Modulation of human cardiac transient outward potassium current by EGFR tyrosine kinase and Src-family kinases

Zhang, Yan-Hui; Wu, Wei; Sun, Hai‐Ying; Deng, Xingming; Cheng, Lin; Li, Xin; Tse, Hung‐Fat; Lau, Chu‐Pak; Li, Gui-Rong

doi:10.1093/cvr/cvr347

Cited by 20 publications

(21 citation statements)

References 48 publications

(85 reference statements)

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“…The established HEK 293 cell lines stably expressing hKv4.3 ( KCND3 ) [22], hKv1.5 ( KCN5A ) [23], [24], hERG (Kv11.1 or KNCH2 ) [25], hKCNQ1/hKCNE1 [26], and hKir2.1 [27], respectively, were cultured in Dulbecco’s modified eagle’s medium (DMEM, Invitrogen, Hong Kong) supplemented with 10% fetal bovine serum and corresponding selective antibiotics. The hKv4.3 channel mutants, T366A, T367A, V392A, I395A and V399A generated using the QuickChange Site-directed Mutagenesis kit (Stratagene, La Jolla, CA, USA), were transiently expressed in HEK 293 cells with a 36-mm culture dish using 10 µl of Lipofectamine 2000 with 4 µg of hKv4.3 mutant cDNA in pcDNA3 vector.…”

Section: Methodsmentioning

confidence: 99%

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Allitridi Inhibits Multiple Cardiac Potassium Channels Expressed in HEK 293 Cells

Sun

Zhang

et al. 2012

PLoS ONE

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Allitridi (diallyl trisulfide) is an active compound (volatile oil) from garlic. The previous studies reported that allitridi had anti-arrhythmic effect. The potential ionic mechanisms are, however, not understood. The present study was designed to determine the effects of allitridi on cardiac potassium channels expressed in HEK 293 cells using a whole-cell patch voltage-clamp technique and mutagenesis. It was found that allitridi inhibited hKv4.3 channels (IC50 = 11.4 µM) by binding to the open channel, shifting availability potential to hyperpolarization, and accelerating closed-state inactivation of the channel. The hKv4.3 mutants T366A, T367A, V392A, and I395A showed a reduced response to allitridi with IC50s of 35.5 µM, 44.7 µM, 23.7 µM, and 42.4 µM. In addition, allitridi decreased hKv1.5, hERG, hKCNQ1/hKCNE1 channels stably expressed in HEK 293 cells with IC50s of 40.2 µM, 19.6 µM and 17.7 µM. However, it slightly inhibited hKir2.1 current (100 µM, inhibited by 9.8% at −120 mV). Our results demonstrate for the first time that allitridi preferably blocks hKv4.3 current by binding to the open channel at T366 and T367 of P-loop helix, and at V392 and I395 of S6 domain. It has a weak inhibition of hKv1.5, hERG, and hKCNQ1/hKCNE1 currents. These effects may account for its anti-arrhythmic effect observed in experimental animal models.

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Section: Methodsmentioning

confidence: 99%

“…The whole cell patch-clamp technique was used as described previously [22]–[27]. The whole-cell membrane currents were measured using an EPC-10 amplifier and Pulse software (Heka Elektronik, Lambrecht, Germany).…”

Section: Methodsmentioning

confidence: 99%

Allitridi Inhibits Multiple Cardiac Potassium Channels Expressed in HEK 293 Cells

Sun

Zhang

et al. 2012

PLoS ONE

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“…Data are presented as mean ± SEM. A group size of number of 5 or more was determined based on previous experience Zhang et al, 2012) with SigmaPlot 12.5 (SPSS Science, Chicago, IL, USA). Paired and/or unpaired Student's two-tailed t-test was used as appropriate to determine the statistical significance of differences between two group means.…”

Section: Data and Statistical Analysismentioning

confidence: 99%

“…Figure 5A shows the family of voltage-dependent I Kur recorded in a typical experiment in the absence and presence of 1 μM PP2 [a concentration 200 times higher (Hanke et al, 1996) than the EC 50 for inhibiting Src-related kinases]. A high concentration of PP2 is usually required for Src family kinase inhibition in experiments at cellular level (Du et al, 2004;Zhang et al, 2008;Zhang et al, 2012), perhaps because PP2 does not easily cross the cell membrane. It is interesting to note that I Kur was slightly increased by PP2 (7 min superfusion), and the enhancement was also reversed by coapplication of 1 mM orthovanadate.…”

