西安交通大学学报（医学版）2016，Vol.37Issue(3)：353-357,371,6.DOI:10.7652/jdyxb201603011
HEK293细胞中杂合状态下无义突变体L539fs/47对野生型HERG电流的作用
The mutant L539fs/47 has the effect of an incomplete dominant negative inhibition on wild type HERG current in HEK293 cells
摘要
Abstract
Objective To study the effect of nonsense mutation of L539fs/47,when coexpressed with wild type human ether-a-go-go-related gene (HERG)current in HEK293 cells in the heterozygous state.Methods Thirty-six hours after transfection by liposome,wild-type HERG and its mutant HERG-L539fs/47 were transfected into HEK2 9 3 cells. HERG mRNA expressions were detected by real-time PCR;its protein localization and quantitative expression were detected by immunofluorescence and Western blotting,respectively.Current densities of HERG were detected by whole cell patch clamp.Results The expression of HERG mRNA in wild-type group was higher than that in L539fs/47 mutation group (1.066 ± 0.612 vs. 0.254 ± 0.397, P < 0.01 ). Immunofluorescence results showed that HERG protein was more than that in mutant group.The wild-type HERG protein was mainly distributed in the cell membrane;but retention of most mutant HERG protein L539fs/47 cytoplasm was detected.Western blotting showed that,different from the wild type HERG 135ku-and 155ku-bands,only one 60ku-band was detected in mutant group.Through the whole cell patch clamp test,the current density of HERG in heterozygous group (WT+MT group)decreased by 5 5 .2 3% compared with WT group (2 2 .0 3 ±2.62 vs.49.20±2.31 pApF,P<0.01).Conclusion In the heterozygous state,the truncation mutant L539fs/47 has the effect of incomplete dominant negative inhibition on wild type HERG current in HEK293 cells.关键词
HERG/无义突变/不完全负显性抑制作用/HEK293 细胞Key words
human ether-a-go-go-related gene (HERG)/nonsense mutation/incomplete dominant negative inhibition/HEK2 9 3 cell分类
医药卫生引用本文复制引用
吕颖,张军波,张爱峰,刘仲伟,王军奎,韩稳琦,潘军强,李国良,孙超峰..HEK293细胞中杂合状态下无义突变体L539fs/47对野生型HERG电流的作用[J].西安交通大学学报（医学版）,2016,37(3):353-357,371,6.基金项目
国家自然科学基金资助项目(No.30800473) Supported by the National Natural Science Foundation of China (No.30800473) （No.30800473）
