Dong Ming-Qing, Sun Hai-Ying, Tang Qiang, Tse Hung-Fat, Lau Chu-Pak, Li Gui-Rong
Department of Medicine and Research Centre of Heart, Brain, Hormone and Healthy Aging, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China.
Biochim Biophys Acta. 2010 May;1798(5):995-1001. doi: 10.1016/j.bbamem.2010.01.010. Epub 2010 Jan 18.
The aim of the present study was to investigate whether/how the recombinant human cardiac I(Ks) could be regulated by epidermal growth factor receptor kinase in HEK 293 cells stably expressing hKCNQ1/hKCNE1 genes using the approaches of perforated patch clamp technique, immunoprecipitation and Western blot analysis. It was found that the broad spectrum isoflavone tyrosine kinase inhibitor genistein and the selective epidermal growth factor receptor kinase inhibitor tyrphostin AG556 suppressed the recombinant I(Ks), and their inhibition was countered by the protein tyrosine phosphatase inhibitor orthovanadate. The Src-family kinase inhibitor PP2 reduced the current, but the effect was not antagonized by orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hKCNQ1 protein was decreased by genistein or AG556, but not by PP2. These results provide the novel information that epidermal growth factor receptor kinase, but not Src-family kinases, regulates the recombinant cardiac I(Ks) stably expressed in HEK 293 cells via phosphorylating KCNQ1 protein of the channel.
本研究的目的是采用穿孔膜片钳技术、免疫沉淀和蛋白质免疫印迹分析方法，研究在稳定表达hKCNQ1/hKCNE1基因的HEK 293细胞中，重组人心脏I(Ks)是否/如何受表皮生长因子受体激酶调控。研究发现，广谱异黄酮酪氨酸激酶抑制剂染料木黄酮和选择性表皮生长因子受体激酶抑制剂 tyrphostin AG556可抑制重组I(Ks)，蛋白酪氨酸磷酸酶抑制剂原钒酸盐可对抗其抑制作用。Src家族激酶抑制剂PP2可降低电流，但原钒酸盐不能拮抗其作用。免疫沉淀和蛋白质免疫印迹分析显示，染料木黄酮或AG556可降低hKCNQ1蛋白的酪氨酸磷酸化水平，但PP2无此作用。这些结果提供了新的信息，即表皮生长因子受体激酶而非Src家族激酶，通过磷酸化通道的KCNQ1蛋白来调控稳定表达于HEK 293细胞中的重组心脏I(Ks)。
