivity, Ca²⁺ and voltage dependence, kinetics) is fairly well established, the molecular identity of CaCCs is still unresolved. CLCA, a family of proteins of which four members have so far been identified in humans, has been proposed to mediate calcium-activated chloride currents. However, the biophysical profile and expression pattern of endogenous CaCCs differ from those of the CLCA proteins. Another family of membrane proteins, the bestrophins, has recently been shown in transfected HEK293 cells to confer anion-selective currents that are activated by submicromolar Ca²⁺ concentrations. Second, pharmacologic tools to manipulate CaCCs are poorly selective. This lack of specificity not only hampers the structural and functional characterization of these channels but also restricts therapeutic options for altering CaCC function. Finally, potential pitfalls with respect to CaCCs as molecular targets for cystic fibrosis therapy are discussed.

Key Words: CFTR • ion channel • airway

Anion channels form a structurally heterogeneous group of channel proteins with a common functional characteristic: the formation of a transmembrane-conductive pathway for anions. Because Cl^- is the most abundant permeable anion under physiologic conditions, these channels mostly mediate Cl^- currents. Anion channels are subdivided according to their gating mechanism: ligand-gated channels (such as -aminobutyric acid and glycine receptors) that open after binding of an extracellular ligand (-aminobutyric acid and glycine, respectively), voltage-gated Cl^- channels (CLC), the phosphorylation-regulated cystic fibrosis (CF) transmembrane conductance regulator (CFTR) channel, volume-regulated anion channels (VRACs), and Ca²⁺-activated Cl^- channels (CaCC*). The first three types of anion channels are relatively well understood with respect to their molecular identity, channel properties, and physiologic role (for a comprehensive review of anion channels, see Reference 1). In contrast, the molecular identity of VRAC and CaCC is still unresolved and/or controversial.

CaCCs are anion-selective channels that are activated by increases in cytosolic Ca²⁺. CaCCs were probably first described in Xenopus oocytes almost 20 years ago (2), and they have subsequently been identified in epithelial cells, vascular endothelial cells, neurons, and smooth and cardiac muscle cells. In line with their widespread expression, CaCCs have been implicated in a variety of cellular functions such as fertilization of the oocyte, transepithelial fluid transport, repolarization and action potential duration in cardiac myocytes, olfactory transduction, and regulation of smooth muscle tone (for reviews, see References 3–5). This article highlights some current problems with respect to CaCC, that is, molecular identity, pharmacology, and suitability as a target for CF therapy.

	THE CACC PHENOTYPE

TOP ABSTRACT THE CACC PHENOTYPE MOLECULAR IDENTITY OF CACC LACK OF SPECIFIC PHARMACOLOGIC... CACC AS A MOLECULAR... REFERENCES

 
The characteristic hallmark of CaCC is the activation by cytosolic Ca²⁺, which experimentally can be induced by stimulating cells with Ca²⁺-mobilizing agonists, by Ca²⁺ ionophores such as ionomycin, by loading cells with Ca²⁺-containing pipette solutions, or finally, by adding Ca²⁺ to the bath solution in excised inside-out patches. It appears, however, that the spatial distribution of the Ca²⁺ signal is an important parameter for CaCC activation because there are indications that CaCC mainly reacts to local Ca²⁺ changes in the subplasma membrane space (6).

CaCC has a characteristic biophysical fingerprint (5). (1) It is activated by cytosolic Ca²⁺ with half-maximal concentrations for activation (K_Ca) in the submicromolar range: 165 nM in Ehrlich ascites tumor cells (7), 283 nM in calf pulmonary artery endothelial cells (8) for currents at +100 mV, and 0.9 µM in Xenopus oocytes at +120 mV (9). (2) Anion and cation substitution experiments reveal an anion selective channel (10). The channel poorly discriminates between anions (e.g., I^- > Cl^-) and has an anion permeability sequence that closely resembles the Eisenman type I sequence. This indicates that the anion-binding site in the pore is weak and that anion selection is mainly governed by the ease of dehydration. (3) CaCC displays time- and voltage-dependent currents. At subsaturating Ca²⁺ concentrations, the current shows slow (> 100 ms) activation at positive potentials and fast deactivation at negative potentials (8, 9). As a result, the steady-state current–voltage relationship is steeply outwardly rectifying. However, at saturating Ca²⁺ concentrations (> 1 µM), the current–voltage plot becomes progressively linear (11).

