Multiple Cellular Mechanisms Underlie the Trafficking-deficient Phenotype for Kv11.1 (hERG) Mutations Linked to Long QT Syndrome

Type 2 Long QT syndrome (LQT2) is commonly caused by missense mutations that disrupt the trafficking of Kv11.1 channels. The goal of this study is to determine the cellular mechanisms that underlie the trafficking-deficient phenotype for LQT2-linked mutations located in different channel domains. We expressed the pore-domain mutation G601S and the cyclic nucleotide binding domain mutation F805C in human embryonic kidney 293 (HEK293) cells or human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). HEK293 cells or hiPSC-CMs expressing G601S showed a diffuse intracellular anti-Kv11.1 staining pattern that colocalized with the transitional endoplasmic reticulum (ER) marker BAP31, but not markers for the rough ER (calnexin), ER-associated degradation compartment (derlin1), ER exit sites (Sec31), or the ER-Golgi intermediate compartment (ERGIC53). Culturing cells in the drug E-4031 (10 μM, 24 hrs), which corrects the G601s trafficking-deficient phenotype, decreased its colocalization with BAP31. Unlike G601S, the trafficking-deficient phenotype for F805C is not corrected by culturing cells in E-4031. Expressing F805C in HEK293 cells or hiPSC-CMs showed a very different anti-Kv11.1 staining pattern that consisted of several discrete aggregates. In hiPSC-CMs, the F805C aggregates partially overlapped with derlin1 staining and co-transfected green fluorescent protein (gfp) in the cytosol. Incubating cells in the proteasome inhibitor bortezomib (10 μM, 4 hrs) dispersed the F805C aggregates to generate a diffuse intracellular staining pattern. We conclude that G601S and F805C localize to distinct ER subcompartments: G601S is sequestered in the transitional ER and F805C localizes to an ER associated degradation subcompartment that is sensitive to proteasome inhibition.