Pakladok Tatsiana, Almilaji Ahmad, Munoz Carlos, Alesutan Ioana, Lang Florian
Department of Physiology, University of Tübingen, Gmelinstrasse 5, Tübingen, Germany.
Cell Physiol Biochem. 2013;31(6):785-94. doi: 10.1159/000350096. Epub 2013 May 31.
BACKGROUND/AIMS: Human ether-a-go-go (hERG) channels contribute to cardiac repolarization and participate in the regulation of tumor cell proliferation. Mutations in hERG channels may cause long QT syndrome and sudden cardiac death due to ventricular arrhythmias. HERG channel activity is up-regulated by the serum- and glucocorticoid-inducible kinase isoforms SGK1 and SGK3. Related kinases are protein kinase B (PKB/Akt) isoforms. SGK´s and PKB/Akt´s activate phosphatidylinositol-3-phosphate-5-kinase PIKfyve, which in turn up-regulates several carriers and channels. An effect of PIKfyve on hERG channels, has, however, never been shown. The present study thus explored the putative influence of PIKfyve on hERG channel expression and activity.
hERG channels were expressed in Xenopus oocytes with or without PIKfyve and/or PKB, expression of endogenous and injected hERG quantified by RT-PCR, and hERG channel activity determined utilizing dual electrode voltage clamp. Moreover, hERG protein abundance in the cell membrane was visualized utilizing specific antibody binding and subsequent confocal microscopy and quantified by chemiluminescence.
Coexpression of wild type PIKfyve increased hERG channel activity in hERG-expressing Xenopus oocytes. hERG channel activity was further increased by coexpression of PKB, an effect augmented by additional coexpression of PIKfyve, but not by additional coexpression of PKB/Akt-resistant PIKfyve mutant PIKfyve(S318A). Coexpression of PIKfyve increased hERG channel protein abundance in the cell membrane. Inhibition of hERG channel insertion into the cell membrane by Brefeldin A (5 µM) resulted in a decline of current, which was similar in Xenopus oocytes expressing hERG together with PIKfyve and in Xenopus oocytes expressing hERG alone.
hERG is up-regulated by PIKfyve, which is in turn activated by PKB/Akt.
背景/目的：人醚 - 去极化通道（hERG）对心脏复极有作用，并参与肿瘤细胞增殖的调节。hERG通道的突变可能导致长QT综合征以及因室性心律失常引发的心源性猝死。hERG通道活性受血清和糖皮质激素诱导激酶同工型SGK1和SGK3上调。相关激酶是蛋白激酶B（PKB/Akt）同工型。SGK和PKB/Akt激活磷脂酰肌醇 - 3 - 磷酸 - 5 - 激酶PIKfyve，进而上调多种载体和通道。然而，PIKfyve对hERG通道的作用从未被证实。因此，本研究探讨了PIKfyve对hERG通道表达和活性的潜在影响。
在非洲爪蟾卵母细胞中表达hERG通道，分别加入或不加入PIKfyve和/或PKB，通过逆转录聚合酶链反应（RT-PCR）定量内源性和注射的hERG表达，利用双电极电压钳测定hERG通道活性。此外，利用特异性抗体结合及随后的共聚焦显微镜观察细胞膜中hERG蛋白丰度，并通过化学发光进行定量。
野生型PIKfyve共表达增加了表达hERG的非洲爪蟾卵母细胞中的hERG通道活性。PKB共表达进一步增加了hERG通道活性，PIKfyve的额外共表达增强了该效应，但PKB/Akt抗性PIKfyve突变体PIKfyve(S318A)的额外共表达则无此作用。PIKfyve共表达增加了细胞膜中hERG通道蛋白丰度。布雷菲德菌素A（5 μM）抑制hERG通道插入细胞膜导致电流下降，这在共表达hERG和PIKfyve的非洲爪蟾卵母细胞以及单独表达hERG的非洲爪蟾卵母细胞中相似。
PIKfyve上调hERG，而PIKfyve又被PKB/Akt激活。
