Lamothe Shawn M, Guo Jun, Li Wentao, Yang Tonghua, Zhang Shetuan
From the Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario K7L 3N6, Canada.
From the Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario K7L 3N6, Canada
J Biol Chem. 2016 Sep 23;291(39):20387-401. doi: 10.1074/jbc.M116.743138. Epub 2016 Aug 8.
The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr), which is important for cardiac repolarization. Dysfunction of hERG causes long QT syndrome and sudden death, which occur in patients with cardiac ischemia. Cardiac ischemia is also associated with activation, up-regulation, and secretion of various proteolytic enzymes. Here, using whole-cell patch clamp and Western blotting analysis, we demonstrate that the hERG/IKr channel was selectively cleaved by the serine protease, proteinase K (PK). Using molecular biology techniques including making a chimeric channel between protease-sensitive hERG and insensitive human ether-a-go-go (hEAG), as well as application of the scorpion toxin BeKm-1, we identified that the S5-pore linker of hERG is the target domain for proteinase K cleavage. To investigate the physiological relevance of the unique susceptibility of hERG to proteases, we show that cardiac ischemia in a rabbit model was associated with a reduction in mature ERG expression and an increase in the expression of several proteases, including calpain. Using cell biology approaches, we found that calpain-1 was actively released into the extracellular milieu and cleaved hERG at the S5-pore linker. Using protease cleavage-predicting software and site-directed mutagenesis, we identified that calpain-1 cleaves hERG at position Gly-603 in the S5-pore linker of hERG. Clarification of protease-mediated damage of hERG extends our understanding of hERG regulation. Damage of hERG mediated by proteases such as calpain may contribute to ischemia-associated QT prolongation and sudden cardiac death.
人类醚 - 去极化相关基因（hERG）编码快速激活延迟整流钾通道（IKr）的孔形成亚基，这对心脏复极化很重要。hERG功能障碍会导致长QT综合征和猝死，这些情况发生在心脏缺血患者中。心脏缺血还与各种蛋白水解酶的激活、上调和分泌有关。在这里，我们使用全细胞膜片钳和蛋白质免疫印迹分析表明，hERG/IKr通道被丝氨酸蛋白酶——蛋白酶K（PK）选择性切割。通过分子生物学技术，包括构建蛋白酶敏感的hERG和不敏感的人类醚 - 去极化（hEAG）之间的嵌合通道，以及应用蝎毒素BeKm - 1，我们确定hERG的S5 - 孔连接区是蛋白酶K切割的靶结构域。为了研究hERG对蛋白酶独特敏感性的生理相关性，我们表明兔模型中的心脏缺血与成熟ERG表达减少以及包括钙蛋白酶在内的几种蛋白酶表达增加有关。使用细胞生物学方法，我们发现钙蛋白酶 - 1被主动释放到细胞外环境中，并在S5 - 孔连接区切割hERG。使用蛋白酶切割预测软件和定点诱变，我们确定钙蛋白酶 - 1在hERG的S5 - 孔连接区的甘氨酸 - 603位置切割hERG。阐明蛋白酶介导的hERG损伤扩展了我们对hERG调节的理解。由钙蛋白酶等蛋白酶介导的hERG损伤可能导致缺血相关的QT延长和心源性猝死。