Kuruma and Hartzell have carefully analyzed the voltage dependence and kinetic behavior of the endogenous CaCC in Xenopus oocytes, taking advantage of the macropatch technique (9). This study has revealed that channel activation is Ca²⁺ dependent but virtually voltage independent. In contrast, channel inactivation is voltage dependent with the probability of channel closure increasing upon hyperpolarization. This model accounts both for the outward rectification at intermediary Ca²⁺ concentrations (< 1 µM in the Xenopus oocyte system) and for the linear current–voltage relationship at high Ca²⁺ concentration (> 1 µM Ca²⁺). Indeed, at intermediary concentrations, the Ca²⁺ signal is sufficiently strong to open the channel at positive potentials, whereas the voltage-dependent closing step outweighs at negative potentials. At higher Ca²⁺ concentrations, the Ca²⁺-dependent opening predominates, even at negative potentials, thereby resulting in channel opening over the entire voltage range. However, other kinetic models have been proposed in which channel opening depends on both Ca²⁺ binding and voltage (12).

The Ca²⁺-dependent step is still incompletely understood at the molecular level. Some authors have invoked direct binding of Ca²⁺ to the channel, for example, in Xenopus oocytes, vascular endothelial cells, and parotid acinar cells (8, 9, 12), whereas other have provided evidence for Ca²⁺-dependent phosphorylation by calmodulin kinase II as the actual trigger for channel opening, for example, in T84 intestinal epithelial cells (13). In contrast, phosphorylation by calmodulin kinase II results in CaCC inactivation in tracheal smooth muscle (14). These cell-type–dependent differences suggest the existence of more than one CaCC isoform and/or of different regulatory pathways.

	MOLECULAR IDENTITY OF CACC

TOP ABSTRACT THE CACC PHENOTYPE MOLECULAR IDENTITY OF CACC LACK OF SPECIFIC PHARMACOLOGIC... CACC AS A MOLECULAR... REFERENCES

 
A major outstanding question is the molecular identity of CaCC. To outsiders, it may seem odd that this is still a controversial issue, but there are some specific technical problems associated with the cloning of CaCC. First, expression cloning of Cl^- channels in Xenopus oocytes is complicated by the background Cl^- currents present in these oocytes. Second, there are currently no highly selective blockers of CaCC, which could be used to purify the CaCC complex biochemically. Third, apart from the anti-CLCA antibodies (discussed later here), there are no well documented antibodies against CaCC that could be reliably used in an immunoscreening assay. Fourth, model organisms such as Drosophila melanogaster or Caenorhabditis elegans have only been minimally investigated with respect to CaCC. For example, it is not immediately clear which phenotype should be looked for in a genetic screen to detect CaCC mutants in C. elegans. This situation sharply contrasts with the field of cation channels in which one or more of the strategies mentioned previously here (expression cloning, channel purification, immunoscreening, or model organism) have allowed the cloning of a wide variety of ion channels.

The CLCA Family
In spite of these problems, molecular candidates for CaCC have been forwarded over the past years, the most notable being the CLCA proteins (15, 16). The first CLCA cDNA clone was isolated in 1995 from a bovine tracheal cDNA library, which was immunoscreened with an antibody against a Ca²⁺-sensitive anion channel (16). Transfection of the bovine CLCA in COS-7 cells resulted in the expression of an anion-selective membrane current that was activated by the Ca²⁺ ionophore ionomycin (16). Further research has shown that CLCA proteins form a multigene family with several isoforms (hCLCA1–4 in humans, mCLCA1–4 in mice; note that the numbering reflects the time sequence of cloning, which differs from species to species) (17). According to a recent model (18), CLCA proteins are synthesized as 125-kD precursor proteins with five transmembrane domains (extracellular amino [NH₂]-terminus, intracellular carboxyl [COOH] terminus), which is then cleaved in a 90-kD NH₂-terminal fragment containing membrane spanning domains 1 to 3 and a COOH-terminal 38-kD fragment with two transmembrane domains. Heterologous expression of various CLCA isoforms have generated membrane currents that could be activated by either ionomycin or by high Ca²⁺ (2 mM) in the pipette solution and that were blocked by chloride channel blockers such as niflumic acid and 4,4'-di-isothiocyanostilbene-2,2'-disulfonic acid. A distinguishing feature of the CLCA currents is their sensitivity to reducing agents such as dithiothreitol (15, 16). Because these experiments were performed under Cl^--selective conditions (i.e., absence of Na⁺ and K⁺ in intracellular and extracellular solutions), these data were interpreted in favor of CLCA being Ca²⁺-activated Cl^- channels. However, this conclusion has not been universally approved because of these observations.

