Abstract

The human Ether-a-go-go Related Gene (hERG) encodes the voltage-gated K+ channel α-subunit that forms the pore of the rapidly activating delayed rectifier K+ current. hERG mutations are associated with type 2 Long QT syndrome (LQT2). Most missense LQT2 mutations are trafficking-deficient, and reduce complex-glycosylation in the Golgi apparatus (Golgi processing) and plasmalemmal expression. Golgi processing and plasmalemmal expression of LQT2 channels can be increased by culturing cells in drugs that block hERG current (IhERG) (pharmacological correction) or by culturing cells at 27°C (temperature correction). LQT2 channels have different ‘patterns’ of correction, for example, G601S-hERG undergoes pharmacological and temperature correction, whereas R752W-hERG only undergoes temperature correction. These data suggest that these mutations may disrupt different steps in hERG trafficking. To test this, we used confocal microscopy to examine the localization of WT-hERG, G601S-hERG, or R752W-hERG, stably expressed in HEK293 cells. We stained cells using intracellular protein markers for the ER (calnexin), the Golgi (58K), or the endosomes (mannose-6-phosphate receptor or M6PR). We found that cells expressing WT-hERG showed plasmalemmal and intracellular staining that co-localized with calnexin, 58K, and M6PR. Cells expressing G601S- or R752W-hERG showed primarily intracellular hERG staining, but their staining patterns were different. G601S-hERG showed diffused intracellular staining that colocalized with calnexin, but not 58K or M6PR. Cells expressing R752W-hERG showed strong co-localization with the calnexin and M6PR, but not 58K. We conclude G601S-hERG appears to be retained primarily with the ER, whereas R752W-hERG is retained in the ER and endosomes. These data are surprising because they suggest that R752W-hERG may exit the ER and traffic to the endosomes without undergoing Golgi processing. These are the first data to show that trafficking-deficient LQT2-linked mutations, with different patterns of correction, colocalize to different intracellular compartments.

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