Abstract
Corneal endothelium secretes HCO 3 − from basolateral (stroma) to apical (anterior chamber) compartments. Apical HCO 3 − permeability can be enhanced by increasing [Ca 2+] i. We hypothesized that the bovine calcium-activated chloride channel 1 (bCLCA1), shown previously by PCR screening to be expressed in corneal endothelium, is involved in Ca 2+ activated apical HCO 3 − permeability. bCLCA1 expression in cultured bovine corneal endothelial cells (CBCEC) was examined by in situ hybridization analysis, immunoblotting, immunofluorescence and confocal microscopy. Rabbit polyclonal antibodies were generated using a 14 aa polypeptide (417–430) from the predicted sequence of bCLCA1. The small interference RNA (siRNA) knock down technique was used to evaluate the functional involvement of bCLCA1 in apical HCO 3 − permeability. In situ hybridization confirmed prominent bCLCA1-specific mRNA expression in CBCEC. bCLCA1 antiserum detected the heterologously expressed bCLCA1 in HEK293 cells and a 90 kDa band in CBCEC, which was absent when using the pre-immune serum or antigen absorption of serum. Immunofluoresence staining with anti-bCLCA1 antibody and confocal microscopy indicates an apical membrane location in CBCEC. In CBCEC transfected with bCLCA1 specific siRNA, bCLCA1 expression was reduced by 80%, while transfection with siControl scrambled sequence had no effect. Increasing [Ca 2+ i] by application of ATPγS or cyclopiazonic acid (CPA) increased apical HCO 3 − permeability in siControl transfected CBCEC, while having no effect on apical HCO 3 − permeability in bCLCA1 specific siRNA transfected cells. Baseline HCO 3 − permeability, however, was not different between controls and siRNA treated cells. We conclude that the calcium-activated chloride channel (bCLCA1) is expressed in bovine corneal endothelial cells and can contribute to Ca 2+ dependent apical HCO 3 − permeability, but not resting permeability, across the corneal endothelium.
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