Molecular expression and functional involvement of the bovine calcium-activated chloride channel 1 (bCLCA1) in apical HCO3- permeability of bovine corneal endothelium.
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 90kDa 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(i)(2+)] by application of ATPgammaS 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.
Zhang
Yan
Y
School of Optometry, Indiana University, 800 East Atwater Avenue, Bloomington, IN 47405, USA.
Li
Jinhua
J
Xie
Qiang
Q
Bonanno
Joseph A
JA
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001639:Bicarbonates / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002463:Cell Membrane Permeability / Q000502:physiology; D002478:Cells, Cultured; D018118:Chloride Channels / Q000276:immunology* / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism; D004728:Endothelium, Corneal / Q000378:metabolism*; D005455:Fluorescent Antibody Technique / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering / Q000378:metabolism
D000906:Antibodies; D001639:Bicarbonates; D018118:Chloride Channels; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D002118:Calcium