Abstract
Abstract: :
Purpose: We previously found that capacitative calcium entry can regulate bicarbonate transport in bovine corneal endothelial cells. Transient receptor potential channels (TRPCs) have been reported to be candidates for capacitative calcium entry. In this study we examined the expression of TRPC4 by using immunoassays. Methods: The C–terminus of TRPC4 was PCR amplified and fused to the GST vector. The fusion protein (GST–TRPC4C) was induced in bacteria and purified by glutathione agarose beads. Purified GST–TRPC4 was used to generate antibodies against TRPC4 in rabbits. Western blot and immunostaining were used to evaluate the expression of TRPC4 in corneal endothelial cells. Results: In Western blots, TRPC4 antiserum recognizes the expressed fusion protein, while the pre–immune serum does not. We used bovine brain membrane protein as a positive control. Two bands at around 75 and 80 kD were seen from the bovine brain proteins. A band at around 80 kD was detected for membrane proteins from both the fresh and cultured bovine corneal endothelial cells. Pre–immune serum could not detect bands at the same sizes in either brain nor corneal endothelial cell samples. This band appears specific since incubation of the antigen (GST–TRPC4C) with the antibody abolished it, while incubation of GST protein alone did not absorb it. Immunostaining with the TRPC4 antibody showed plasma membrane and cytoplasmic staining. Conclusions:These results showed that TRPC4 protein is expressed in the bovine corneal endothelial cells.
Keywords: calcium • cornea: endothelium • ion channels