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Y. Zhang, Q. Xie, J.A. Bonanno; Molecular Characterization of a Bovine Calcium–Activated Chloride Channel (bCLCA1) Expressed in Corneal Endothelium . Invest. Ophthalmol. Vis. Sci. 2004;45(13):426.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In corneal endothelial cells, apical HCO3– permeability can be enhanced by increasing [Ca2+]i via either activation of purinergic receptor or inhibition of the sarcoendoplasmic reticulum calcium–ATPase ( SERCA ). Our goal is to identify the molecular candidate of the calcium–activated anion channel. Methods: Fresh and cultured bovine corneal endothelial cells ( FBCEC, CBCEC) were used in the study. A probe containing the first 457bp of the bCLCA1 open reading frame was used for in situ hybridization analysis. A 15 aa polypeptide (417–431) conjugate was used to generate polyclonal antibodies in rabbit. The immunoblot and immunofluoresence assay were performed to detect the protein expression of bCLCA1. Results: In situ hybridization with the bCLCA1–specific probe confirmed prominent mRNA expression in CBCEC. In immunoblot assay, the antiserum detects the heterologously expressed bCLCA1in HEK293 cell line, which was transfected with pcDNA3.1–bCLCA1 (a gift from Dr. Fuller in University of Alabama). Furthermore, the antiserum identifies a band around 90kD in FBCEC, which is absent in preimmune serum and can be absorbed by the polypeptide conjugate. The immunofluoresence with the specific antibody and confocal microscopy suggests the protein might be located on the apical membrane of CBCEC. Conclusion: The Calcium–activated chloride channel (bCLCA1) is expressed in bovine cornea endothelial cells and may be involved in anion transport across the BCEC.
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