The Mechanism Of Chaperone Calnexin In HERG-A561V Mutation Channel Protein Trafficking Defective

Background and objective: Hereditary long QT syndrome(hLQTS)is a diease of ionic channel dysfunction in cardiomyocyte membrane caused by mutations in the gene encoding cardiac ionic channel protein.In clinic which is characterized as QT interval prolongation、ST-T change、Ventricular Tachycardia(VT)、Torsade de points(TdP)、syncope and sudden cardiac death.hLQTS has a high mortality rate and particularly in children and teenagers.It has been demonstrated that protein trafficking-defect plays an important role in pathogenesis of hLQTS.Mutant channel proteins retained in endoplasmic reticulum and cannot be transported to the cell membrane to form mature and functional HERG channels,which finally leading to hLQTS.Molecular chaperones play an important role in this process.However,it is unclear that what’s the mechanism of the chaperone and whether it can influences the transport of HERG protein by regulating the expression of molecular chaperones.This paper mainly discuss that do regulation the expression of CNX could restore the trafficking defect of HERG-A561 V mutant protein.And confirm the role of CNX in the folding and trafficking defective in this process.To provide a new research direction for the treatment of LQTS at the molecular level.Method: Transfected HEK293 T cells with pcDNA3-hERG,pcDNA3-A561V-hERG and pcDNA3-A561V-hERG,pcDNA3-HERG plasmid to construct wild-type 、 mutant and heteromeric cell models respectively.Then we transfect CNX and shRNA-CNX plasmids into each groups respectively.The expression level of hERG protein was detected by western bolt to determine the mechanism of chaperone CNX.Result:After we transfected with overexpression CNX plasmid into each groups,the expression of mature HERG protein(155KDa)had no difference in each groups.However,in heterozygous cells,the expression of 155 KDa band slightly increased compared with the control group;After transfection of shRNA-CNX plasmid,we found that the expression of wild-type HERG did not differ from with control group,while in mutant and heterozygous cell models,the expression of 155 KDa band increased more than that of the control group,and the increase was more obviously in the heterozygous group.Conclusion: Molecular chaperone CNX plays an important role in protein folding and trafficking induced by HERG-A561 V mutation.Overexpression of CNX does not restore the transport of wild-type and mutant cells,but it can partly restore the trafficking-deficient of heterozygous cells.In addition,knockdown of the CNX expression has no effect on the transport of wild-type cells.However,it can make mutant and heterozygous proteins which retained in the endoplasmic reticulum move out of endoplasmic reticulum,especially heterozygous cells.Therefore,CNX plays an important role in the traffickingdeficient of HERG mutant channel proteins,and the translocation of HERG mutant channel proteins can be restored by regulating the expression level of chaperone CNX.