Journal of Pharmacological Reports

Journal of Pharmacological Reports
Open Access



The Association of Endoplasmic Reticulum Stress Response and Clearance Pathways with Trafficking Deficiency of HERG Mutation and Pharmacological Rescue

Lian JF, Fang, Zheng M, Huang X, Yang X, Guo J, Ba Y, Zhou J, Huang G, Wang Y, Chen J, Chen B and Zhuang K

Background: The human ether-a-go-go related gene (HERG) gene mutation leads to type II hereditary long QT syndrome (LQTS2), which is a main cause of sudden cardiac death of young teenagers. Previous studies have demonstrated that most Type 2 Long QT syndrome caused by trafficking deficiency of HERG Mutation. Although many studies have been performed to elucidate the mechanisms of mutant HERG channel trafficking defects, the players and their exact roles in these process is still largely unknown. Herein, we investigated the HERG-A561V and HERG-L539fs/47, two HERG mutants with different trafficking process leading to LQTS2, studied the roles that chaperones Calnexin/Calreticulin and ATF6 play and proposed to treat LQTS through restoring protein trafficking as well as discuss if these molecules are involved in these process.

Methods: We transiently transfected HEK293 cells with pcDNA3-HERG-WT, pcDNA3-HERG-A561V, pcDNA3- HERG-L539fs/47, pcDNA3-HERG-WT/A561V, and pcDNA3-HERG-WT/L539fs/47 plasmids respectively and analyzed protein expression by Western blotting. Confocal imaging and immunoprecipitation were used to detect the localization and protein-protein interaction of different HERGs and chaperones Calnexin/Calreticulin. Cycloheximide was used to check the dynamic change of the different protein levels. Transfected HEK293 cells were also treated with or without lactacystin (LACT), a irreversible proteasomal inhibitor, followed by interaction, expression and localization analyses by immunoprecipitation, Western blotting and Confocal imaging.

Results: The immature form of HERG-A561V and HERG-WT/A561V had a stronger association with Calnexin/ Calreticulin, while HERG-L539fs/47 had a weaker. HERG-A561V and HERG-A561V/WT activated ATF6, while HERG-L539fs/47 did not. Upon cycloheximide treatment, HERG-A561V had an obvious reduction with time, whereas HERG-L539fs/47, similar to HERG-WT, had a less. Furthermore, the interaction between HERG-A561V or WT/A561V with Calnexin/Calreticulin increased significantly after LACT treatment for 24 hours, and the transport process of HERG-WT/A561V and HERG-A561V was rescued, which was also demonstrated through detecting the localization of HERG-WT/A561V and HERG-A561V both on plasma membrane and in cytoplasm.

Conclusion: The trafficking deficient HERG-A561V mutant protein can activate UPR by activating ATF6 and get degraded by the proteasome pathway, while the HERG-L539fs/47 with a normal trafficking process does not. LACT rescue the trafficking deficiency of HERG-A561V and WT/A561V mutant protein in vitro, which may provide some reference in the treatment of LQTS. In addition, Calnexin/Calreticulin and ATF6 may be involved in the trafficking deficiency, ERAD and LACT rescue of HERG-A561V mutant protein, and play important role in that process.