The Role Of Endoplasmic Reticulum Stress Playing In The Misfolding And Trafficking Defect Of HERG-A561V Mutations

Background and objective: Sudden cardiac death(SCD)is one of the major reasons for human death.Congenital long QT syndrome(c LQTs)is a major cause of SCD,which is characterized by prolonged QT intervals,ventricular arrhythmias,tortuous ventricular tachycardia,episodic syncope and SCD.HERG gene encodes the alpha subunit of rapidly activating component of rectifier potassium current(Ikr),the mutant of HERG gene leads to type 2 long QT syndrome and is the important reason for the juvenile SCD.The HERG-A561V mutant protein can’t be trafficked to the cell membrane surface due to the self-misfolded,which reduces the number of mature HERG channels on the cell membrane surface.These mutant proteins,especially the heterozygous channels,will still play some compensatory function if could be partially recovered its trafficking to cell membrane after regulation.Recently,studies have found that the quality control system,which function is known as folding and transporting channel proteins,plays an important role in the trafficking of ion channels.Endoplasmic reticulum(ER)is an important organelle in eukaryotic cells.Endoplasmic reticulum stress(ERS)is a coordinating and protective mechanism that is triggered by the accumulation of misfolded proteins in the ER.This study aims to investigate whether ERS plays a role in the trafficking of HERG-A561V mutant protein.Moreover,we focus on whether drug intervention of ERS can rectify the mutant protein trafficking defects and improve the function of HERG channels.Method:HERG-A561V mutant gene was studied as research object,we transiently transfected pc DNA3-HERG,pc DNA3-HERG/pc DNA3-HERG-A561V and pc DNA3-HERG-A561V into HEK-293 cells to build wild,heterozygous and mutant cell lines,respectively.Western blot was used to explore the expression of ERS associated downstream proteins and HERG proteins.Immunofluorescence was used to locate the downstream proteins of ERS associated pathways,and whole cell patch clamp technique was applied to detect the difference of Ikr current between each groups.ERS related drugs,thapsigargin(TG)and tauroursodeoxycholic acid(TUDCA)were used to incubate heterozygous cell lines,respectively.Plasma membrane separation method was used to extract the membrane proteins and cytoplasmic proteins.Western blot was used to detect the expression of ERS related downstream proteins and HERG protein in the cytoplasm and cell membrane,respectively.Whole cell patch clamp technique was applied to detect the changes of Ikr current after intervened by TG and TUDCA.Result:Western blot shows that the expression of molecular chaperone Bip,ERS associated three pathways’ downstream protein XBP-1,ATF6,ATF4 in mutant and heterozygous cells were significantly increased,comparing with wild type cells,respectively.Immunofluorescence was used to locate ATF6 and XBP-1 in three group cells.Whole cell patch clamp technique shows that the tail current density of Ikrin heterozygous cells was reduced obviously compared with wild type cells.Then TG was used to incubate heterozygous cells,WB showed that the expression levels of ATF6 and Bip proteins were up-regulated,the expression levels of mature and immature HERG proteins were decreased on the cell membrane surface while up-regulated in cytoplasm.However,no significant changes in Ikr were detected.We used TUDCA to intervene heterozygous cells,no changes were observed in the expression of HERG protein on both cell membrane and cytoplasm,but the tail current parameters of Ikr had changed.Conclusion: Our study found that the expression levels of molecular chaperone Bip and ERS associated three pathways downstream proteins were up-regulated in both mutational and heterozygous cells,which demonstrated that ERS plays important roles in the trafficking of A561V-HERG mutant proteins.Heterozygous cells incubated with TG could enhance ERS levels which showing the increasing expression of molecule chaperone Bip and ATF6,thereby inhibited the trafficking of A561V-HERG mutant protein from endoplasmic reticulum to cell membrane.After intervened heterozygous cells with TUDCA,the expression levels of mature and immature HERG proteins on the cell membrane witnessed no change,however,the tail current parameters of Ikrhad changed.