The chloride channel ClC-2 has been implicated in neonatal airway chloride secretion. To assess its role in secretion by the small intestine, we assessed its subcellular expression in ileal segments obtained from mice and studied the chloride transport properties of this tissue. Chloride secretion across the mucosa of murine ileal segments was assessed in Ussing chambers as negative short-circuit current (I(sc)). If ClC-2 contributed to chloride secretion, we predicted on the basis of previous studies that negative I(sc) would be stimulated by dilution of the mucosal bath and that this response would depend on chloride ion and would be blocked by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid but not by DIDS. In fact, mucosal hypotonicity did stimulate a chloride-dependent change in I(sc) that exhibited pharmacological properties consistent with those of ClC-2. This secretory response is unlikely to be mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) channel because it was also observed in CFTR knockout animals. Assessment of the native expression pattern of ClC-2 protein in the murine intestinal epithelium by confocal and electron microscopy showed that ClC-2 exhibits a novel distribution, a distribution pattern somewhat unexpected for a channel involved in chloride secretion. Immunolabeled ClC-2 was detected predominantly at the tight junction complex between adjacent intestinal epithelial cells.

It has been previously determined that ClC-2, a member of the ClC chloride channel superfamily, is expressed in certain epithelial tissues. These findings fueled speculation that ClC-2 can compensate for impaired chloride transport in epithelial tissues affected by cystic fibrosis and lacking the cystic fibrosis transmembrane conductance regulator. However, direct evidence linking ClC-2 channel expression to epithelial chloride secretion was lacking. In the present studies, we show that ClC-2 transcripts and protein are present endogenously in the Caco-2 cell line, a cell line that models the human small intestine. Using an antisense strategy we show that ClC-2 contributes to native chloride currents in Caco-2 cells measured by patch clamp electrophysiology. Antisense ClC-2-transfected monolayers of Caco-2 cells exhibited less chloride secretion (monitored as iodide efflux) than did mock transfected monolayers, providing the first direct molecular evidence that ClC-2 can contribute to chloride secretion by the human intestinal epithelium. Further, examination of ClC-2 localization by confocal microscopy revealed that ClC-2 contributes to secretion from a unique location in this epithelium, from the apical aspect of the tight junction complex. Hence, these studies provide the necessary rationale for considering ClC-2 as a possible therapeutic target for diseases affecting intestinal chloride secretion such as cystic fibrosis.

No abstract

We have used a mouse model to study the ability of human CFTR to correct the defect in mice deficient of the endogenous protein. In this model, expression of the endogenous Cftr gene was disrupted and replaced with a human CFTR cDNA by a gene targeted 'knock-in' event. Animals homozygous for the gene replacement failed to show neither improved intestinal pathology nor survival when compared to mice completely lacking CFTR. RNA analyses showed that the human CFTR sequence was transcribed from the targeted allele in the respiratory and intestinal epithelial cells. Furthermore, in vivo potential difference measurements showed that basal CFTR chloride channel activity was present in the apical membranes of both nasal and rectal epithelial cells in all homozygous knock-in animals examined. Ussing chamber studies showed, however, that the cAMP-mediated chloride channel function was impaired in the intestinal tract among the majority of homozygous knock-in animals. Hence, failure to correct the intestinal pathology associated with loss of endogenous CFTR was related to inefficient functional expression of the human protein in mice. These results emphasize the need to understand the tissue-specific expression and regulation of CFTR function when animal models are used in gene therapy studies.

Cystic Fibrosis (CF) is caused by mutations in the CF gene that lead, for the most part, to mislocalization of the protein product, the cystic fibrosis transmembrane conductance regulatory (CFTR). CFTR is a chloride channel normally situated in the apical membrane of epithelial cells where it contributes to transepithelial ion transport. In this study we demonstrated the feasibility of in vivo transfer of purified CFTR protein via phospholipid liposomes into the apical membrane of nasal epithelia of CFTR knockout mice. Membrane incorporation of immunogold-labeled CFTR could be visualized by electron microscopy and correction of CF-related defects in ion transport measured by nasal potential difference (PD) measurements in about one-third of the animals treated. Although these initial results are promising, effectiveness of this therapeutic approach appears to be limited by the inefficient incorporation of CFTR into the apical epithelial cell membrane.