First, it is not clear which part of the protein is responsible for the channel properties (Figure 1). Initial biochemical experiments had indicated that the CaCC channel consisted of a multimeric 140-kD complex, which under reducing conditions fell apart in 38-kD monomers (21). Immunoscreening of a bovine trachea cDNA library with an antibody against the 38-kD monomer yielded a cDNA clone (later called bCLCA1) encoding a 100-kD peptide, which after glycosylation and proteolytic cleavage is processed in a NH₂-terminal 90-kD and COOH-terminal 40-kD fragment (16). However, Ji and colleagues generated a deletion mutant from the 90-kD bCLCA1 fragment and expressed it in Xenopus oocytes (20). This fragment yielded identical currents as expressing the full-length fragment. Thus, based on immunologic and biochemical evidence, one would predict that the channel activity is located in the COOH-terminal 40-kD domain. In contrast, the mutagenesis study identifies the 90-kD subfragment as the channel protein. This discrepancy should be addressed by further studies. Moreover, although the first CLCA cDNA was cloned in 1995, we still have to wait for additional mutagenesis studies to probe the structure–function relationship. A final problem related to the CLCA structure is the discovery of human CLCA3, which is a NH₂ terminally truncated variant without any of the transmembrane domains and which appears to be secreted in the extracellular medium (19). The relationship between this secreted protein and the Cl^- channel function of CLCA proteins remains to be elucidated.

View larger version (13K): [in this window] [in a new window]   Figure 1. Schematic structure of the hCLCA2 protein (943 amino acids) as proposed by Gruber and colleagues (18). Proposed transmembrane domains (TM1–5) are represented by black diamonds with the NH2 terminus situated extracellular and the COOH (carboxyl) terminus intracellular. The tree-like structures represent predicted N-glycosylation sites in extracellular regions. The proteolytic cleavage site is indicated by the vertical arrow. The location of the secreted hCLCA3 (19) and of the truncated bCLCA1 expressed by Ji and colleagues (20) is indicated by the horizontal arrows underneath.

To conclude, the molecular identification of CaCC is far from resolved. Although CLCA proteins are valid candidates, further data are needed before definitely establishing CLCA as Ca²⁺-activated Cl^- channels. First, in spite of their being cloned more than 7 years ago, a detailed analysis of the biophysical fingerprint of CLCAs (ion selectivity, Ca²⁺ dependence, and time and voltage dependence) is still urgently needed to allow a more refined comparison with endogenous CaCC. Second, a structure–function relationship should be worked out. A crucial experiment here is to correlate the initial observation that the channel function resides in a 40-kD fragment with the later observations that channel function resides in a larger 90-kD fragment. Third, part of the controversy could be explained by the possibility that CLCA proteins are regulators of Cl^- channels rather than ion channels, as suggested by some recent experiments (23, 24). Finally, one should bear in mind that CLCA proteins can serve functions completely different from ion transport. This is suggested by the cloning of a secreted CLCA isoform (hCLCA3) incapable of transmembrane ion transport. Furthermore, several findings link CLCA to mucus production and to cell adhesion. Indeed, mCLCA3 (or gob-5) localizes to intracellular mucin granule membranes in the intestine and the lung (25). Moreover, expression of mCLCA3 promotes mucus production both in vivo (in the trachea of airway-hyperresponsive mice) and in vitro (transfection in a mucoepidermoid cell line) (26). These data suggest a role for mCLCA3 (and its human ortholog hCLCA1) in the control of mucus production and as potential therapeutic targets in diseases with mucus overproductions such as asthma. On the other hand, hCLCA2, which is expressed on the plasma membrane of pulmonary endothelial cells, mediates binding of tumor cells via a ß4 integrin–hCLCA2 interaction, thereby facilitating tumor metastasis to the lung (27). Similarly, bovine CLCA2 (originally cloned as Lu-Ecam-1) is expressed in the pulmonary venous endothelium and has been shown to promote adhesion of melanoma cells (28). Thus, irrespective of their Cl^- channel function, CLCA proteins are bound to generate much interest in view of their pleiotropic and clinically relevant effects.

Other Molecular Candidates for CaCC
Sun and colleagues recently showed that transient expression of human bestrophin 2, a membrane protein mutated in vitelliform macular dystrophy (Best disease), generates Ca²⁺-sensitive anion currents (29). An extensive homology search of the human genome has identified four bestrophin isoforms, all of which generate membrane currents in transfected HEK293 cells (30). Moreover, specific point mutations in bestrophins alter the current phenotype, suggesting that bestrophins are genuine channel components (29, 30). Furthermore, Qu and colleagues have recently examined the biophysical properties of two bestrophins cloned from Xenopus laevis oocytes. In transfected HEK293 cells, they detected Ca²⁺ activated Cl^- currents with a K_Ca of 210 nM and an I^- > Br^- > Cl^- > aspartate permeability ratio (31). Thus, bestrophins are the first cloned anion channels that are activated by physiologic Ca²⁺ concentrations. Based on these data, bestrophins are very promising candidates for Ca²⁺-activated Cl^- channels, but there still remain many questions to answer. For example, do all or only some of four identified bestrophins function as Ca²⁺-activated Cl^- channels? Are there are interacting proteins that could affect the functional properties such as Ca²⁺ activation and voltage dependence? Which region in the protein sequence is responsible for pore formation and gating?

