HERG K⁺ Channel Conductance Promotes H₂O₂-Induced Apoptosis in HEK293 Cells: Cellular Mechanisms

Abstract

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 (ñ4-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 H₂O₂ (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 H₂O₂ at a concentration-dependent fashion, starting at 200 µM and reaching maximum at 1 mM. Selective HERG channel inhibitors, dofetilide or E-4031 (5 µM) 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 ñ4-fold lower in HERG cells vs. non-transfected cells in the absence of H₂O₂ and was slightly increased (ñ2-fold) after H₂O₂ exposure. We conclude that HERG channels facilitate cellular DNA fragmentation in HEK cells via concomitant activation of MAP kinases and inactivation of Akt.

The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes

“…The protein samples were extracted from H9c2 cells and cultured neonatal rat ventricular cells, with the procedures essentially the same as described in detail elsewhere (Han et al, 2001;Han et al, 2004a;Han et al, 2004b;Luo et al, 2007;Wang et al, 2002). Protein samples (~50 g) were fractionated by SDS-PAGE (7.5-10% polyacrylamide gels).…”

Human ether-�-go-go-related gene expression is essential for cisplatin to induce apoptosis in human gastric cancer

“…When the HERG was blocked, H 2 O 2 -induced apoptosis was also inhibited. Han et al (30) reached a similar conclusion that HERG facilitates cellular DNA fragmentation in HEK cells via concomitant activation of MAP kinases and inactivation of Akt. In our study, when the gastric cancer cells were treated with cisplatin, HERG expression increased in both cell lines and silencing HERG by siRNA inhibited the apoptosis induced by cisiplatin, indicating that HERG expression may be essential for cisplatin to induce apoptosis in human gastric cancer.…”

Downregulation of the HERG (KCNH2) K+ channel by ceramide: evidence for ubiquitin-mediated lysosomal degradation

“…The I HERG decrement was attributed to unspecified changes at the functional level . ROS modulation of I HERG was shown to occur through changes in the voltage dependence of activation and inactivation, with ROS generation enhancing I HERG (Taglialatela et al, 1997;Han et al, 2004). I HERG kinetics were unchanged by TNF-␣ , whereas previously ROS was demonstrated to accelerate I HERG deactivation (Taglialatela et al, 1997).…”