奎尼丁对HEK293细胞hERG电流和hERG蛋白的影响

Effects of quinidine on hERG potassium current and hERG channel protein in HEK293 cell line

目的观察奎尼丁对hERG电流和hERG通道蛋白表达的影响,进一步明确其导致长QT间期综合征的机制。方法用脂质体转染法将含有hERG基因的质粒转入HEK293细胞,全细胞膜片钳技术记录电流和Western blot技术观察hERG通道蛋白表达的影响。实验分组:(1)HEK293细胞转染进质粒48 h时加入不同浓度(0,1,3,10μmol/L)的奎尼丁,并进行膜片钳实验观察奎尼丁的瞬时作用。(2)HEK293细胞转染进质粒24 h时往培养基加入不同浓度(1,3,10μmol/L)的奎尼丁,继续培养24 h洗脱奎尼丁后立即进行膜片钳及Western blot实验观察奎尼丁的慢性作用。结果 (1)1μmol/L、3μmol/L及10μmol/L组的最大尾电流分别为(55.9±3.8)pA/pF、(35.7±3.2)pA/pF及(15.5±1.4)pA/pF,均明显小于对照(0μmol/L)组(均P<0.05)。(2)不同浓度的奎尼丁孵育HEK293细胞后hERG电流无明显变化。Western blot观察到不同浓度奎尼丁作用下hERG蛋白表达无明显变化。结论奎尼丁对hERG电流有瞬时直接抑制作用,但并非影响hERG通道蛋白的表达来改变hERG电流。奎尼丁对hERG通道的直接抑制作用可能为其引起长QT间期综合征的根本机制。

Objective To explore the effect of quinidine on ether-a-go-go related gene（hERG） current and hERG channel protein and its possible mechanism of quinidine-induced long QT syndrome. Methods HEK293 cells were transiently transfected with plasmid containing hERG gene via Lipofectamine. The hERG current was observed by whole-cell patch-clamping and the expression of hERG channel protein was detected by Western blot analysis.（1）HEK293 cells were transfected with plasmid,and after 48 h the cells were treated with different concentrations（0,1,3,10 μmol/L） of quinidine and patch clamp experiments were operated for instantaneous effect of quinidine.（2）HEK293 cells were transfected into plasmid for 24 h,then treated with different concentrations of quinidine（1,3,10 μmol/L） for 24 h,and then the patch clamp and Western blot were operated to observe the chronic effect of quinidine. Results After HEK293 cells were transfected into the plasmid for 48 h,the hERG current was significantly inhibited after treated with different concentrations of quinidine. The maximal density of tail currents were（55. 9 ± 3. 8） pA/pF,（35. 7 ± 3. 2） pA/pF and（15. 5 ± 1. 4） pA/pF in 1 μmol/L,3 μmol/L and 10 μmol/L quinidine groups,respectively（P〈0. 05 vs 0 μmol/L）. HEK293 cells were transfected into plasmid for 24 h and cultured with different concentrations of quinidine for 24 h,the hERG current showed no significant change. Western blot result showed that there was no significant change in the expression of hERG protein in HEK293 cells cultured with different concentrations of quinidine. Conclusion Quinidine shows an immediate and direct inhibitory effect on hERG potassium current,but it does not affect the expression of hERG channel protein. The direct inhibition of hERG channel is the basic mechanism of long QT syndrome caused by quinidine.