It has also been reported that ClC-3 may function as a calmodulin kinase II–regulated anion channel in the plasma membrane (32). At present, it is not clear how this observation relates to the intracellular location of ClC-3 or to its presumed link to VRACs (1).

	LACK OF SPECIFIC PHARMACOLOGIC TOOLS

TOP ABSTRACT THE CACC PHENOTYPE MOLECULAR IDENTITY OF CACC LACK OF SPECIFIC PHARMACOLOGIC... CACC AS A MOLECULAR... REFERENCES

 
Specific pharmacologic tools, often in the form of biological toxins with a high affinity and selectivity, have been (and still are) an invaluable asset in the identification of cation channels and in the study of their structure and function. Sadly, there are no equivalent tools for CaCC. At one time, chlorotoxin, an insectotoxin purified from the venom of the scorpion Leiurus quinquestriatus, seemed to be a promising candidate for a chloride channel-specific toxin (33). However, Maertens and colleagues showed that chlorotoxin does not inhibit CaCC in T84 intestinal epithelial cells or other Cl^- channels such as VRAC or CFTR (34). In addition, the chemical compounds that block CaCC suffer from lack of selectivity and/or low affinity. This is poignantly illustrated by Table 2, which compares the inhibitory effect of some commonly used compounds on endogenous CaCC and VRAC. Clearly, none of the compounds are specific for one or the other channel. Even niflumic acid, which is often used to prove the involvement of CaCC, cannot be considered as a CaCC-specific probe since it also blocks VRAC in some cell types albeit at higher (10-fold) concentration (35).

	CACC AS A MOLECULAR TARGET IN CF

TOP ABSTRACT THE CACC PHENOTYPE MOLECULAR IDENTITY OF CACC LACK OF SPECIFIC PHARMACOLOGIC... CACC AS A MOLECULAR... REFERENCES

 
Rationale of a CaCC-targeted CF Therapy
The basic defect in CF is the lack of functional CFTR proteins in the apical membrane of secretory epithelia (44). In the airway epithelium, this defect has a dual effect: (1) an increased fluid reabsorption because of deinhibition of the epithelial Na⁺ channel. Indeed, wild-type CFTR exerts a tonic inhibitory influence on epithelial Na⁺ channel, thereby limiting fluid absorption across the epithelium. (2) Decreased fluid secretion because of loss of the apical Cl^- efflux via CFTR acting as a Cl^- channel. The combined result of the loss of CFTR function is a depletion of airway surface liquid (ASL), compaction of the mucus layer on the apical cilia, and reduced mucociliary clearance leading to colonization and infection of the airways (for a review, see Reference 45). Given that CFTR and CaCC are both apical Cl^- channels, it seems logical to advance CaCC as a molecular target for CF therapy. However, one should bear in mind that CaCC and CFTR are not functionally equivalent. Indeed, there is no experimental evidence that CaCC negatively interacts with epithelial Na⁺ channel. Thus, even if Cl^- secretion can be fully restored by activating CaCC, fluid hyperabsorption would not be corrected. CaCC targeting can therefore be better described as a bypass therapy (shunting the increased fluid reabsorption by increasing fluid secretion) rather than replacement therapy.

A second point to bear in mind when discussing CaCC as a potential molecular target for CF is that CFTR and CaCC functionally interact. Indeed, transient expression of CFTR in a cell line without endogenous CFTR currents reduced the endogenous CaCC currents (46). The inhibition was independent of the mode of CaCC activation, as it was observed in cells dialyzed with high Ca²⁺ via the pipette solution and in cells stimulated with ATP as a Ca²⁺ mobilizing agonist. The precise mode of interaction is not understood, but the point of interaction lies downstream of the Ca²⁺ signal because heterologous expression of CFTR did not affect the magnitude of the Ca²⁺ transient in ATP-stimulated cells. The inhibition is partly activity dependent, as indicated by the observation that CaCC downregulation by CFTR is more pronounced if CFTR has been preactivated by a cAMP increasing cocktail. Conversely, Tarran and colleagues observed an upregulation of CaCC in polarized mouse tracheal epithelial cells of CF mice, although in these experiments the higher CaCC currents could partly be ascribed to an increased Ca²⁺ signal in CF cells (47). Taken together, these data indicate an inhibitory interaction between CFTR and CaCC with an increase in CFTR expression leading to CaCC downregulation and vice versa. Unfortunately, the lack of molecular identification hampers further experiments to nail down the precise interaction points between CFTR and CaCC.

