EDITORIAL FOCUS
Chloride channels and tight junctions.
Focus on "Expression of the chloride channel ClC-2 in the murine small intestine epithelium"

Kevin L. Kirk

Department of Physiology and Biophysics and Department of Neurobiology, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005


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A MAJOR GOAL in cystic fibrosis (CF) research is the identification of alternate chloride channels that might substitute for the cystic fibrosis transmembrane conductance regulator (CFTR), the anion channel that is encoded by the CF gene (2, 10). CFTR localizes primarily to the luminal surfaces of epithelial cells, where it mediates transcellular chloride and bicarbonate transport (1, 5). Activation of CFTR by cyclic nucleotide-dependent phosphorylation leads to the secretion of salt and water in intestine and exocrine glands. Excessive CFTR activity causes secretory diarrhea (induced, for example, by bacterial toxins that elevate cyclic nucleotide production in the gut). Reduced CFTR activity causes CF. The severest cases of CF are characterized by airway infection and inflammation, pancreatic insufficiency, and intestinal obstruction (4). Whether all of these pathologies are attributable to a loss of chloride channel activity vs. other possible functions of CFTR is not clear (8). However, it seems reasonable to search for additional epithelial chloride channels that might serve as proxies for CFTR, since the anion channel activity of CFTR is its most well-accepted functional property.

The study by Gyömörey et al., the current article in focus (Ref. 7, see p. C1787 in this issue), provides evidence for a chloride channel other than CFTR that can mediate chloride secretion in the mouse small intestine. The authors show that segments of ileum isolated from CFTR knockout mice are capable of exhibiting chloride-dependent secretion and that the secretory rate of this tissue is increased by modest dilution of the mucosal (i.e., luminal) bath. The activation of this pathway by hypotonic shock, as well as its pharmacological profile [inhibited by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), but not by DIDS], are characteristics of ClC-2, a member of the ClC family of chloride channels (9, 12, 13). In support of this interpretation, the authors provide solid evidence for the expression of ClC-2 message and protein in mouse small intestine.

ClC-2 is an interesting candidate for an alternate chloride channel because of its relatively broad tissue distribution and its capacity to be activated by several factors including hypotonic shock, low pH, and protein kinases (9, 12, 13). However, the most surprising finding of the study by Gyömörey et al. is the predominant localization of ClC-2 protein to the tight junctions between epithelial cells in the surface villi of the small intestine. CFTR is most abundant in the intestinal crypts, which are the major sites of fluid secretion in this tissue (14, 16). The localization of ClC-2 to the villi rather than to the crypts does not necessarily exclude the possibility that activation of this channel could substitute for the missing CFTR activity in the CF intestine. For example, Zhou et al. (17) showed that the expression of human CFTR in the intestines of CFTR knockout mice reversed the intestinal pathology exhibited by these animals even though the human CFTR protein was localized primarily to the villus epithelial cells. What is more surprising is the apparent localization of ClC-2 to the tight junctions, which form continuous belts between adjacent epithelial cells. This localization of ClC-2 to tight junctions may be unique to the small intestine, since it has not been observed for fetal airway epithelial cells (which express ClC-2 along the luminal membrane, Ref. 9) or for the large intestine (Gyömörey K and Bear CE, unpublished observations). Thus ClC-2 may serve a specialized function at epithelial tight junctions in the small intestine.

What are the possible implications of localizing a chloride channel to the region of the tight junctions between epithelial cells? Two possibilities seem worth considering. First, ClC-2 channels may be localized to the luminal aspect of the tight junction by protein-protein interactions as a means to facilitate regulatory interactions between these channels and signaling molecules. Junctional complexes are "hot spots" for cell signaling molecules and PDZ domain-mediated protein interactions (reviewed in Ref. 15). Although ClC-2 appears to lack a canonical PDZ recognition signal, it could be localized to junctions by another mechanism and thereby interact with kinases and phosphatases that are concentrated in this region of the cell. According to this scenario, ClC-2 could still mediate chloride flow across the luminal membrane; its localization near the tight junction would simply increase its potential for regulation by signaling molecules.

A second possibility is that ClC-2 channels mediate chloride transport across epithelial tight junctions in the small intestine. Tight junctions are permselective, regulatable barriers to the flow of solutes between epithelial cells (i.e., the paracellular pathway). Although there has been considerable progress in our understanding of the molecular architecture of epithelial tight junctions (15), the mechanisms by which specific ions permeate this barrier are largely unknown. One exception is the recent identification of paracellin-1 as a mediator of magnesium reabsorption across tight junctions in the thick ascending limb of Henle (11). In an exciting convergence of genetics and epithelial physiology, the paracellin-1 gene was identified in patients with hereditary hypomagnesemia by positional cloning and was shown to encode a member of the claudin family of tight junction-associated proteins (11, 15). Paracellin-1 localizes specifically to the tight junctions in the thick ascending limb of the nephron, where it is essential for transepithelial magnesium reabsorption (11). Conceivably, ClC-2 channels that are localized to the tight junctions in small intestine could serve as conduits for paracellular chloride flow across this tissue. In this regard, one of the major functions of the villi of the small intestine is to reabsorb large quantities of sodium, chloride, and fluid (6). Sodium reabsorption occurs across the cells and is driven by the Na+-K+-ATPase at the basolateral membrane. Chloride reabsorption occurs at least in part via the paracellular pathway and is driven by the electrical gradient generated by active sodium reabsorption. Factors that increase the paracellular chloride permeability would increase the reabsorption of sodium as well as chloride by shunting the transepithelial voltage generated by sodium transport. It may be relevant that the chloride permeability of the paracellular pathway in mouse ileum is stimulated by agonists that also stimulate ClC-2 channel activity in heterologous expression systems (3, 12). Perhaps ClC-2 provides a regulated pathway for the paracellular flow of chloride in small intestine as a means to modulate the rates of salt (and fluid) reabsorption across this tissue.

In summary, the results of Gyömörey et al. remind us that CFTR is not the only interesting chloride channel in epithelial cells. Whether ClC-2 will become a useful target for drugs to circumvent the CF defect depends on the extent to which this channel can mediate chloride transport across the luminal membranes of airway and intestinal epithelial cells and on the identification of specific factors that increase its activity. However, irrespective of any possible connection to CF, the localization of ClC-2 to epithelial tight junctions raises interesting possibilities about the functional role of this chloride channel in the small intestine.


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REFERENCES

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Am J Physiol Cell Physiol 279(6):C1675-C1676
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