摘要
目的观察增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因与h ERG(human ether-a-go-go-related gene)基因融合后是否影响h ERG通道的电生理特性。方法 HEK293细胞分别被转入pc DNA3-WT-h ERG和/或pc DNA3-G604S-h ERG,p EGFP-WT-h ERG和/或p EGFP-G604S-h ERG。激光共聚焦观察h ERG通道蛋白在细胞内的表达定位;膜片钳实验记录h ERG电流。结果在转染pc DNA3-WT-h ERG或p EGFP-WT-h ERG的细胞中,h ERG蛋白表达于胞质与胞膜;在转染pc DNA3-G604S-h ERG或p EGFP-G604S-h ERG的细胞中,h ERG蛋白仅在胞质表达。HEK293细胞转染pc DNA3-WT-h ERG后,h ERG电流的最大尾电流密度为(75.4±2.2)p A/p F,明显高于转染p EGFP-WT-h ERG的最大尾电流密度[(15.1±0.7)p A/p F,P<0.05]。共转染pc DNA3-WT-h ERG与pc DNA3-G604S-h ERG的最大h ERG尾电流密度明显高于p EGFP-WT-h ERG/p EGFP-G604S-h ERG的最大h ERG尾电流密度[(23.1±0.8)p A/p F vs(12.6±0.9)p A/p F,P<0.05]。此外,EGFP基因与野生型或突变h ERG基因融合后,明显改变了h ERG通道的电压依赖性激活及去失活特性。结论 EGFP基因与野生型或突变h ERG基因融合不影响h ERG通道蛋白在细胞内的表达定位,但明显改变了h ERG电流的大小、激活及去失活特性,因此检测突变h ERG通道的电生理功能时应避免使用绿色荧光蛋白基因质粒作为表达载体。
Objective To explore the effect of EGFP tagged to h ERG channels on the electrophysiological properties of h ERG channels. Methods HEK293 cells were transfected with pc DNA3-WT-h ERG and / or pc DNA3-G604S-h ERG,p EGFP-WT-h ERG and / or p EGFP-G604S-h ERG. The subcellular location of h ERG channels in HEK293 cells was analyzed under confocal microscopy. And h ERG currents were recorded using the voltage clamp technique. Results In HEK293 cells transfected with pc DNA3-WT-h ERG or p EGFP-WT-h ERG,h ERG channels expressed both on the cell surface and within the cytoplasm. In cells transfected with pc DNA3-G604S-h ERG or p EGFP-G604S-h ERG,h ERG channels only expressed in the cytoplasm. The maximal density of tail currents was（ 75. 4 ± 2. 2） p A/p F and（ 15. 1 ± 0. 7） p A/p F in cells expressing pc DNA3-WT-h ERG and p EGFP-WT-h ERG,respectively（ P〈0. 05）. The maximal density of tail currents in cells co-expressing pc DNA3-WT-h ERG and pc DNA3-G604S-h ERG was（ 23. 1 ± 0. 8）p A / p F,which was significantly higher than in cells co-expressing p EGFP-WT-h ERG and p EGFP-G604S-h ERG[（ 12. 6 ± 0. 9） p A / p F,P〈0. 05]. In addition,EGFP / h ERG fusion protein altered the voltage-dependent h ERG channel activation and deactivation. Conclusion The enhanced green fluorescent protein shows no effect on the transport and localization of wild-type or mutant h ERG channel protein,but significantly alters electrophysiological function of h ERG current. Thus,it should be avoided using EGFP as a reporter to study the electrophysiological function of h ERG channel.
作者
霍建华
马爱群
郭雪艳
强华
刘平
白玲
HUO Jianhua MA Aiqun GUO Xueyan QIANG Hua LIU Ping BAI Ling(Department of Cardiovascular Medicine, First Affiliated Hospital of Xi＇ an Jiaotong University, Xi＇ an 710061, China Department of Digestive Diseases, Shaanxi Provincial People＇ s Hospital)
出处
《山西医科大学学报》
CAS
2016年第9期785-790,共6页
Journal of Shanxi Medical University
基金
国家自然科学基金资助项目(30801133)
陕西省自然科学基础研究计划基金资助项目(2014JM2-8154)
