Role Of The ER Chaperones Calnexin And Calreticulin In The Long Qt Syndrome-Associated G572R And E637K Mutations

Background:Long QT syndrome type 2 is caused by KCNH2 or human ether-a-go-go related gene (hERG) mutations. Among these hERG mutations, trafficking deficient mutations represent the most dominant mechanism for the loss of hERG function in LQT2. These trafficking deficient proteins are retained in the endoplasmic reticulum (ER) as the core-glycosylated immature form, prolonged association with the ER chaperone protein and are rapidly degraded by the ubiquitin proteasome pathway.Aim:We investigated the role of the ER chaperone protein calnexin and calreticulin in the folding and degradation of G572R-hERG and E637K-hERG protein.Method and results:WT- hERG,G572R-hERG and E637K-hERG were transient transfection in HEK293 cells respectively by using a lipofectamine method. Confocal microscopy and western blotting were used to detect the expression and subcellular location of WT-hERG , G572R-hERG , E637K-hERG, calnexin and calreticulin. Interaction of WT- hERG and mutant hERG with calnexin and calreticulin was observed by coimmunoprecipitation. ATF6 was used to detect the hERG channel folding. In additional, proteasome inhibitor studies were performed by treating transfected HEK 293 cells with or without ALLN and LACT. Our results showed that the immature forms of WT- hERG, G572R- hERG and E637K- hERG associated with calnexin and calreticulin, however the mutant hERG associated more with chaperone protein. The two mutant proteins activated UPR. Morever, proteasome inhibition increases the proteins level of immature form of WT- hERG, E637K-hERG and G572R-hERG.Conclusion:Defective folding E637K-hERG and G572R-hERG proteins display increased association with calnexin and calreticulin in the ER. This observation might illustrate the attempt of the calnexin and calreticulin to arrest the mutant proteins in the ER and re-fold them into a correct native conformation. Morever, proteasome is involved in the degradation of the immature form of hERG protein, and proteasome inhibition result to chaperone protein have a prolonged association with E637K-hERG and G572R-hERG. Therefore, the ER chaperone protein calnexin and calreticulin have a important role in the properly folding and degradation of E637K-hERG and G572R-hERG proteins.