Section: Inhibition Of Hk V 15 Current By Genistein and Ag556mentioning

confidence: 99%

Genistein and tyrphostin AG556 decrease ultra‐rapidly activating delayed rectifier K⁺ current of human atria by inhibiting EGF receptor tyrosine kinase

Xiao

Zhang

et al. 2017

British J Pharmacology

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BACKGROUND AND PURPOSEThe ultra-rapidly activating delayed rectifier K + current I Kur (encoded by K v 1.5 or KCNA5) plays an important role in human atrial repolarization. The present study investigates the regulation of this current by protein tyrosine kinases (PTKs). EXPERIMENTAL APPROACHWhole-cell patch voltage clamp technique and immunoprecipitation and Western blotting analysis were used to investigate whether the PTK inhibitors genistein, tyrphostin AG556 (AG556) and PP2 regulate human atrial I Kur and hKv1.5 channels stably expressed in HEK 293 cells. KEY RESULTSHuman atrial I Kur was decreased by genistein (a broad-spectrum PTK inhibitor) and AG556 (a highly selective EGFR TK inhibitor) in a concentration-dependent manner. Inhibition of I Kur induced by 30 μM genistein or 10 μM AG556 was significantly reversed by 1 mM orthovanadate (a protein tyrosine phosphatase inhibitor). Similar results were observed in HEK 293 cells stably expressing hK v 1.5 channels. On the other hand, the Src family kinase inhibitor PP2 (1 μM) slightly enhanced I Kur and hK v 1.5 current, and the current increase was also reversed by orthovanadate. Immunoprecipitation and Western blotting analysis showed that genistein, AG556, and PP2 decreased tyrosine phosphorylation of hK v 1.5 channels and that the decrease was countered by orthovanadate. CONCLUSION AND IMPLICATIONSThe PTK inhibitors genistein and AG556 decrease human atrial I Kur and cloned hK v 1.5 channels by inhibiting EGFR TK, whereas the Src kinase inhibitor PP2 increases I Kur and hK v 1.5 current. These results imply that EGFR TK and the soluble Src kinases may have opposite effects on human atrial I Kur .Abbreviations EGFR, EGF receptor; I Kur , ultra-rapidly activating delayed rectifier potassium current; PP2, 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo [3,4-d] pyrimidin-4-amine; PTK, protein tyrosine kinase; TK, protein kinase; tyrphostin AG556, AG556

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“…The protein tyrosine phosphatase inhibitor sodium orthovanadate (SOV) (50 µmol/L) was added to ascertain if genistein-induced effects on SRs are related to protein tyrosine kinase (PTK) inhibition, in which case SOV should antagonize these PTK-dependent effects [28]. Western blot analysis (figure 4) demonstrated that…”

Section: Vsmcsmentioning

confidence: 99%

Genistein suppresses smooth muscle cell-derived foam cell formation through tyrosine kinase pathway

Lin

Zhao

et al. 2015

Biochemical and Biophysical Research Communications

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Modulation of human cardiac transient outward potassium current by EGFR tyrosine kinase and Src-family kinases

Cited by 20 publications

References 48 publications

Allitridi Inhibits Multiple Cardiac Potassium Channels Expressed in HEK 293 Cells

Allitridi Inhibits Multiple Cardiac Potassium Channels Expressed in HEK 293 Cells

Genistein and tyrphostin AG556 decrease ultra‐rapidly activating delayed rectifier K⁺ current of human atria by inhibiting EGF receptor tyrosine kinase

Genistein suppresses smooth muscle cell-derived foam cell formation through tyrosine kinase pathway

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Modulation of human cardiac transient outward potassium current by EGFR tyrosine kinase and Src-family kinases

Cited by 20 publications

References 48 publications

Allitridi Inhibits Multiple Cardiac Potassium Channels Expressed in HEK 293 Cells

Allitridi Inhibits Multiple Cardiac Potassium Channels Expressed in HEK 293 Cells

Genistein and tyrphostin AG556 decrease ultra‐rapidly activating delayed rectifier K+ current of human atria by inhibiting EGF receptor tyrosine kinase

Genistein suppresses smooth muscle cell-derived foam cell formation through tyrosine kinase pathway

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Genistein and tyrphostin AG556 decrease ultra‐rapidly activating delayed rectifier K⁺ current of human atria by inhibiting EGF receptor tyrosine kinase