Large T-antigen up-regulates Kv4.3 K+ channels through Sp1, and Kv4.3 K+ channels contribute to cell apoptosis and necrosis through activation of calcium/calmodulin-dependent protein kinase II

2012 ◽  
Vol 441 (3) ◽  
pp. 859-869 ◽  
Author(s):  
Qi Li ◽  
Ying Zhang ◽  
Yue Sheng ◽  
Rong Huo ◽  
Bo Sun ◽  
...  

Down-regulation of Kv4.3 K+ channels commonly occurs in multiple diseases, but the understanding of the regulation of Kv4.3 K+ channels and the role of Kv4.3 K+ channels in pathological conditions are limited. HEK (human embryonic kidney)-293T cells are derived from HEK-293 cells which are transformed by expression of the large T-antigen. In the present study, by comparing HEK-293 and HEK-293T cells, we find that HEK-293T cells express more Kv4.3 K+ channels and more transcription factor Sp1 (specificity protein 1) than HEK-293 cells. Inhibition of Sp1 with Sp1 decoy oligonucleotide reduces Kv4.3 K+ channel expression in HEK-293T cells. Transfection of pN3-Sp1FL vector increases Sp1 protein expression and results in increased Kv4.3 K+ expression in HEK-293 cells. Since the ultimate determinant of the phenotype difference between HEK-293 and HEK-293T cells is the large T-antigen, we conclude that the large T-antigen up-regulates Kv4.3 K+ channel expression through an increase in Sp1. In both HEK-293 and HEK-293T cells, inhibition of Kv4.3 K+ channels with 4-AP (4-aminopyridine) or Kv4.3 small interfering RNA induces cell apoptosis and necrosis, which are completely rescued by the specific CaMKII (calcium/calmodulin-dependent protein kinase II) inhibitor KN-93, suggesting that Kv4.3 K+ channels contribute to cell apoptosis and necrosis through CaMKII activation. In summary, we establish: (i) the HEK-293 and HEK-293T cell model for Kv4.3 K+ channel study; (ii) that large T-antigen up-regulates Kv4.3 K+ channels through increasing Sp1 levels; and (iii) that Kv4.3 K+ channels contribute to cell apoptosis and necrosis through activating CaMKII. The present study provides deep insights into the mechanism of the regulation of Kv4.3 K+ channels and the role of Kv4.3 K+ channels in cell death.

2001 ◽  
Vol 354 (3) ◽  
pp. 717-725 ◽  
Author(s):  
Zongming ZHANG ◽  
Yufang TANG ◽  
Michael Xi ZHU

Drosophila transient receptor potential (Trp) and its mammalian homologues are postulated to form capacitative Ca2+ entry or store-operated channels. Here we show that expression of murine Trp4 in HEK 293 cells also leads to an increase in inwardly rectifying K+ currents. No similar increase was found in cell lines expressing Trp1, Trp3 or Trp6. Consistent with typical characteristics of inward rectifiers, the K+ currents in Trp4-expressing cells were blocked by low millimolar concentrations of Cs+ and Ba2+, but not by 1.2mM Ca2+, and were only slightly inhibited by 5mM tetraethylammonium. Single channel recordings of excised inside-out patches revealed the presence of two conducting states of 51pS and 94pS in Trp4-expressing cells. The outward current in the excised patches was blocked by 1mM spermine, but not by 1mM Mg2+. How Trp4 expression causes the increase in the K+ currents is not known. We propose that Trp4 either participates in the formation of a novel K+ channel or up-regulates the expression or activity of endogenous inwardly rectifying K+ channels.


2003 ◽  
Vol 553 (1) ◽  
pp. 13-19 ◽  
Author(s):  
David C. H. Benton ◽  
Alan S. Monaghan ◽  
Ramine Hosseini ◽  
Parmvir K. Bahia ◽  
Dennis G. Haylett ◽  
...  

2000 ◽  
Vol 129 (7) ◽  
pp. 1323-1332 ◽  
Author(s):  
Murali Gopalakrishnan ◽  
Eduardo J Molinari ◽  
Char-Chang Shieh ◽  
Lisa M Monteggia ◽  
Jean-Marc Roch ◽  
...  

PLoS ONE ◽  
2017 ◽  
Vol 12 (8) ◽  
pp. e0183708 ◽  
Author(s):  
Xiu-Ling Deng ◽  
Yan Wang ◽  
Guo-Sheng Xiao
Keyword(s):  
Hek 293 ◽  

1998 ◽  
Vol 76 ◽  
pp. 239
Author(s):  
Takuma Oku ◽  
Susumu Ohya ◽  
Mamiko Tanaka ◽  
Minoru Watanabe ◽  
Yuji Imaizumi

2000 ◽  
Vol 129 (5) ◽  
pp. 991-999 ◽  
Author(s):  
Dorte Strøbaek ◽  
Tino D Jørgensen ◽  
Palle Christophersen ◽  
Philip K Ahring ◽  
Søren-Peter Olesen

2017 ◽  
Vol 8 ◽  
Author(s):  
Kui-Hao Chen ◽  
Hui Liu ◽  
Hai-Ying Sun ◽  
Man-Wen Jin ◽  
Guo-Sheng Xiao ◽  
...  

Autophagy ◽  
2013 ◽  
Vol 9 (9) ◽  
pp. 1407-1417 ◽  
Author(s):  
Patience Musiwaro ◽  
Matthew Smith ◽  
Maria Manifava ◽  
Simon A. Walker ◽  
Nicholas T. Ktistakis
Keyword(s):  
Hek 293 ◽  

2005 ◽  
Vol 103 (6) ◽  
pp. 1156-1166 ◽  
Author(s):  
Kevin J. Gingrich ◽  
Son Tran ◽  
Igor M. Nikonorov ◽  
Thomas J. Blanck

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.


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