分子伴侣Rer1参与人胚胎肾上皮细胞系HEK293T中hERG钾通道蛋白转运

Chaperone Rer1 involves in transport of hERG potassium channel protein in cell line HEK293T

目的探讨分子伴侣Rer1在hERG钾通道蛋白转运中的作用机制,并进行可能恢复hERG转运异常的药物Baf A1研究。方法将A561V突变体及野生型hERG载体瞬时转染人胚胎肾上皮细胞系(HEK293T),建立野生、突变及混合转染细胞模型;运用免疫荧光、蛋白免疫印迹检测hERG及Rer1的表达;采用小干扰RNA技术敲低Rer1表达后检测hERG蛋白的表达;V-ATP酶抑制剂Bafilomycin A1(Baf A1)以1 mmol/L孵育各组细胞6 h后Western blot检测hERG蛋白表达;用膜片钳对有潜在功能的混转细胞给予Baf A1孵育后测定膜电流。结果与野生组相比,A561V突变可致hERG蛋白转运缺陷并表现不同条带水平,突变组hERG蛋白的膜表达明显减少(P<0.01);Rer1在突变、混转组中表达明显下降(P<0.01),进一步敲低Rer1后可促进部分相对成熟hERG蛋白的顺向转运;此外,与对照组相比,Baf A1孵育可使野生、突变组中成熟hERG蛋白明显增加(P<0.05),混转组中未成熟hERG蛋白则显著上调(P<0.05);而在混转细胞中,Baf A1孵育可使尾电流相对密度明显增强(P<0.05)。结论Rer1参与hERG钾通道蛋白的转运,并能抑制部分异常hERG蛋白在细胞中的顺向转运,Baf A1能明显增强混转细胞的尾电流密度。

Objective To investigate the role of Rer1 in hERG potassium channel protein transport,and to study the medicine Baf A1 that may restore abnormal hERG transport.Methods HEK293T cells were transiently transfected with constructed A561V mutant and WT plasmids,the wild-type,mutant and mixed transfection cell models were established.The expressions of hERG and Rer1 were detected by immunofluorescence and Western blot.siRNA was used to knockdown the expression of Rer1 and detect the expression of hERG;Bafilomycin A1,a V-ATPase inhibitorwas incubated with 1 mmol/L for 6 hours.Western blot was used to detect the changes of hERG protein before and after the incubation.Patch clamp was used to measure the current of potential functional in mixed transfection cells.Results Compared with the WT group,A561V-mutation could cause trafficking deficient of hERG protein.The PM expression of hERG in mutant group was significantly reduced(P<0.01).The expression of Rer1 in mutation and mixed transfection groups decreased significantly(P<0.01).Knockdown of Rer1 promoted the forward transport of mature hERG.Besides,after incubation with Baf A1,the mature hERG protein in WT and mutant groups increased significantly(P<0.05),while the immature hERG in the mixed transformation group was significantly increased(P<0.05).Relative density of tail current was elevated following incubation with Baf A1 compared with control.Conclusions Rer1 is involved in the transport of hERG potassium channel and inhibits the forward transport of some abnormal hERG.Baf A1 significantly enhances the tail current density of mixed transfection cells.