Expression and functional evaluation of transient receptor potential channel 4 in bovine corneal endothelial cells

Abstract

We previously found that activation of purinergic receptors mobilizes Ca 2+ and enhances bicarbonate transport in bovine corneal endothelial cells (BCEC). Since transient receptor potential channel 4 (TRPC) has been reported to be a candidate for capacitative calcium entry (CCE) and receptor operated calcium entry (ROC), we examined the expression of TRPC4 and evaluated the potential involvement of TRPC4 in CCE or ROC in BCEC. The C-terminus of TRPC4 was fused into the glutathione S-transferase (GST) expression vector. The fusion protein GST-TRPC4c was induced in bacteria and purified by affinity chromatography. An antibody was raised in rabbit by using the purified GST-TRPC4c antigen. In Western blotting, the TRPC4 antibody recognized the fusion protein while the pre-immune IgG did not. The TRPC4 antibody recognized a band at around 80 kD for membrane proteins from both the fresh and cultured BCEC. The pre-immune IgG could not detect bands at the same size. Incubation with the TRPC4c antigen abolished the 80 kD band. Immunofluorescence using the TRPC4 antibody stained both fresh and cultured BCEC, while pre-immune IgG did not. RNAi knocked down the expression of TRPC4 in cultured BCEC. Ca 2+ entry induced by the purinergic receptor agonist ATP, was increased in TRPC4-siRNA transfected cells compared with the scrambled siRNA control, while Ca 2+ entry induced by store depletion through blocking the endoplasmic reticulum Ca 2+ pump, did not differ between the siRNA and scrambled siRNA-treated cells. Taken together, these results show that TRPC4 protein is expressed in the bovine corneal endothelial cells and may be a negative regulator in ROC stimulated by purinergic activation, but not by store depletion itself.