Modulation of hERG potassium currents in HEK-293 cells by protein kinase C. Evidence for direct phosphorylation of pore forming subunits
Modulation of hERG potassium currents in HEK-293 cells by protein kinase C. Evidence for direct phosphorylation of pore forming subunits
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DOI：
10.1113/jphysiol.2006.123414
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发表时间：
2007-06-01
影响因子：
5.5
通讯作者：
Mitcheson, J. S.
中科院分区：
文献类型：
--
作者：
Cockerill, S. L.;Tobin, A. B.;Mitcheson, J. S.
人类以太-a-go-go相关基因（hERG）钾通道在多种组织中表达，包括心脏、神经元和一些癌细胞。hERG通道受多种细胞内信号通路的调节，这些通路为调节细胞兴奋性提供了重要的机制。在这项研究中，我们研究了毒毒碱对HEK-293细胞中hERG电流的调节，以及在生理相关温度下通过蛋白激酶C （PKC）直接磷酸化表达的通道亚基。G（α q/11）偶联m -3毒蕈碱受体与甲胆碱的激活，降低了所有电位下的电流振幅，对激活和失活的电压依赖性影响较小。对甲胆碱的反应对细胞内BAPTA不敏感，但通过300 nM双吲哚马来酰亚胺-1 （bis1）对PKC的急性抑制或用phorbol 12-肉豆酸13-乙酸酯（PMA）预处理细胞24小时慢性下调PKC同种异型，可以减弱对甲基胆碱的反应。用二酰基甘油（DAG）的类似物1-油基2-乙酰甘油（OAG）刺激PKC，模拟毒蕈碱受体刺激的作用。用抗hERG抗体对免疫沉淀蛋白进行[P-32]正磷酸盐标记，检测hERG的直接磷酸化。在未受刺激的细胞中，基础磷酸化水平较高，并在OAG的作用下进一步升高。OAG依赖性的增加被bis-1和PKC的下调所消除，但磷酸化的基础水平不变。hERG氨基末端的缺失阻止了OAG对通道活性的调节和磷酸化的增加。我们的研究结果与PKC的钙和/或DAG敏感同型调节hERG电流的机制一致，该机制涉及hERG氨基端位点的直接磷酸化。
The human ether-a-go-go related gene (hERG) potassium channel is expressed in a variety of tissues including the heart, neurons and some cancer cells. hERG channels are modulated by several intracellular signalling pathways and these provide important mechanisms for regulating cellular excitability. In this study, we investigated muscarinic modulation of hERG currents and direct phosphorylation of channel subunits expressed in HEK-293 cells at physiologically relevant temperatures by protein kinase C (PKC). Activation of G(alpha q/11)-coupled M-3-muscarinic receptors with methacholine, reduced current amplitudes at all potentials with minor effects on the voltage dependence of activation and inactivation. The response to methacholine was insensitive to intracellular BAPTA, but was attenuated by either acute inhibition of PKC with 300 nM bisindolylmaleimide-1 (bis-1) or chronic down-regulation of PKC isoforms by 24 h pretreatment of cells with phorbol 12-myristate 13-acetate (PMA). Stimulation of PKC with 1-oleoyl 2-acetylglycerol (OAG), an analogue of diacylglycerol (DAG), mimicked the actions of muscarinic receptor stimulation. Direct phosphorylation of hERG was measured by [P-32]orthophosphate labelling of immunoprecipitated protein with an anti-hERG antibody. Basal phosphorylation was high in unstimulated cells and further increased by OAG. The OAG dependent increase was abolished by bis-1 and down-regulation of PKC, but basal levels of phosphorylation were unchanged. Deletion of the amino-terminus of hERG prevented both the modulation of channel activity and the increase of phosphorylation by OAG. Our results are consistent with calcium and/or DAG sensitive isotypes of PKC modulating hERG currents through a mechanism that involves direct phosphorylation of sites on the amino terminus of hERG.