Nanduri Jayasri, Bergson Pamela, Wang Ning, Ficker Eckhard, Prabhakar Nanduri R
Center for Systems Biology, Department of Medicine, The University of Chicago, Chicago, IL 60637, USA.
Biochem Biophys Res Commun. 2009 Oct 16;388(2):212-6. doi: 10.1016/j.bbrc.2009.07.149. Epub 2009 Aug 3.
We previously reported that reactive oxygen species (ROS) generated during hypoxia decrease hERG current density and protein expression in HEK cells stably expressing hERG protein. In the present study, we investigated the molecular mechanisms involved in hypoxia-induced downregulation of hERG protein. Culturing cells at low temperatures and addition of chemical chaperones during hypoxia restored hERG expression and currents to normoxic levels while antiarrhythmic drugs, which selectively block hERG channels, had no effect on hERG protein levels. Pulse chase studies showed that hypoxia blocks maturation of the core glycosylated form in the endoplasmic reticulum (ER) to the fully glycosylated form on the cell surface. Co-immunoprecipitation experiments revealed that hypoxia inhibited interaction of hERG with Hsp90 chaperone required for maturation, which was restored in the presence of ROS scavengers. These results demonstrate that ROS generated during hypoxia prevents maturation of the hERG protein by inhibiting Hsp90 interaction resulting in decreased protein expression and currents.
我们之前报道过，缺氧期间产生的活性氧（ROS）会降低稳定表达hERG蛋白的HEK细胞中的hERG电流密度和蛋白表达。在本研究中，我们调查了缺氧诱导hERG蛋白下调所涉及的分子机制。在低温下培养细胞以及在缺氧期间添加化学伴侣可将hERG表达和电流恢复到常氧水平，而选择性阻断hERG通道的抗心律失常药物对hERG蛋白水平没有影响。脉冲追踪研究表明，缺氧会阻断内质网（ER）中核心糖基化形式向细胞表面完全糖基化形式的成熟过程。免疫共沉淀实验显示，缺氧会抑制hERG与成熟所需的Hsp90伴侣的相互作用，而在存在ROS清除剂的情况下这种相互作用得以恢复。这些结果表明，缺氧期间产生的ROS通过抑制Hsp90相互作用来阻止hERG蛋白的成熟，从而导致蛋白表达和电流降低。
