Abstract
The alpha subunit of the voltage gated human ether-a-go-go-related (hERG) potassium channel regulates cell excitability in a broad range of cell lines. HERG channels are also expressed in a variety of cancer cells and control cell proliferation and apoptosis. Hypoxia, a common feature of tumors, alters gating properties of hERG currents in SH-SY5Y neuroblastoma cells. In the present study, we examined the molecular mechanisms and physiological significance underlying hypoxia-altered hERG currents in SH-SY5Y neuroblastoma cells. Hypoxia reduced the surface expression of 150kDa form and increased 125kDa form of hERG protein expression in the endoplasmic reticulum (ER). The changes in protein expression were associated with ~50% decrease in hERG potassium conductance. ER retention of hERG 125kDa form by CH was due to defective trafficking and was rescued by exposing cells to hypoxia at low temperatures or treatment with E-4031, a hERG channel blocker. Prolonged association of hERG with molecular chaperone Hsp90 resulting in complex oligomeric insoluble aggregates contributed to ER accumulation and trafficking defect. Hypoxia increased reactive oxygen species (ROS) levels and manganese (111) tetrakis (1methyl-4-pyridyl) porphyrin pentachloride, a membrane-permeable antioxidant prevented hypoxia-induced degradation of 150kDa and accumulation of 125kDa forms. Impaired trafficking of hERG by hypoxia was associated with reduced cell proliferation and this effect was prevented by antioxidant treatment. These results demonstrate that hypoxia through increased oxidative stress impairs hERG trafficking, leading to decreased K+ currents resulting in cell cycle arrest in SH-SY5Y cells.
Highlights
The human ether-a-go-go-related gene, the α subunit of a voltage gated potassium channel encodes a rapidly activating delayed rectifier current (Ikr) [1]
Major findings of the present study are: a) continuous hypoxia (CH) reduces membrane expression with concomitant increase in endoplasmic reticulum (ER) accumulation of human ether-a-go-go-related gene (hERG) protein in SH-SY5Y cells, b) reduced membrane expression was due to defective trafficking of hERG protein from ER, c) the effects of Continuous hypoxia (CH) on hERG protein were associated with reduced hERG K+ conductance and inhibition cell proliferation, and d) CH increased reactive oxygen species (ROS) abundance and anti-oxidant treatment prevented the trafficking defect and restored the membrane expression of the protein, K + conductance and cell proliferation
CH-induced trafficking defect was rescued by culturing cells at low temperatures and E-4031 [5, 25, 26], b) hERG 125kDa form was resistant to trypsin digestion as opposed to 150kDa form which is sensitive to trypsin suggesting that CH results in misfolding of the hERG protein form in the ER, c) misfolding contributed to prolonged association of 125kDa band with molecular chaperone Hsp90 as evidenced by coimmunoprecipitation experiments and d) resistance of 125kDa form to detergent extraction suggests significant aggregation and ER retention similar to that observed with trafficking-deficient long QT syndrome type 2 (LQT2) mutants
Summary
The human ether-a-go-go-related gene (hERG), the α subunit of a voltage gated potassium channel encodes a rapidly activating delayed rectifier current (Ikr) [1]. Hypoxia impaired hERG-trafficking leads to cell cycle arrest endoplasmic reticulum (ER), as an immature core glycosylated protein (cg) of about 125kDa, is exported to the Golgi apparatus for complex glycosylation and eventually inserted into the plasma membrane as fully glycosylated mature protein (fg) of ~150kDa [8, 9]. HERG potassium channels, originally identified as promoters of cardiac action potential repolarization, are shown to serve as regulators of proliferation and apoptosis in cancer cells [11,12,13]. Silencing hERG or selective hERG channel blockade by pharmacological inhibitors lead to reduced proliferation, cell cycle arrest and increased apoptosis in cancerous cells [15, 16] [17]
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