敲低IK1钾通道抑制ClC-3氯通道的表达和功能

 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。

Knockdown of IK1 potassium channel inhibits expression and function of ClC-3 chloride channel

AIM: To investigate whether the ClC-3 chloride channel is an acting target of the IK1 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 IK1 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 hypotonicity-induced current was reduced in the cells which successfully transfected with IK1 siRNA. CONCLUSION: The knockdown of IK1 potassium channels inhibits the expression and function of ClC-3 chloride channel.