Instability of KCNE1-D85N that Causes Long QT Syndrome: Stabilization by Verapamil
Instability of KCNE1-D85N that Causes Long QT Syndrome: Stabilization by Verapamil
复制标题
导致长 QT 综合征的 KCNE1-D85N 不稳定：维拉帕米的稳定作用
DOI：
10.1111/pace.12360
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发表时间：
2014
期刊：
影响因子：
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通讯作者：
Hisatome I.
中科院分区：
文献类型：
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作者：
Sakata S;Kurata Y;Li P;Notsu T;Morikawa K;Miake J;Higaki K;Yamamoto Y;Yoshida A;Shirayoshi Y;Yamamoto K;Horie M;Ninomiya H;Kanzaki S;Hisatome I.
背景KCNE1基因D85 N多态性可导致长QT综合征（LQTS），并伴有缓慢激活的延迟整流钾通道电流（IKs）降低。方法将KCNE1-D85N或野生型KCNE1蛋白与KCNQ1共表达于COS7细胞中，构建KCNE1-D85N/KCNQ1-KCNE1-D85N/KCNE1-KCNQ1-KCNE1-D85N/KCNE1-KCNQ1-KCNE1-KCNQ1-KCNE1-K KCNE1蛋白的表达，降解和细胞内定位，以及由KCNQ1/KCNE1复合物赋予的电流，使用免疫印迹，免疫荧光和膜片钳techniques. ResultsThe蛋白水平的KCNE1-D85 N低于野生型，尽管其mRNA的水平相当。与野生型相比，KCNE1-D85 N高度泛素化并迅速降解;蛋白酶体抑制剂MG 132抑制其降解并增加其稳态水平。KCNE1-D85 N和野生型蛋白质均与KCNQ1共免疫沉淀。KCNE1-D85 N的免疫荧光信号在内质网和高尔基体中积累，细胞膜上的水平降低。膜片钳实验表明，表达KCNE1-D85 N的细胞中对应于IKs的膜电流比表达野生型的细胞小得多。维拉帕米（0.5 - 10 μ M）增加KCNE1-D85 N的蛋白水平，降低其泛素化，减缓其降解，并增强KCNQ1/KCNE1-D85 N通道电流。结论KCNE1 ‐ D85 N蛋白的稳定性较野生型差，在泛素-蛋白酶体系统中被迅速降解。维拉帕米可能通过防止KCNE1-D85N降解而对LQTS患者具有治疗价值。
BackgroundA KCNE1 polymorphism, D85N, causes long QT syndrome (LQTS) with a decrease in the slowly activating delayed‐rectifier K+channel current (IKs). We examined impacts of D85N polymorphism on KCNE1 protein stability and functions, and tested the ability of various drugs to modify them.MethodsKCNE1‐D85N or the wild‐type protein was coexpressed in COS7 cells with KCNQ1 to form K+channels. Expression, degradation, and intracellular localization of KCNE1 proteins, as well as the currents conferred by KCNQ1/KCNE1 complexes, were determined using immunoblots, immunofluorescence, and patch‐clamp techniques.ResultsThe protein level of KCNE1‐D85N was lower than that of the wild‐type, in spite of the comparable levels of their mRNA. KCNE1‐D85N was highly ubiquitinated and rapidly degraded as compared to the wild‐type; a proteasome inhibitor, MG132, inhibited its degradation and increased its steady‐state level. Both KCNE1‐D85N and the wild‐type proteins were co‐immunoprecipitated with KCNQ1. Immunofluorescent signals of KCNE1‐D85N accumulated in the endoplasmic reticulum and Golgi apparatus, with reduced levels on the cell membrane. Patch‐clamp experiments demonstrated that the membrane current corresponding to IKswas much smaller in cells expressing KCNE1‐D85N than in those expressing the wild‐type. Verapamil (0.5–10 μM) increased the protein level of KCNE1‐D85N, decreased its ubiquitination, slowed its degradation, and enhanced KCNQ1/KCNE1‐D85N channel currents. Pretreatment with amiodarone abolished these effects of verapamil.ConclusionKCNE1‐D85N is less stable than the wild‐type protein, and is rapidly degraded through the ubiquitin‐proteasome system. Verapamil may be of a therapeutic value in LQTS patients via preventing degradation of KCNE1‐D85N.