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),mutant-type(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.
Heat-shock protein 90Congenital long QT syndromeHuman ether-a-go-go-related geneA561V mutationProtein transport
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