敲低 IK1钾通道抑制 ClC－3氯通道的表达和功能

摘要

目的：探讨ClC－3氯通道是否为IK1钾通道的调节靶点，重点研究鼻咽癌细胞IK1钾通道对ClC－3氯通道功能及蛋白表达的影响。方法：采用siRNA转染技术抑制低分化鼻咽癌上皮细胞（ CNE－2Z） IK1基因的表达；real－time PCR技术检测ClC－3 mRNA的表达；Western blot检测ClC－3的蛋白表达；细胞免疫荧光结合激光共聚焦显微镜技术检测ClC－3和IK1蛋白在细胞内分布；全细胞膜片钳记录细胞氯电流。结果：IK1 siRNA可以成功转染CNE－2Z细胞，有效抑制鼻咽癌细胞IK1钾离子通道的表达；用IK1 siRNA抑制鼻咽癌细胞IK1钾离子通道的表达后， ClC－3的mRNA表达上调而ClC－3蛋白却表达减少：在低分化鼻咽癌上皮细胞，低渗刺激可激活氯通道，产生一个较大的氯电流，在成功转染IK1 siRNA的细胞，此氯电流明显减弱。结论：敲低IK1钾离子通道可抑制ClC－3氯离子通道的表达和功能。%[ ABSTRACT] AIM:To investigate whether the ClC-3 chloride channel is an acting target of the IK 1 potassium channel, and to study the action of IK1 potassium channel on the functional activities and expression of ClC-3 chloride channels.METHODS: IK1 gene was silenced by IK1 siRNA in poorly-differentiated nasopharyngeal carcinoma cells (CNE-2Z).Real-time PCR and Western blot were used to detect the expression of ClC-3 at mRNA and protein levels.The distribution of ClC-3 protein in the cells was observed under confocal immunofluorescence microscope .The chloride current was recorded by the patch-clamp technique.RESULTS: IK1 siRNA was successfully transfected into the CNE-2Z cells and knocked down the expression of IK 1 potassium.The mRNA expression of ClC-3 was increased by the IK1 siRNA.IK1 siRNA inhibited the expression of ClC-3 protein.A chloride current was activated by hypotonic challenges , and the hypoto-nicity-induced current was reduced in the cells which successfully transfected with IK 1 siRNA.CONCLUSION: The knockdown of IK 1 potassium channels inhibits the expression and function of ClC-3 chloride channel .