High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel K(V)11.1.
High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel K(V)11.1.
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DOI：
10.1016/j.hrthm.2020.05.041
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发表时间：
2020-12
期刊：
影响因子：
5.5
通讯作者：
Kroncke BM
中科院分区：
文献类型：
--
作者：
Kozek KA;Glazer AM;Ng CA;Blackwell D;Egly CL;Vanags LR;Blair M;Mitchell D;Matreyek KA;Fowler DM;Knollmann BC;Vandenberg JI;Roden DM;Kroncke BM
KCHN2 编码负责 IKr 的 KV11.1 钾通道，IKr 是心肌细胞动作电位期间的主要复极电流。导致 IKr 降低的 KCNH2 变异与 2 型长 QT 综合征 (LQT2) 相关。 LQT2 的机制最常见的是诱导 KV11.1 运输至细胞表面的丧失。准确区分正常和异常贩运的变体将有助于了解这些变体的有害性质；然而，报道的非同义 KCNH2 变体的数量妨碍了使用常规方法进行功能研究。我们报告了一种针对 KCNH2 遗传变异的高通量、多重筛选方法，能够测量所得 KV11.1 通道中数百个错义变异的细胞表面丰度。我们开发了一种方法来定量 S5 螺旋中 11 个残基的试点区域上的 KV11.1 变体贩运。我们为试点地区的 220/231 错义变体生成了贩运分数。对于 5/5 变体，在单变体流式细胞术和共焦显微镜实验中进行测试时，高通量运输分数得到验证。我们通过平面贴片电生理学进一步探讨了这些结果，发现丢失的运输变体确实不产生 IKr。相反，但正如预期的那样，一些正常流量的变体在功能上仍然受到损害。在这里，我们描述了一种在多重检测中检测 KV11.1 运输缺陷变体的新方法。这种新方法准确地生成了 KV11.1 变体的运输数据，并且可扩展到 Kv11.1 中的所有残基和其他细胞表面蛋白。
KCHN2 encodes the KV11.1 potassium channel responsible for IKr, a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that lead to decreased IKr have been associated with Type 2 Long QT syndrome (LQT2). The mechanism of LQT2 is most often induced loss of KV11.1 trafficking to the cell surface. Accurately discriminating between variants with normal and abnormal trafficking would aid in understanding the deleterious nature of these variants; however, the volume of reported nonsynonymous KCNH2 variants precludes the use of conventional methods for functional study. We report a high-throughput, multiplexed screening method for KCNH2 genetic variants capable of measuring the cell surface abundance of hundreds of missense variants in the resulting KV11.1 channel. We developed a method to quantitate KV11.1 variant trafficking on a pilot region of 11 residues in the S5 helix. We generated trafficking scores for 220/231 missense variants in the pilot region. For 5/5 variants, high-throughput trafficking scores validated when tested in single variant flow cytometry and confocal microscopy experiments. We further explored these results with planar patch electrophysiology and found that loss-of-trafficking variants indeed do not produce IKr. Conversely, but expectedly, some variants that traffic normally were still functionally compromised. Here, we described a new method for detecting KV11.1 trafficking-deficient variants in a multiplexed assay. This new method accurately generated trafficking data for variants in KV11.1 and is extendable both to all residues in Kv11.1 and to other cell surface proteins.
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影响因子：
5.5
作者：
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Shendure, Jay
影响因子：
64.5
作者：
Wang W;MacKinnon R
通讯作者：
MacKinnon R
影响因子：
64.8
作者：
Itzhaki, Ilanit;Maizels, Leonid;Gepstein, Lior
通讯作者：
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