Is CaCC Present in the Airway Epithelium?
The first question to answer when considering CaCC as a molecular target for CF is whether CaCC is present in the airway epithelium. Several experimental approaches (transepithelial short-circuit measurements or whole-cell patch clamping) point to the presence of a Ca²⁺-activated Cl^- conductance in airway epithelial cells, in addition to the well characterized protein kinase A–activated, CFTR-mediated Cl^- conductance (48). Moreover, by selectively permeabilizing the basolateral membrane of murine tracheal epithelial cells, Gabriel and colleagues provided functional evidence for the apical location of CaCC (49).

A major shortcoming with respect to our understanding of CaCC expression in the airways is the molecular identity. The first member of the CLCA family was originally cloned from bovine trachea (16), but as discussed previously here, it remains controversial whether CLCA proteins correspond to CaCC.

Does CaCC Contribute to Airway Fluid Secretion and in Particular to ASL Volume?
A second question relates to the functional role of CaCC in the apical membrane of airway epithelium. Functional studies have indicated a Ca²⁺-dependent increase in Cl^- secretion across airway epithelial cells. Specifically, using a murine tracheal epithelial cell line, Tarran and colleagues showed that apical application of uridine triphosphate (UTP), a Ca²⁺-mobilizing agonist acting on P2Y purinergic receptors, increased the short-circuit current and ASL height (47). The UTP effect was more pronounced in a CF cell line than in a wild-type cell line, again underscoring the negative interaction between CFTR and CaCC. Moreover, it was concluded that CaCC does not contribute to basal ASL homeostasis, which is predominantly determined by CFTR. In contrast, CaCC seems to be an acute regulator of Cl^- secretion and ASL height and therefore integrates extracellular stimuli and ASL height. The contribution of CaCC to ASL homeostasis together with the prominent expression of CaCC in murine airway epithelium probably explains the lack of a lung phenotype in the various CF mice models (50).

Outward Rectification of CaCC: Implications for Efficient Cl^- Secretion
Another point to consider is the biophysical profile of CaCC, which does not immediately suggest a role in secretion. Indeed, the steep outward rectification of CaCC implies tiny inward currents and hence minimal Cl^- efflux. Thus, one might ask whether CaCC is a good target after all to bypass the cellular defect in CF. One should bear in mind that the rectification results from the opposing effects of Ca²⁺-dependent channel opening versus voltage-dependent channel closing (9). Outward rectification is observed at intermediary Ca²⁺ concentrations (opening predominating at positive potentials, closing at negative potentials), whereas at saturating Ca²⁺ concentrations, the voltage-independent Ca²⁺-dependent opening is the major event, thereby resulting in a linear current–voltage relationship and hence significant Cl^- efflux. This biophysical property is directly relevant to the problem of CF therapy. Therapeutic interventions that activate CaCC via Ca²⁺-mobilizing agonists should provoke (near) maximal stimulation of the airway epithelium with a micromolar Ca²⁺ transient, thereby ensuring significant Cl^- efflux via CaCC. However, continuously exposing cells to high intracellular Ca²⁺ levels may trigger unwanted side effects, including apoptosis (51). A possible strategy to simultaneously activate CaCC and to avoid side effects due to a global increase in cytosolic Ca²⁺ would be the application of an agonist, which induces a Ca²⁺ signal that is spatially limited to the apical subplasma membrane zone. Mucosal administration of nucleotide agonists that act on apically located P2Y receptors may fulfill this criterion (6). Alternatively, one could look for Ca²⁺-independent channel openers, but the current lack of selective CaCC ligands seriously impairs this quest.

Epithelium-selective Activation of CaCC
A final point to consider is the epithelium-selective activation of CaCC in the airways. Indeed, CaCC is also expressed in airway smooth muscle, where opening of CaCC causes so-called depolarizing spontaneous transient inward currents and hence smooth muscle contraction (52). Thus, epithelium-selective activation of CaCC is required to avoid bronchoconstriction due to smooth muscle CaCC activation.

CONCLUSIONS
Although CaCC has been known for nearly two decades, it remains difficult to appreciate fully its functional role and how this channel can be exploited as a therapeutic target. The main hurdle to be taken at the moment is the molecular identification. Indeed, clarification of the genetic and protein structure will allow the development of specific molecular tools to probe CaCC structure and function at the molecular, cellular, and organ level.

	FOOTNOTES

 
CaCC will be used as a generic abbreviation referring to Ca²⁺ activated Cl^- channels in general. CLCA refers to a gene family containing several members (hCLCA1-4 in humans, mCLCA1-4 in mice) that have been proposed as molecular candidates for CaCC.

Supported by the Forton Foundation (Koning Boudewijn Stichting, Belgium).

