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

基础医学与临床 ›› 2021, Vol. 41 ›› Issue (10): 1403-1409.

• 研究论文 • 下一篇

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

陈邦盛¹, 余小玲¹, 毛飞燕², 廉姜芳³, 俞徐云^1*

1.宁波市鄞州第二医院急救医学中心,浙江宁波 315192;
2.中国科学院大学宁波华美医院(宁波市第二医院) 普外科,浙江宁波 315010;
3.宁波市医疗中心李惠利医院兴宁院区心血管内科,浙江宁波 315040

收稿日期:2021-02-02 修回日期:2021-06-28 发布日期:2021-09-29
通讯作者: *8339684@qq.com
基金资助:
国家自然科学基金(81870255);浙江省自然科学基金(LY21H020001);宁波市自然科学基金(2018A610398);宁波市鄞州区科技局项目([2017]110号)

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

CHEN Bang-sheng¹, YU Xiao-ling¹, MAO Fei-yan², LIAN Jiang-fang³, YU Xu-yun^1*

1. Emergency Medical Center,Ningbo Yinzhou No.2 Hospital, Ningbo 315192;
2. Department of General Surgery, HwaMei Hospital, Ningbo No.2 Hospital, University of Chinese Academy of Sciences, Ningbo 315010;
3. Department of Cardiovascular Disease, Ningbo Medical Center Lihuili Xingning Hospital, Ningbo 315040, China

Received:2021-02-02 Revised:2021-06-28 Published:2021-09-29
Contact: *8339684@qq.com

摘要/Abstract

摘要： 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。

关键词: 分子伴侣, Rer1, hERG, LQTS

Abstract: 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 inhibitor was 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.

Key words: chaperone, Rer1, hERG, LQTS

中图分类号:

R54

陈邦盛, 余小玲, 毛飞燕, 廉姜芳, 俞徐云. 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[J]. 基础医学与临床, 2021, 41(10): 1403-1409.

CHEN Bang-sheng, YU Xiao-ling, MAO Fei-yan, LIAN Jiang-fang, YU Xu-yun. Chaperone Rer1 involves in transport of hERG potassium channel protein in cell line HEK293T[J]. Basic & Clinical Medicine, 2021, 41(10): 1403-1409.

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