KCNE1胞外N段功能的研究

Function of extracellular N region in KCNE1

目的:研究电压依赖性钾离子通道-IKs通道的调节亚基KCNE1胞外肽段(氨基端肽段,简称N段)的功能,探究胞外N段在KCNE1调控IKs通道中的作用。方法:通过分子生物学技术制备KCNE1的N段缺失突变体;利用Western Blot方法了解突变体的蛋白表达情况;以细胞免疫染色方法观察突变体的亚细胞定位情况;以全细胞膜片钳记录技术检测突变体(与KCNQ1共表达)的通道电流特征。结果:成功构建KCNE1胞外N段缺失的突变体KCNE1-ΔN,突变体体外表达正常;与野生型相比:KCNE1-ΔN在细胞内的分布出现蛋白集聚现象;通道的电流密度减小,激活电压相比较出现了明显的左移,即通道的激活速率变快。结论:KCNE1的N段对其蛋白表达无明显影响,但对蛋白的转运起重要作用;N段参与了KCNE1对通道电流大小的调节,以及对通道激活特征的调控。

Objective： To investigate the function of N region in KCNE1, which is the regulatory subunit of voltage gated potassium channel-IKs, in order to find out the role of N region on KCNE1 modulating channel. Methods： With molecular technologies, we created N-truncated form named KCNE1-ΔN and then analyse its protein expression using Western Blot;immunocytochemistry was employed to detect subcellular localization of the truncation; while whole cell patch-calmp was used to detect the characteristics of KCNE1-ΔN expressed with KCNQ1. Results： KCNE1-ΔN was constructed successfully,and expressed normally in vivo. Comparing to wide type of KCNE1, KCNE1-ΔN showed intracellular aggression; KCNE1-ΔN also showed significant reduction on whole cell current densities and displayed a significant shift of the voltage-dependent curve towards left, which means speeded activation. Conclusions： N region does not have effect on protein expression but playes a significant role on protein trafficking; N region plays a role on KNCE1 modulating IKschannel＇s whole cell current densities and channel characteristics on activation.