HERG K channel conductance promotes H2O2-induced apoptosis in HEK293 cells: cellular mechanisms.
The human ether-a-go-go-related gene (HERG) encodes a delayed rectifier K(+) channel, which is expressed in a variety of tissues and cells. Besides its well-recognized function in cellular electrophysiology, HERG channels have also been implicated in neuronal differentiation and cell cycle regulation. We have recently found that HERG regulates apoptosis. To elucidate the signaling pathways, we performed studies in HEK293 cells stably expressing HERG channels. ELISA was used to quantify DNA fragmentation, a biochemical hallmark of apoptosis. In HERG-transfected HEK cells, the degree of DNA fragmentation was found consistently higher (approximately 4-times) than in non-transfected cells. Correspondingly, remarkable activation of caspase 3, caspase 9 and cleavage of PARP were seen in HERG-expressing cells, which were otherwise minimal in non-transfected cells. Exposure of cells to H(2)O(2) (10 hrs) at concentrations up to 1 mM, which is known to induce apoptosis in a variety of cells, caused minimal DNA fragmentation in non-transfected cells. HERG expression facilitates DNA fragmentation induced by H(2)O(2) at a concentration-dependent fashion, starting at 200 microM and reaching maximum at 1 mM. Selective HERG channel inhibitors, dofetilide or E-4031 (5 microM) prevented DNA fragmentation. Inhibition of p38 by SB-203580 alleviated DNA-F and PD-98059, which inhibited activation of ERKs, nearly abolished DNA-F. Immunoblotting analysis demonstrated that p38, SAPKs and ERKs MAP kinases were all substantially activated (>10-fold higher) in HERG-expressing cells vs. non-transfected cells. Akt activity was approximately 4-fold lower in HERG cells vs. non-transfected cells in the absence of H(2)O(2) and was slightly increased (approximately 2-fold) after H(2)O(2) exposure. We conclude that HERG channels facilitate cellular DNA fragmentation in HEK cells via concomitant activation of MAP kinases and inactivation of Akt.
Han
Hong
H
Research Center, Montreal Heart Institute, Quebec, Canada.
Wang
Jingxiong
J
Zhang
Yiqiang
Y
Long
Hong
H
Wang
Huizhen
H
Xu
Donghui
D
Wang
Zhiguo
Z
D000889:Anti-Arrhythmia Agents; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D053938:DNA Fragmentation / Q000187:drug effects*; D000072237:ERG1 Potassium Channel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051638:Ether-A-Go-Go Potassium Channels; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005419:Flavonoids / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000889:Anti-Arrhythmia Agents; D000072237:ERG1 Potassium Channel; D004791:Enzyme Inhibitors; D051638:Ether-A-Go-Go Potassium Channels; D005419:Flavonoids; D007093:Imidazoles; C000606913:KCNH2 protein, human; D010880:Piperidines; D024642:Potassium Channels, Voltage-Gated; D011725:Pyridines; C063968:E 4031; D006861:Hydrogen Peroxide; D011065:Poly(ADP-ribose) Polymerases; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one