Department of Physiology & Biophysics, Virginia Commonwealth University, Richmond, VA, USA.
Institute of Medicinal biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
J Physiol. 2021 Jul;599(13):3337-3361. doi: 10.1113/JP281773. Epub 2021 Jun 1.
In adult ventricular myocytes, the slow delayed rectifier (I ) channels are distributed on the surface sarcolemma, not t-tubules. In adult ventricular myocytes, KCNQ1 and KCNE1 have distinct cell surface and cytoplasmic pools. KCNQ1 and KCNE1 traffic from the endoplasmic reticulum to the plasma membrane by separate routes, and assemble into I channels on the cell surface. Liquid chromatography/tandem mass spectrometry applied to affinity-purified KCNQ1 and KCNE1 interacting proteins reveals novel interactors involved in protein trafficking and assembly. Microtubule plus-end binding protein 1 (EB1) binds KCNQ1 preferentially in its dimer form, and promotes KCNQ1 to reach the cell surface. An LQT1-associated mutation, Y111C, reduces KCNQ1 binding to EB1 dimer.
Slow delayed rectifier (I ) channels consist of KCNQ1 and KCNE1. I functions as a 'repolarization reserve' in the heart by providing extra current for ventricular action potential shortening during β-adrenergic stimulation. There has been much debate about how KCNQ1 and KCNE1 traffic in cells, where they associate to form I channels, and the distribution pattern of I channels relative to β-adrenergic signalling complex. We used experimental strategies not previously applied to KCNQ1, KCNE1 or I , to provide new insights into these issues. 'Retention-using-selected-hook' experiments showed that newly translated KCNE1 constitutively trafficked through the conventional secretory path to the cell surface. KCNQ1 largely stayed in the endoplasmic reticulum, although dynamic KCNQ1 vesicles were observed in the submembrane region. Disulphide-bonded KCNQ1/KCNE1 constructs reported preferential association after they had reached cell surface. An in situ proximity ligation assay detected I channels in surface sarcolemma but not t-tubules of ventricular myocytes, similar to the reported location of adenylate cyclase 9/yotiao. Fluorescent protein-tagged KCNQ1 and KCNE1, in conjunction with antibodies targeting their extracellular epitopes, detected distinct cell surface and cytoplasmic pools of both proteins in myocytes. We conclude that, in cardiomyocytes, KCNQ1 and KCNE1 traffic by different routes to surface sarcolemma where they assemble into I channels. This mode of delayed channel assembly helps I fulfil its function of repolarization reserve. Proteomic experiments revealed a novel KCNQ1 interactor, microtubule plus-end binding protein 1 (EB1). EB1 dimer (active form) bound KCNQ1 and increased its surface level. An LQT1 mutation, Y111C, reduced KCNQ1 binding to EB1 dimer.
在成年心室肌细胞中，缓慢延迟整流（I）通道分布在表面肌质网，而不是 T 小管上。在成年心室肌细胞中，KCNQ1 和 KCNE1 具有独特的细胞表面和细胞质池。KCNQ1 和 KCNE1 通过不同的途径从内质网运输到质膜，并在细胞表面组装成 I 通道。应用于亲和纯化的 KCNQ1 和 KCNE1 相互作用蛋白的液相色谱/串联质谱揭示了参与蛋白运输和组装的新相互作用蛋白。微管末端结合蛋白 1（EB1）优先结合二聚体形式的 KCNQ1，并促进 KCNQ1 到达细胞表面。与 LQT1 相关的突变 Y111C 减少了 KCNQ1 与 EB1 二聚体的结合。
缓慢延迟整流（I）通道由 KCNQ1 和 KCNE1 组成。I 作为心脏中的“复极化储备”，通过在β-肾上腺素能刺激期间提供额外的电流来缩短心室动作电位缩短，从而发挥作用。关于 KCNQ1 和 KCNE1 在细胞中的运输方式、它们在何处结合形成 I 通道以及 I 通道相对于β-肾上腺素能信号复合物的分布模式，一直存在很多争议。我们使用了以前未应用于 KCNQ1、KCNE1 或 I 的实验策略，为这些问题提供了新的见解。“使用选定钩保留”实验表明，新翻译的 KCNE1 持续通过常规分泌途径运输到细胞表面。KCNQ1 主要停留在内质网中，尽管在亚膜区域观察到动态 KCNQ1 囊泡。二硫键结合的 KCNQ1/KCNE1 构建体在到达细胞表面后报告优先结合。原位邻近连接测定法检测到心室肌细胞表面肌质网中的 I 通道，但未检测到 T 小管中的 I 通道，这与报道的腺苷酸环化酶 9/yotiao 的位置相似。荧光蛋白标记的 KCNQ1 和 KCNE1 与靶向其细胞外表位的抗体结合，在肌细胞中检测到这两种蛋白的独特细胞表面和细胞质池。我们得出的结论是，在心肌细胞中，KCNQ1 和 KCNE1 通过不同的途径运输到表面肌质网，在那里它们组装成 I 通道。这种延迟通道组装的模式有助于 I 发挥其复极化储备的功能。蛋白质组学实验揭示了一种新的 KCNQ1 相互作用蛋白，微管末端结合蛋白 1（EB1）。EB1 二聚体（活性形式）结合 KCNQ1 并增加其表面水平。LQT1 突变 Y111C 减少了 KCNQ1 与 EB1 二聚体的结合。
