Cav1.3L-型钙通道K228E突变体的构建及功能研究

Construction and function analysis of K228E mutation of Cav1.3 L-type calcium channel

目的通过构建Cav1.3基因K228E突变,观察K228E突变对L-型钙通道电生理特性及蛋白表达和转运的影响。确定钙通道α1亚基结构域ⅠS4段上N-端带正电荷的氨基酸对该通道电生理学特性的作用,从而为创建钙通道起搏器奠定基础。方法①一步法快速定点诱变构建Cav1.3基因K228E突变体的真核表达载体;②膜片钳观察突变钙通道电流,激光共聚焦技术观察突变钙通道蛋白在细胞内的表达和定位。结果①检测表达于HEK293细胞上的L-型钙电流:转染野生型Cav1.3质粒和相关亚基质粒,可检出L-型钙电流,其半激活电压V1/2=(-36.56±2.08)mV、半失活电压V1/2=(-42.93±0.14)mV(5mmolBa2+);而转染K228E-Cav1.3质粒和相关亚基质粒的HEK293T细胞上未检测到钙电流;②检测K228E突变通道蛋白在细胞内的表达和定位:转染野生型和突变型Cav1.3质粒及相关亚基质粒,48h后观察到野生型与突变型钙通道蛋白在细胞膜上都有表达。结论Cav1.3基因K228E突变虽不影响L-型钙通道蛋白质的表达与转运,但却不表达钙通道电流,是一种功能丧失性突变。

Objective The study is to construct the K228E mutation of Cavl.3 and evaluate its effect on electrophysiological characteristics and protein expression and transport of L-type calcium channel. The effect of amino acid with positive charges in N-terminal of segment I S4 of Cav1. 3 on electrophysiological characteristics of L-type calcium channel is also investigated so as to create a calcium channel pacemaker. Methods O The eukaryotic expression vector for K228E-Cav1. 3 mutant was constructed by rapid site-directed mutagenesis; ② Ca^2＋ current was measured by patch-clamp technique; the expression and subcellular localization of Ca^2＋ channel protein were observed by laser confocal microscopy. Results O Patch-clamp recordings of Ca^2＋ current： There was obvious current from HEK293T cells transfected with pCDNA6-WT-Cavl. 3 and related subunit, V1/2 from steady state-activation curve was （- 36.56 ±2.08）mV; V1/2 from steady state-inactivation curve was （- 42.93 ± 0.14）mV （5 mmol/L Ba^2＋）. However, there was no current from HEK293T cells transfected with pCDNA6-K228E-Cav1. 3 and related subunit. ② The expression and subcellular localization of Ca2＋ channel protein： 48 hours after the HEK293T cells were transfected with WT-Cav1.3 or K228E-Cavl.3 plasmids, both the channel protein of WT-Cav1. 3 and K228E-Cav1.3 were expressed on cell membrane surface. Conclusion The K228E mutation of Cav1. 3 channel has no effect on the protein expression and transport of L-type calcium channel. It is a functional loss mutation which has no expression of Ca^2＋ current.