(Received in original form June 19, 2003; accepted in final form September 5, 2003)

	REFERENCES

TOP ABSTRACT THE CACC PHENOTYPE MOLECULAR IDENTITY OF CACC LACK OF SPECIFIC PHARMACOLOGIC... CACC AS A MOLECULAR... REFERENCES

 

1. Jentsch TJ, Stein V, Weinreich F, Zdebik AA. Molecular structure and physiological function of chloride channels. Physiol Rev 2002;82:503–568.[Abstract/Free Full Text]
2. Barish ME. A transient calcium-dependent chloride current in the immature Xenopus oocyte. J Physiol 1983;342:309–325.[Abstract/Free Full Text]
3. Large WA, Wang Q. Characteristics and physiological role of the Ca²⁺-activated Cl^- conductance in smooth muscle. Am J Physiol Cell Physiol 1996;40:C435–C454.
4. Begenisch T, Melvin JE. Regulation of chloride channels in secretory epithelia. J Membr Biol 1998;163:77–85.[CrossRef][Medline]
5. Frings S, Reuter D, Kleene SJ. Neuronal Ca²⁺-activated Cl^- channels: homing in on an elusive channel species. Prog Neurobiol 2000;60:247–289.[CrossRef][Medline]
6. Paradiso AM, Ribeiro CM, Boucher RC. Polarized signaling via purinoceptors in normal and cystic fibrosis airway epithelia. J Gen Physiol 2001;117:53–67.
7. Papassotiriou J, Eggermont J, Droogmans G, Nilius B. Ca²⁺-activated Cl^- channels in Ehrlich ascites tumor cells are distinct from mCLCA1, 2 and 3. Pflügers Arch 2001;442:273–279.[CrossRef][Medline]
8. Nilius B, Prenen J, Voets T, Vandenbremt K, Eggermont J, Droogmans G. Kinetic and pharmacological properties of the calcium activated chloride current in macrovascular endothelial cells. Cell Calcium 1997;22:53–63.[CrossRef][Medline]
9. Kuruma A, Hartzell HC. Bimodal control of a Ca²⁺-activated Cl^- channel by different Ca²⁺ signals. J Gen Physiol 2000;115:59–80.
10. Qu ZQ, Hartzell HC. Anion permeation in Ca²⁺-activated Cl^- channels. J Gen Physiol 2000;116:825–844.[Abstract/Free Full Text]
11. Evans MG, Marty A. Calcium-dependent chloride currents in isolated cells from rat lacrimal glands. J Physiol 1986;378:437–460.[Abstract/Free Full Text]
12. Arreola J, Melvin JE, Begenisch T. Activation of calcium-dependent chloride channels in rat parotid acinar cells. J Gen Physiol 1996;108:35–47.[Abstract/Free Full Text]
13. Worrell RT, Frizzell RA. CaMKII mediates stimulation of chloride conductance by calcium in T84 cells. Am J Physiol 1991;260:C877–C882.
14. Wang YX, Kotlikoff MI. Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase. Proc Natl Acad Sci USA 1997;94:14918–14923.[Abstract/Free Full Text]
15. Gruber AD, Elble RC, Ji HL, Schreur KD, Fuller CM, 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 1998;54:200–214.[CrossRef][Medline]
16. Cunningham SA, Awayda MS, Bubien JK, Ismailov II, Arrate MP, Berdiev BK, Benos DJ, Fuller CM. Cloning of an epithelial chloride channel from bovine trachea. J Biol Chem 1995;270:31016–31026.[Abstract/Free Full Text]
17. Fuller CM, Ji H-L, Tousson A, Elble RC, Pauli BU, Benos DJ. Ca²⁺-activated Cl^- channels: a newly emerging anion transport family. Pflügers Arch 2001;443:S107–S110.
18. Gruber AD, Schreur KD, Ji HL, Fuller CM, Pauli BU. Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland. Am J Physiol Cell Physiol 1999;45:C1261–C1270.
19. Gruber AD, Pauli BU. Molecular cloning and biochemical characterization of a truncated, secreted member of the human family of Ca²⁺-activated Cl^- channels. Biochim Biophys Acta 1999;1444:418–423.[Medline]
20. Ji HL, DuVall MD, Patton HK, Satterfield CL, Fuller CM, Benos DJ. Functional expression of a truncated Ca²⁺-activated Cl^- channel and activation by phorbol ester. Am J Physiol Cell Physiol 1998;274:C455–C464.[Abstract/Free Full Text]
21. Ran S, Fuller CM, Arrate MP, Latorre R, Benos DJ. Functional reconstitution of a chloride channel protein from bovine trachea. J Biol Chem 1992;267:20630–20637.[Abstract/Free Full Text]
22. Greenwood IA, Miller LJ, Ohya S, Horowitz B. The large conductance potassium channel ß-subunit can interact with and modulate the functional properties of a calcium-activated chloride channel, CLCA1. J Biol Chem 2002;277:22119–22122.[Abstract/Free Full Text]
23. Loewen ME, Smith NK, Hamilton DL, Grahn BH, Forsyth GW. CLCA protein and chloride transport in canine retinal pigment epithelium. Am J Physiol Cell Physiol 2003;285:C1314–C1321.[Abstract/Free Full Text]
24. Loewen ME, Bekar LK, Gabriel SE, Walz W, Forsyth GW. pCLCA1 becomes a cAMP-dependent chloride conductance mediator in Caco-2 cells. Biochem Biophys Res Commun 2002;298:531–536.[CrossRef][Medline]
25. Leverkoehne I, Gruber AD. The murine mCLCA3 (alias gob-5) protein is located in the mucin granule membranes of intestinal, respiratory, and uterine goblet cells. J Histochem Cytochem 2002;50:829–838.[Abstract/Free Full Text]
26. Nakanishi A, Morita S, Iwashita H, Sagiya Y, Ashida Y, Shirafuji H, Fujisawa Y, Nishimura O, Fujino M. Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma. Proc Natl Acad Sci USA 2001;98:5175–5180.[Abstract/Free Full Text]
27. Abdel-Ghany M, Cheng HC, Elble RC, Pauli BU. The breast cancer ß₄ integrin and endothelial human CLCA2 mediate lung metastasis. J Biol Chem 2001;276:25438–25446.[Abstract/Free Full Text]
