小G蛋白在心肌钠通道蛋白转运中的作用

Role of small GTP-binding proteins in cardiac voltage-gated sodium channel transport

目的研究小G蛋白Sar1和Arf1对钠通道蛋白(Nav1.5)转运的调控作用。方法以HEK293细胞作为宿主细胞,将人SCN5A基因转染到HEK293细胞中,通过G418筛选稳定表达SCN5A基因的细胞系;为了检测小G蛋白对Nav1.5的调节效应,野生型或突变型的小G蛋白Sar1和ARF1分别瞬转到HEK293-Nav1.5细胞。利用免疫印迹对目标蛋白进行定量,应用免疫荧光对细胞内的目标蛋白进行定位。结果过表达突变的Sar1减少了细胞膜上Nav1.5的表达和抑制了Nav1.5转运到细胞膜上。另外,过表达ARF1对于Nav1.5在细胞的表达及定位无明显影响。结论小G蛋白Sar1可能参与调节Nav1.5在细胞内的运输。

Objective To investigate the role of small GTPases Sar1 and Arf1 in the transcription of Nav1.5 protein.Methods In this experiment,we used HEK 293 cells as the host cells to co-express Nav1.5 with the wild-type(WT)or mutant form of the small GTPases Sar1 and Arf1.Then in order to check the effect that small GTPase had on Nav1.5 regulation,two techniques were used to analyze the results.Western blot was performed to measure the expression level of Nav1.5,and Confocal microscopy was used to visualize the cellular localization of Nav1.5.Results We found that mutant forms of Sar1 decreased Nav1.5 expression level significantly and inhibited its localization at the cell membrane.On the other hand,Arf1 only showed small effect on the expression level and localization of Nav1.5.Conclusion This study provides evidences that Nav1.5 intracellular transport is regulated by small GTPases Sar1 in particular.