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C.J. Connon, S. Kawasaki, K. Yamasaki, A. Quantock, S. Kinoshita; Understanding the Function and Location of a Calcium Activated Chloride Channel in Ocular Surface Epithelium . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1038.
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Purpose: Calcium activated chloride channels (CLCA) are a recently discovered family of proteins with multi–functional capabilities, having the potential to act as anion channels and/or as cell adhesion molecules. The distribution and function of the human isoform CLCA–2 (hCLCA2) within ocular epithelium is presently unknown but may prove to be an important component of diseased ocular epithelium. This study endeavors to understand the role of hCLCA2 in human ocular epithelium. Methods: Snap frozen samples of human ocular surface epithelium embedded in OCT compound were sectioned at 6µm and subjected to an indirect fluorescent immunohistochemical analysis. Immunolabelling was facilitated by a novel anti–hCLCA2 primary antibody, raised against a synthetic polypeptide from hCLCA2 protein. Immunogold labelling was also conducted on human donor corneal tissue fixed in 4% paraformaldehyde, embedded in Unicryl resin, sectioned and viewed by TEM. Furthermore, RNAs were extracted with TRIzol from different types of ocular epithelia and subjected to RT– PCR and competitive PCR against hCLCA2 gene expression. Results: Immunofluorescent microscopy revealed a predominant expression of cytosolic hCLCA2 in basal cells adjacent to the basement membrane in corneal and limbal epithelium. The increased resolution gained by immunogold microscopy found hCLCA2 expression in the basal cells to be closely associated with hemidesmosomes and double labelling experiments revealed a coexpression with hemidesmosome component, integrin ß6. The immunohistochemical results were supported by hCLCA2 mRNA expression and quantification which revealed a significantly increased expression in the cornea. Conclusions: This study reports the cellular distribution of hCLCA2 in human ocular epithelia, and suggests a possible involvement in epithelial stratification and cell–basement membrane adhesion due to the close association of hCLCA2 with hemidesmosomes and the cell adhesion molecule integrin ß6. View OriginalDownload SlideView OriginalDownload Slide
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