摘要
目的评价西沙比利对转染HERG基因表达的快速激活延迟整流钾电流(IKr)和蛋白的影响,探讨其致心律失常的机制。方法使用脂质体介导的瞬时转染法把野生型HERG基因转染进人胚肾细胞(HEK293),采用全细胞膜片钳技术评价西沙比利对IKr通道电流和动力学的影响;使用G418筛选出稳定转染HERG的细胞,并用西沙比利进行干预,应用免疫蛋白印迹技术评价药物对蛋白的影响。结果西沙比利对IKr通道的刺激电流(IHERG)和尾电流(Itail)具有浓度和电压依赖性抑制作用,半数最大抑制浓度分别14.5和3.9nmol/L。西沙比利使IHERG和Itail的最大峰值电位前移,但不改变激活电位;使激活曲线左移并加快通道的失活,但不改变通道失活后的恢复时间;西沙比利对转染后的HEK293细胞上的IKr通道蛋白无明显抑制。结论西沙比利通过作用于通道的失活态抑制HERG钾电流,但不影响HERG通道蛋白的表达。
Objective To evaluate the electrophysiogical effects of cisapride on the rapidly activating delayed rectifier potassium current (IKr) and its gene HERG-encoded protein trafficking. Methods Wild-type HERG cDNA plasmids were transfected into human embryonic kidney ( HEK293 ) cells by lipofectamine method. To build up a stably transfected HERG-HEK 293 cells line by G418 treatment. The whole-cell patch clamp method was used to record the IKr and kinesics of channel gating and to assess the effects of cisapride on the protein expression by Western blot analysis. Results Cisapride produced concentration-dependent and voltage-dependent blockage of the IHERG and Itail with 50% inhibitory concentra- tion of 14.5 and 3.9 nmol/L, respectively. Cisapride degraded peak current potential of IHERG and Itail without altered activation threshold potential. The activation curve was shifted in a negative direction and accelerated channel inactivation by eisapride, but the time constants of recovery from inactivation did not significantly change. Cisapride did not present significant influences on the IKr channel protein trafficking. Conclusion Cisapride blocks HERG K^+ channel via inactivated state, but dose not influenc on the protein trafficking.
出处
《中国心脏起搏与心电生理杂志》
北大核心
2009年第4期337-342,共6页
Chinese Journal of Cardiac Pacing and Electrophysiology
基金
中央级公益性科研院所基本科研业务费专项资金(基金编号2006F002)
关键词
电生理学
膜片钳
西沙比利
离子通道
钾通道
HERG基因
人胚肾细胞
免疫蛋白印迹
Electrophysiology
Patch clamp
Cisapride
Ion channel
Potassium channel
HERG gene
Human embryonic kidney ( HEK293 ) cells
Western blot