心脏传导阻滞SCN5A基因L1001Q突变体构建和功能研究

目的对作者发现的一个中国3代人心脏传导阻滞家系SCN5A基因新突变L1001Q,构建突变表达载体,利用分子生物学方法和全细胞膜片钳技术观察L1001Q突变的功能。方法一步定点诱变法构建pEGFP-L1001Q-SCN5A突变体;激光共聚焦和Western blotting技术检测突变蛋白定位和表达;全细胞膜片钳技术观察突变体钠电流。结果野生型和L1001Q突变均表达在细胞膜上,且表达量无明显变化。二者最大激活电流分别为(148.34±0.77)pA/pF和(132.59±0.96)pA/pF(P>0.05),L1001Q突变半失活电压V1/2为(-73.25±0.87)mV,较野生型正向转移约3.5mVV1/2为(-76.71±0.73)mV,P<0.05],L1001Q突变失活后恢复时间常数较野生型稍减小,差异无统计学意义(WT tau=16.8ms,L1001Qtau=14.1ms,P>0.05)。结论 L1001Q突变未影响钠通道蛋白的表达和转运,主要通过改变钠通道门控特性引起钠通道功能减弱进而导致心脏传导阻滞。

Construction and function of SCN5A gene L1001Q mutant identified in a cardiac conduction defect family

Objective To construct SCN5A gene expression vectors of SCN5A-L1001Q mutant identified in a Chinese cardiac conduction defect family so as to evaluate the biological and electrophysiological properties of HEK293 cells caused by SCN5A-L1001Q mutation.Methods The mutation pEGFP-L1001Q-SCN5A was generated by site-directed mutagenesis.The expected mutation was confirmed by direct sequencing.Wild type(WT) and L1001Q mutations were transfected into HEK293 cells for immunochemical staining and visualized by confocal microscopy,and the channel protein was examined by Western blotting.Sodium current of mutant was measured by whole-cell patch clamp technique.Results Both WT and L1001Q mutations were expressed in the cell membrane,and the expression quantity of mutant L1001Q channels protein was similar to that of WT channels.The maximum current density of L1001Q mutation (132.59±0.96)pA/pF] did not obviously differ from that of WT mutation (148.34±0.77)pA/pF,P>0.05];the voltage to achieve half inactivation(V1/2) was(-76.71±0.73)mV for WT and(-73.25±0.87)mV for L1001Q mutation.The mutation produced a small but significant positive shift of steady state inactivation(P<0.05).The mutant channels showed a slight decrease in the time constants of inactivation(tau=14.1 ms) compared with WT(tau=16.8 ms)(P>0.05).Conclusion The L1001Q mutation does not affect the protein expression and transport of sodium channel,but alters the gate properties of cardiac sodium channel.