Trafficking-Deficient G572R-hERG and E637K-hERG Activate Stress and Clearance Pathways in Endoplasmic Reticulum

Abstract

BackgroundLong QT syndrome type 2 (LQT2) is the second most common type of all long QT syndromes. It is well-known that trafficking deficient mutant human ether-a-go-go-related gene (hERG) proteins are often involved in LQT2. Cells respond to misfolded and trafficking-deficient proteins by eliciting the unfolded protein response (UPR) and Activating Transcription Factor (ATF6) has been identified as a key regulator of the mammalian UPR. In this study, we investigated the role of ER chaperone proteins (Calnexin and Calreticulin) in the processing of G572R-hERG and E637K-hERG mutant proteins.MethodspcDNA3-WT-hERG, pcDNA3-G572R-hERG and pcDNA3-E637K-hERG plasmids were transfected into U2OS and HEK293 cells. Confocal microscopy and western blotting were used to analyze subcellular localization and protein expression. Interaction between WT or mutant hERGs and Calnexin/Calreticulin was tested by coimmunoprecipitation. To assess the role of the ubiquitin proteasome pathway in the degradation of mutant hERG proteins, transfected HEK293 cells were treated with proteasome inhibitors and their effects on the steady state protein levels of WT and mutant hERGs were examined.ConclusionOur results showed that levels of core-glycosylated immature forms of G572R-hERG and E637K-hERG in association with Calnexin and Calreticulin were higher than that in WT-hERG. Both mutant hERG proteins could activate the UPR by upregulating levels of active ATF6. Furthermore, proteasome inhibition increased the levels of core-glycosylated immature forms of WT and mutant hERGs. In addition, interaction between mutant hERGs and Calnexin/Calreticulin was stronger after proteasome inhibition, compared to WT-hERG. These results suggest that trafficking-deficient G572R-hERG and E637K-hERG mutant proteins can activate ER stress pathways and are targeted to the proteasome for degradation. Calnexin and Calreticulin play important roles in these processes.

Translational toxicology and rescue strategies of the hERG channel dysfunction: biochemical and molecular mechanistic aspects

“…When the hERG protein is unfolded or misfolded, QC will activate the unfolded protein response [UPR, also called endoplasmic reticulum stress response (ERS)] to prevent abnormal forward trafficking. Specifically, chaperones are overexpressed to assist protein folding [17] . Mistakenly-folded hERG protein is fated to be marked with the C-terminal of the Hsp70-interacting protein (CHIP) and ubiquitin, and degraded at the proteasome by the endoplasmic reticulum-associated degradation (ERAD) mechanism [1] .…”

“…For example, G572R-hERG and E637K-hERG mutants upregulate the activating transcription factor 6 (ATF6)-one early responder of UPR. Downstream targets of ATF6, calnexin and calreticulin, are further increased to help hERG channel folding [17] . QC is an essential mechanism that guarantees the correction and clearance of abnormal channels.…”

“…For example, ATF-6, Grp78, Grp94, and calreticulin are all activated to assist in I593R-hERG folding [68] . UPR is also activated by the E637K-hERG and G572R-hERG mutations [17] . In addition, some mutations can alter the kinetics of hERG channel.…”

“…E-4031, astemizole, fexofenadine and dofetilide are most often chosen. For example, E-4031 is able to remove trafficking-deficient G601S-hERG from the microtubule-dependent QC compartment and increase ER export of the mutant hERG channel [17] . In addition, astemizole and fexofenadine are both reported to rescue expression deficiency of N470D-hERG [15,92] .…”

“…For example, low temperature (27 °C) is commonly applied as a rescue strategy by stabilizing the conformation of the hERG channel [15,16] . In addition, the hERG channel blocker E-4031 is able to remove the traffickingdeficient G601S-hERG channel from a microtubule-dependent quality control compartment and increase export from the ER [17] . Meanwhile, the hERG channel activator RPR260243 can slow down hERG channel deactivation, increase I [Kr] and reverse dofentilide-induced action potential duration (APD) and prolongation [18][19][20] .…”