Molecular expression and functional involvement of the bovine calcium-activated chloride channel 1 (bCLCA1) in apical HCO3- permeability of bovine corneal endothelium

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 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.

Fig. 1

Detection of bCLCA1 mRNA expression in cultured BCEC by in situ hybridization with biotin-labeled RNA probe. (A) Staining of cultured BCEC by in situ hybridization with 457 bp RNA probe. (B) Negative control, cultured BCEC hybridized without probe.

Fig. 2

Characterization of the anti-bCLCA1 antibody and detection of bCLCA1 in transfected HEK293 cells, fresh and cultured BCEC. (A) Coomassie staining of induced GST protein (26 kDa) and induced fusion protein GST-bCLCA1 (expected size, 31/32 kDa). (B) Immunoblotting using the anti-bCLCA1 antibody (left lane) and pre-immune serum (right lane) to detect the fusion protein antigen. (C) Immunoblotting using the anti-bCLCA1 antibody (left two lanes) or pre-immune serum (right two lanes) to detect bCLCA1 heterologously expressed in HEK293 cells and negative control, mock HEK293 cells. (D) Immunoblotting using the anti-bCLCA1 antibody to specifically detect the bCLCA1 expression in cultured BCEC (lane 1) and heterologously expressed HEK293cells (lane 2).

Fig. 3

Immunostaining and laser scanning confocal microscopy of bCLCA1 in cultured BCEC. (A) Immunofluorescence of cultured BCEC by the rabbit anti-bCLCA1 antibody plus anti-rabbit IgG-conjugated Alexa 488 (lower left panel) and rat anti-ZO-1 Ab plus anti-rat IgG-conjugated Alexa 594 (middle panels). The right panels are the overlay of the left and middle panel. The upper left panel, negative control, excluded addition of anti-bCLCA1 Ab. (B) The upper panel is the confocal montage of cultured BCEC stained with anti-bCLCA1 (Alexa Fluor 488-linked second antibody). Nuclei were counterstained by DAPI. The images are sequential from the most basolateral section (upper left) to the most apical (lower right), the z-axis separation between images is 0.5 μm. The lower panel is an X–Z section that shows the bCLCA1 staining apical to the nuclear staining. (C) ZO-1 staining. The upper panel is a confocal montage of cultured BCEC stained with anti-ZO-1 (Alexa Fluor 594-linked second antibody). Nuclei were counterstained by DAPI. The images are sequential from the most basolateral section (upper left) to the most apical (lower right), the z-axis separation between images is 0.5 μm. The lower panel is an X–Z section showing the ZO-1 relatively apical to nuclear staining. (D) Arrow indicates apical side bCLCA1 staining (green) in fresh frozen section from cow cornea. Nuclei stained with DAPI.

Fig. 4

siRNA knockdown of bCLCA1 expression. (A) Immunoblotting analysis of the effects of three siRNA constructs on the expression of bCLCA1 at the protein level, compared with the vehicle control (oligofectamine) and negative control siRNA, respectively. β-Actin was an equal-loading control. The histogram shows the protein expression percentage relative to the vehicle control. (B) Real-time PCR analysis of the effects of bCLCA1-RNAi-989 (989, arrow) on the mRNA expression of bCLCA1, compared with endothelium alone (e), the vehicle control (o), and negative control siRNA (cs), respectively. The housekeeping gene, GAPDH, was used as an equal loading control. (C) Gel analysis of the effects of bCLCA1-RNAi-989 on the expression of bCLCA1 at the mRNA level compared with negative control siRNA. GAPDH was a loading control.

Fig. 5

Effect of bCLCA1-RNAi-989 on the ATPγS-induced apical ${\text{HCO}}_3^{-}$ permeability in CBCEC. Cultured BCEC grown on permeable membranes were loaded with the pH-sensitive fluorescent dye, BCECF. Apical and basolateral compartments were perfused with ${\text{HCO}}_3^{-}$-rich, Cl⁻-free Ringer’s. Black boxes: LB (low ${\text{HCO}}_3^{-}$) pulse applied to the apical side. (A) Effects of negative control siRNA on ATPγS (100 μM)-induced apical ${\text{HCO}}_3^{-}$ permeability. (B) Summary of the effects of negative control siRNA on the initial rates of pH_i changes (dpH_i/dt) between the control and ATPγS LB pulses. (C) Effects of bCLCA1-RNAi-989 on ATPγS (100 μM)-induced apical ${\text{HCO}}_3^{-}$ permeability. (D) Summary of the effects of bCLCA1-RNAi-989 on the initial rates of pH_i changes (dpH_i/dt) between the control and ATPγS LB pulses.

Fig. 6

Effect of bCLCA1-RNAi-989 on the CPA-induced apical ${\text{HCO}}_3^{-}$ permeability in CBCEC. (A) Effects of negative control siRNA on CPA (20 μM)-induced apical ${\text{HCO}}_3^{-}$ permeability increase. (B) Summary of the effects of negative control siRNA on the initial rates of pH_i changes (dpH_i/dt) between the control and CPA LB pulses. (C) Effects of bCLCA1-RNAi-989 on CPA (20 μM)-induced apical ${\text{HCO}}_3^{-}$ permeability. (D) Summary of the effects of bCLCA1-RNAi-989 on the initial rates of pH_i changes (dpH_i/dt) between the control and CPA LB pulses.