热休克蛋白90对hERG-A561V通道蛋白转运及通道功能影响的研究

The effects of heat-shock protein 90 on the transport and channel function of hERG-A561V channel protein

目的探讨热休克蛋白90(Hsp90)对人类ether-a-go-go相关基因(hERG)-A561V通道蛋白转运及通道功能影响的研究。方法构建野生型(WT)、突变型(A561V)、杂合型(WT/A561V)细胞模型,采用蛋白质印迹法检测各组细胞hERG及Hsp90蛋白表达差异。转染减表达空载(Itf NC)、Hsp90减表达(Hsp90-)、过表达空载(Ovp NC)或Hsp90过表达(Hsp90+)质粒至各细胞模型(即Itf NC组、Hsp90-组、Ovp NC组、Hsp90+组),另设不加载体的对照(Ctl)组,采用蛋白质印迹法检测各组细胞调控Hsp90前后hERG及Hsp90蛋白表达差异。采用免疫荧光法检测Hsp90调控前后杂合型细胞模型hERG及Hsp90蛋白定位表达情况。采用全细胞膜片钳技术检测调控Hsp90前后杂合型细胞模型hERG通道激活电流及尾电流密度。结果突变型、杂合型细胞模型Hsp90及hERG(135 kDa)蛋白表达水平均较野生型明显升高(均P<0.05)。野生型细胞模型Hsp90+组hERG(135 kDa)、hERG(155 kDa)蛋白表达水平较Ctl组、Ovp NC组均明显升高(均P<0.05),突变型细胞模型Hsp90+组hERG(135 kDa)蛋白表达水平较Ctl组、Ovp NC组均明显升高(均P<0.05),杂合型细胞模型Hsp90+组hERG(155 kDa)蛋白表达水平较Ctl组、Ovp NC组均明显升高(均P<0.05)。融合Hsp90和hERG蛋白后,Ctl组hERG表达于细胞膜、细胞质,Hsp90-组细胞膜上hERG蛋白表达减少,Hsp90+组细胞膜上hERG蛋白表达增多。与Ctl组比较,Hsp90+组Ikr曲线左移14.709,斜率因子k值未见明显变化。杂合型细胞模型Ctl组、Hsp90-组、Hsp90+组激活电流及尾电流密度均在50 mV处达到最高;杂合型细胞模型Hsp90+组在20、30 mV处的激活电流密度均明显高于Ctl组、Hsp90-组(均P<0.05),在30、40 mV处的尾电流密度均明显高于Ctl组、Hsp90-组(均P<0.05)。结论Hsp90在hERG-A561V通道蛋白折叠转运中起作用,而上调Hsp90可促进WT/A561V hERG的折叠转运,进而影响通道功能。

Objective To investigate the effects of heat-shock protein 90(Hsp90)on the transport and channel function of human ether-a-go-go-related gene(hERG)-A561V channel protein.Methods Wild-type(WT),mutanttype(A561V),and heterozygous(WT/A561V)cell models were constructed.The differences in hERG and Hsp90 protein expression among the groups were detected using western blotting.Plasmids for transfection of reduced expression empty vector(Itf NC),Hsp90 reduced expression(Hsp90-),overexpression empty vector(Ovp NC),or Hsp90 overexpression(Hsp90+)were transfected into each cell model(i.e,Itf NC group,Hsp90-group,Ovp NC group,Hsp90+group)and the control(Ctl)group without any vector.The differences in hERG and Hsp90 protein expression before and after Hsp90 regulation in each group of cells were detected using western blotting.Immunofluorescence was used to detect the expression of hERG and Hsp90 protein in the heterozygous cell model before and after Hsp90 regulation.Whole-cell patch clamp technique was used to detect the activation current and tail current density of hERG channel in the heterozygous cell model before and after Hsp90 regulation.Results The expression of Hsp90 and hERG(135 kDa)protein in mutant-type and heterozygous cell models were significantly higher than those in the wild-type(both P<0.05).In the wild-type cell model,the expression of hERG(135 kDa)and hERG(155 kDa)proteins were significantly higher in the Hsp90+group than in the Ctl group and Ovp NC group(both P<0.05).In the mutant-type cell model,the expression of hERG(135 kDa)protein was significantly higher in the Hsp90+group than in the Ctl group and Ovp NC group(both P<0.05).In the heterozygous cell model,the expression of hERG(155 kDa)protein was significantly higher in the Hsp90+group than in the Ctl group and Ovp NC group(both P<0.05).After fusion of Hsp90 and hERG proteins,hERG expression in the Ctl group was located on the cell membrane and cytoplasm,while the expression of hERG protein on the cell membrane was reduced in the Hsp90-group and increased in the Hsp90+group.Compared with the Ctl group,the Ikr curve of the Hsp90+group shifted to the left by 14.709,and the slope factor k did not change significantly.In the heterozygous cell model,the activation current and tail current density of the Ctl group,Hsp90-group,and Hsp90+group reached the highest at 50 mV.The activation current density of the Hsp90+group in the heterozygous cell model was significantly higher than that of the Ctl group and Hsp90-group at 20 and 30 mV(both P<0.05),and the tail current density at 30 and 40 mV was significantly higher than that of the Ctl group and Hsp90-group(both P<0.05).Conclusion Hsp90 plays a role in the folding and transport of hERG-A561V channel protein.Upregulation of Hsp90 can promote the folding and transport of WT/A561V hERG,thereby affecting channel function.