Isolation and characterization of a Ca2+-activated chloride channel from human corneal epithelium

Abstract

Purpose. Transparency of the cornea is maintained through the activity of secretory mechanisms in the epithelium and endothelium, which offset the tendency of the stroma to imbibe fluid and swell. These secretory mechanisms establish osmotic gradients thereby providing the osmotic driving forces for coupled fluid transport from the stroma into both the tears and the anterior chamber. To further characterize the mechanism of epithelial Cl secretion, we cloned a cDNA encoding a Ca 2+ -dependent chloride channel, an abundant mRNA in human corneal epithelium. We investigated the abundance of all known human chloride channels in corneal epithelium to identify those responsible for regulating chloride conductance in this tissue. Methods. For the isolation of a full-length cDNA clone, a probe was selected from a set of expressed sequenced tag (EST) clones classified as unique to corneal epithelium (http://bodymap. ims.u-tokyo.ac.jp). The expression patterns of the corresponding gene encoding novel chloride channel gene in human cornea and other tissues were examined by reverse transcription-polymerase chain reaction (RT-PCR). Quantitative PCR was performed to clarify the expression level of the novel chloride channel gene in cornea relative to that in other human tissues. Results. We cloned a new Ca 2+ -activated chloride channel, CLCA2, from corneal epithelium. The full length cDNA clone encoded 943 amino acids with 62% identity to bovine Ca 2+ -activated chloride channel. The CLCA2 gene mapped to human chromosome 1p32. Quantitative expression analysis by RT-PCR showed that it is the most abundant chloride channel in corneal epithelium. Conclusion. High and tissue specific expression of the CLCA2 gene in human corneal epithelium implies an important role in corneal transparency maintenance.