28. Elble RC, Widom J, Gruber AD, AbdelGhany M, Levine R, Goodwin A, Cheng HC, Pauli BU. Cloning and characterization of lung endothelial cell adhesion molecule-1 suggest it is an endothelial chloride channel. J Biol Chem 1997;272:27853–27861.[Abstract/Free Full Text]
29. Sun H, Tsunenari T, Yau K-W, Nathans J. The vitelliform macular dystrophy protein defines a new family of chloride channels. Proc Natl Acad Sci USA 2002;99:4008–4013.[Abstract/Free Full Text]
30. Tsunenari T, Sun H, Williams J, Cahill H, Smallwood P, Yau K-W, Nathans J. Structure-function analysis of the bestrophin family of anion channels. J Biol Chem 2003;278:41114–41125.[Abstract/Free Full Text]
31. Qu Z, Wei RW, Mann W, Hartzell HC. Two bestrophins cloned from Xenopus laevis oocytes express Ca-activated Cl currents. J Biol Chem (In press)
32. Huang P, Liu J, Di A, Robinson NC, Musch MW, Kaetzel MA, Nelson DJ. Regulation of human ClC-3 channels by multifunctional Ca²⁺/calmodulin-dependent protein kinase. J Biol Chem 2001;276:20093–20100.[Abstract/Free Full Text]
33. DeBin JA, Maggio JE, Strichartz GR. Purification and characterization of chlorotoxin, a chloride channel ligand from the venom of the scorpion. Am J Physiol 1993;264:C361–C369.
34. Maertens C, Wei L, Tytgat J, Droogmans G, Nilius B. Chlorotoxin does not inhibit volume-regulated, calcium-activated and cyclic AMP-activated chloride channels. Br J Pharmacol 2000;129:791–801.[CrossRef][Medline]
35. Greenwood IA, Large WA. Properties of a Cl^- current activated by cell swelling in rabbit portal vein vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 1998;44:H1524–H1532.
36. Maertens C, Wei L, Droogmans G, Nilius B. Inhibition of volume-regulated and calcium-activated chloride channels by the antimalarial mefloquine. J Pharmacol Exp Ther 2000;295:29–36.[Abstract/Free Full Text]
37. Yamazaki J, Hume JR. Inhibitory effects of glibenclamide on cystic fibrosis transmembrane regulator, swelling activated, and Ca²⁺ activated Cl^- channels in mammalian cardiac myocytes. Circ Res 1997;81:101–109.[Abstract/Free Full Text]
38. Fan HT, Morishima S, Kida H, Okada Y. Phloretin differentially inhibits volume-sensitive and cyclic AMP- activated, but not Ca-activated, Cl^- channels. Br J Pharmacol 2001;133:1096–1106.[CrossRef][Medline]
39. White MM, Aylwin M. Niflumic and flufenamic acids are potent reversible blockers of Ca²⁺-activated Cl^- channels in Xenopus oocytes. Mol Pharmacol 1990;37:720–724.[Abstract]
40. Pedersen SF, Prenen J, Droogmans G, Hoffmann EK, Nilius B. Separate swelling- and Ca²⁺-activated anion currents in Ehrlich ascites tumor cells. J Membr Biol 1998;163:97–110.[CrossRef][Medline]
41. Nilius B, Sehrer J, Viana F, De Greef C, Raeymaekers L, Eggermont J, Droogmans G. Volume-activated Cl^- currents in different mammalian non-excitable cell types. Pflügers Arch 1994;428:364–371.[CrossRef][Medline]
42. Nilius B, Prenen J, Szucs G, Wei L, Tanzi F, Voets T, Droogmans G. Calcium-activated chloride channels in bovine pulmonary artery endothelial cells. J Physiol 1997;498:381–396.[Medline]
43. Valverde MA, Mintenig GM, Sepulveda FV. Differential effects of tamoxifen and I^- on three distinguishable chloride currents activated in T84 intestinal cells. Pflügers Arch 1993;425:552–554.[CrossRef][Medline]
44. Wine JJ. The genesis of cystic fibrosis lung disease. J Clin Invest 1999;103:309–312.[Medline]
45. Knowles MR, Boucher RC. Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 2002;109:571–577.[CrossRef][Medline]
46. Wei L, Vankeerberghen A, Cuppens H, Eggermont J, Cassiman JJ, Droogmans G, Nilius B. Interaction between calcium-activated chloride channels and the cystic fibrosis transmembrane conductance regulator. Pflügers Arch 1999;438:635–641.[CrossRef][Medline]
47. Tarran R, Loewen ME, Paradiso AM, Olsen JC, Gray MA, Argent BE, Boucher RC, Gabriel SE. Regulation of murine airway surface liquid volume by CFTR and Ca²⁺-activated Cl^- conductances. J Gen Physiol 2002;120:407–418.[Abstract/Free Full Text]
48. Anderson MP, Welsh MJ. Calcium and cAMP activate different chloride channels in the apical membrane of normal and cystic fibrosis epithelia. Proc Natl Acad Sci USA 1991;88:6003–6007.[Abstract/Free Full Text]
49. Gabriel SE, Makhlina M, Martsen E, Thomas EJ, Lethem MI, Boucher RC. Permeabilization via the P2X7 purinoreceptor reveals the presence of a Ca²⁺-activated Cl^- conductance in the apical membrane of murine tracheal epithelial cells. J Biol Chem 2000;275:35028–35033.[Abstract/Free Full Text]
50. Clarke L, Grubb B, Yankaskas J, Cotton C, McKenzie A, Boucher R. Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in Cftr(-/-) mice. Proc Natl Acad Sci USA 1994;91:479–483.[Abstract/Free Full Text]
51. Berridge MJ, Bootman MD, Lipp P. Calcium: a life and death signal. Nature 1998;395:645–648.[CrossRef][Medline]
52. Janssen LJ, Sims SM. Ca²⁺-dependent Cl^- current in canine tracheal smooth muscle cells. Am J Physiol 1995;269:C163–C169.

