小G蛋白Rab5对表达在HEK293细胞上的大电导钙激活钾通道的影响

摘要

目的 探讨小G蛋白Rab5对HEK293细胞大电导钙激活钾通道(BKCa)的影响.方法 将对数生长期HEK293细胞,随机分为Control组、Rab5WT组、Rab5CA组和Rab5DN组.采用脂质体转染法进行转染,Control组转染Flag-hSlo1-GFP质粒和对照质粒pcDNA3.1,Rab5WT组转染Flag-hSlo1-GFP质粒和Rab5WT质粒,Rab5CA组转染Flag-hSlo1-GFP质粒和Rab5CA质粒,Rab5DN组转染Flag-hSlo1-GFP质粒和Rab5DN质粒,每组两种质粒总量为4μg,按质量比1∶1共转染至3.5 mm培养皿中.转染72 h、荧光显微镜下观察转染效率在70％以上时,采用膜片钳、Western blotting法和流式细胞术观察Rab5对HEK293细胞BKCa的作用.结果 与Control组比较,Rab5WT组、Rab5CA组BKCa全细胞电流密度明显增加,而Rab5DN组明显降低;Rab5WT组、Rab5CA组BKCa膜蛋白的相对表达量明显升高,而Rab5DN组明显降低(P均＜0.05);Rab5WT组、Rab5CA组Flag+/GFP+明显增加,而Rab5DN组明显降低(P均＜0.05).结论 Rab5能够明显增加表达在HEK293细胞上的BKCa电流及细胞膜上的表达,并可促进BKCa向质膜的转运过程.%Objective To investigate the effect of Rab5 on large-conductance Ca2+-activated K+ channels (BKCa channels) expressed in HEK293 cells.Methods HEK293 cells in the logarithmic phase were randomly divided to four groups:the control group,Rab5WT group,Rab5CA group and Rab5DN group.HEK293 cells were transfected with FlaghSlo-GFP plasmid in each group and with pcDNA3.1 for control group,Rab5WT for Rab5WT group,Rab5CA for Rab5CA group and Rab5DN for Rab5DN group,respectively.Total 4 μg plasmid with 1∶1 to each plasmid was transfected into 3.5 mm dish.Transfection rate was observed under fluorescence microscopy and the effect of Rab5 on BKCa channels was detected by_patch clamp technique,Western blotting and flow cytometry when the trasfection rate was above 70％.Results Compared with the control group,Rab5 WT and Rab5 CA significantly increased BKCa macroscopic currents,while BKCa currents were decreased in the Rab5DN group.Rab5 WT and Rab5 CA increased the expression of membranous BKCa protein in HEK293 cells,while Rab5DN decreased the expression (all P ＜0.05).Rab5 WT and Rab5 CA increased the Flag+/GFP+ ratio (all P＜0.05),while Rab5DN decreased the ratio (P ＜0.05).Conclusion Rab5 significantly increases BKCa currents and channel expression on cell surface and may promote the transport process of BKCa currents to cell membrane in HEK293 cells.