Abstract
BackgroundCLIC1 is a chloride channel whose cellular role remains uncertain. The distribution of CLIC1 in normal tissues is largely unknown and conflicting data have been reported regarding the cellular membrane fraction in which CLIC1 resides.ResultsNew antisera to CLIC1 were generated and were found to be sensitive and specific for detecting this protein. These antisera were used to investigate the distribution of CLIC1 in mouse tissue sections and three cultured cell lines. We find CLIC1 is expressed in the apical domains of several simple columnar epithelia including glandular stomach, small intestine, colon, bile ducts, pancreatic ducts, airway, and the tail of the epididymis, in addition to the previously reported renal proximal tubule. CLIC1 is expressed in a non-polarized distribution in the basal epithelial cell layer of the stratified squamous epithelium of the upper gastrointesitinal tract and the basal cells of the epididymis, and is present diffusely in skeletal muscle. Distribution of CLIC1 was examined in Panc1 cells, a relatively undifferentiated, non-polarized human cell line derived from pancreatic cancer, and T84 cells, a human colon cancer cell line which can form a polarized epithelium that is capable of regulated chloride transport. Digitonin extraction was used to distinguish membrane-inserted CLIC1 from the soluble cytoplasmic form of the protein. We find that digitonin-resistant CLIC1 is primarily present in the plasma membrane of Panc1 cells. In T84 cells, we find digitonin-resistant CLIC1 is present in an intracellular compartment which is concentrated immediately below the apical plasma membrane and the extent of apical polarization is enhanced with forskolin, which activates transepithelial chloride transport and apical membrane traffic in these cells. The sub-apical CLIC1 compartment was further characterized in a well-differentiated mouse renal proximal tubule cell line. The distribution of CLIC1 was found to overlap that of megalin and the sodium-phosphate cotransporter, NaPi-II, which are markers of the apical endocytic/recycling compartment in proximal tubule.ConclusionThe cell and tissue specific patterns of CLIC1 expression suggest it may play distinct roles in different cell types. In certain polarized columnar epithelia, it may play a role in apical membrane recycling.
Highlights
CLIC1 is a chloride channel whose cellular role remains uncertain
In non-polarized cells such as basal epithelial cells of upper gastrointestinal tract, skeletal muscle, and cultured Panc1 cells, CLIC1 is expressed in a nonpolarized distribution that appears to be throughout the cytoplasm
In the cultured cell model of non-polarized cells, the majority of the CLIC1 is present in a soluble form in the cytoplasm, while the membrane-inserted fraction of CLIC1 is primarily in the plasma membrane
Summary
CLIC1 is a chloride channel whose cellular role remains uncertain. A variety of distinct chloride channel activities have been described that carry out many essential roles in organismal physiology [1]. The proteins responsible for some chloride conductances have been described, there are still multiple physiologically important chloride channel activities for which the molecular basis is not yet understood. Identification of the proteins responsible for these "orphan" channel activities will be an important step in more fully understanding ion transport physiology in health and disease. CLICs are a closely related family of chloride channel proteins [3,4]. The highly conserved CLIC domain has no homology to other known chloride channel proteins; it does show low but significant homology to the family of glutathione-S-transferases [5]
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