This article has been cited by other articles:

				H. C. Hartzell, Z. Qu, K. Yu, Q. Xiao, and L.-T. Chien Molecular Physiology of Bestrophins: Multifunctional Membrane Proteins Linked to Best Disease and Other Retinopathies Physiol Rev, April 1, 2008; 88(2): 639 - 672. [Abstract] [Full Text] [PDF]

				R. De La Fuente, W. Namkung, A. Mills, and A. S. Verkman Small-Molecule Screen Identifies Inhibitors of a Human Intestinal Calcium-Activated Chloride Channel Mol. Pharmacol., March 1, 2008; 73(3): 758 - 768. [Abstract] [Full Text] [PDF]

				A. Saiardi and S. Cockcroft Human ITPK1: A Reversible Inositol Phosphate Kinase/Phosphatase That Links Receptor-Dependent Phospholipase C to Ca2+-Activated Chloride Channels Sci. Signal., January 29, 2008; 1(4): pe5 - pe5. [Abstract] [Full Text] [PDF]

				L.-T. Chien, Z.-R. Zhang, and H. C. Hartzell Single Cl- Channels Activated by Ca2+ in Drosophila S2 Cells Are Mediated By Bestrophins J. Gen. Physiol., August 28, 2006; 128(3): 247 - 259. [Abstract] [Full Text] [PDF]

				A. Gibson, A. P. Lewis, K. Affleck, A. J. Aitken, E. Meldrum, and N. Thompson hCLCA1 and mCLCA3 Are Secreted Non-integral Membrane Proteins and Therefore Are Not Ion Channels J. Biol. Chem., July 22, 2005; 280(29): 27205 - 27212. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Eggermont, J. Search for Related Content PubMed PubMed Citation Articles by Eggermont, J.