Xie Qiang, Zhang Yan, Cai Sun Xing, Zhai Changbin, Bonanno Joseph A
School of Optometry, Indiana University, 800 E Atwater Avenue, Bloomington, IN 47405-3680, USA.
Exp Eye Res. 2005 Jul;81(1):5-14. doi: 10.1016/j.exer.2005.01.003.
We previously found that activation of purinergic receptors mobilizes Ca2+ 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. Ca2+ entry induced by the purinergic receptor agonist ATP, was increased in TRPC4-siRNA transfected cells compared with the scrambled siRNA control, while Ca2+ entry induced by store depletion through blocking the endoplasmic reticulum Ca2+ 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.
我们之前发现，嘌呤能受体的激活可动员钙离子（Ca2+）并增强牛角膜内皮细胞（BCEC）中的碳酸氢盐转运。由于瞬时受体电位通道4（TRPC）已被报道为容量性钙内流（CCE）和受体操纵性钙内流（ROC）的候选通道，我们检测了TRPC4的表达，并评估了TRPC4在BCEC的CCE或ROC中的潜在作用。将TRPC4的C末端融合到谷胱甘肽S-转移酶（GST）表达载体中。融合蛋白GST-TRPC4c在细菌中诱导表达，并通过亲和层析进行纯化。使用纯化的GST-TRPC4c抗原在兔体内制备抗体。在蛋白质免疫印迹中，TRPC4抗体可识别融合蛋白，而免疫前IgG则不能。TRPC4抗体识别来自新鲜和培养的BCEC的膜蛋白中约80 kD处的条带。免疫前IgG无法检测到相同大小的条带。与TRPC4c抗原孵育可消除80 kD的条带。使用TRPC4抗体进行免疫荧光染色可同时标记新鲜和培养的BCEC，而免疫前IgG则不能。RNA干扰（RNAi）降低了培养的BCEC中TRPC4的表达。与乱序siRNA对照相比，嘌呤能受体激动剂ATP诱导的Ca2+内流在TRPC4-siRNA转染的细胞中增加，而通过阻断内质网Ca2+泵导致的内质网钙库耗竭诱导的Ca2+内流在siRNA处理组和乱序siRNA处理组细胞之间没有差异。综上所述，这些结果表明，TRPC4蛋白在牛角膜内皮细胞中表达，并且可能是嘌呤能激活刺激的ROC中的负调节因子，但不是内质网钙库耗竭本身的负调节因子。
