Regulatory and Functional Mechanisms in hERG Ion Channels

人类乙醚-围棋相关基因(HERG)钾通道具有非常重要的临床意义。 HERG通道通过产生使心脏活动重新极化的电流，在心脏中扮演着重要的角色 潜力。HERG基因突变与方剂非靶向作用对HERG通道的抑制作用 药物导致遗传性和获得性长QT的Herg电流减少 综合征(LQTS)，一种易发生心律失常的症状。HERG的疾病相关性强调了 这些通道在正常生理功能中的重要性。HERG通道具有高度专业化的门控 属性(打开和关闭)，优化它们在心脏和专化亚单位中的细胞角色 也控制通道选通的程序集属性。Herg(也称为初级异构体，hERG1a) 与另一种‘α’亚基异构体hERG1b相关联。亚基联合的机制是一种主要的 感兴趣的领域，了解异构体hERG1a/hERG1b通道是如何调节和门控的。这个 拟议中的实验的目标是了解这些现象背后的分子机制。 专门化及其如何控制同聚体hERG1a和异构体hERG1a/hERG1b通道。我们会 用新的荧光方法和方法检测hERG1a和hERG1b亚单位蛋白质-蛋白质相互作用 蛋白质生化分析。我们将测试静态N-末端和C-末端结构域相互作用的最新结构 在hERG1a中，并测试这些域交互如何控制组装和在 频道选通。我们的方法是尖端的，因为我们将使用电生理记录来研究 通道构象变化和荧光显微镜研究结构相互作用如何控制 通道选通和调节。我们将利用非规范的氨基酸生物学来设计小的 HERG1a的探针和在最近结构引导的位置上引入金属结合位点的探针和测试 带有过渡金属振荡器和电压的机芯。我们还将使用一个功能强大的工具箱来 研究hERG1a和hERG1b亚基的结构和功能相互作用以及HERG1a和hERG1b的细胞作用 人类诱导的多能干细胞来源的心肌细胞中的致病突变，具有 由hERG1a和hERG1b通道亚基形成的强健的心脏IKR电流。 这些研究的完成将使我们更好地了解 同源hERG1a和异构体hERGa1a/hERG1b通道门控，深入了解细胞内结构域 调节hERG1a和hERG1b亚单位的组装以及突变如何扰乱这些 互动。基于我们对机制的深刻理解，我们已经开发并将测试hERG1a 编码hERG1a功能区的多肽用于挽救hERG1a和hERG1b LQTS突变体 频道。我们的结果有望导致合理的生物医学策略，以抵消或增强 导致心律失常的hERG1a和hERG1b亚基功能丧失突变。

Human ether á go-go related gene (hERG) potassium channels are of extraordinary clinical importance. hERG channels play a prominent role in the heart by generating a current that repolarizes cardiac action potentials. Mutations in the hERG gene and inhibition of hERG channels by the off-target action of prescription drugs cause a reduction in hERG current that accounts for both inherited and acquired forms of long QT syndrome (LQTS), a predisposition to cardiac arrhythmias. The disease relevance of hERG emphasizes the importance of these channels in normal physiological function. hERG channels have highly specialized gating properties (opening and closing) that optimize them for their cellular roles in the heart and specialized subunit assembly properties that also control channel gating. hERG (also known as the primary isoform, hERG1a) associates with another `alpha' subunit isoform, hERG1b. The mechanisms of subunit association are a major area of interest for understanding how heteromeric hERG1a/hERG1b channels are regulated and gated. The goal of the proposed experiments is to understand the molecular mechanisms that underlie these specializations and how they control homomeric hERG1a and heteromeric hERG1a/hERG1b channels. We will examine hERG1a and hERG1b subunit protein-protein interactions using novel fluorescence methods and protein biochemistry assays. We will test recent structures of static N- and C-terminal domain interactions within hERG1a and test how these domain interactions control assembly and dynamically rearrange during channel gating. Our approach is cutting-edge as we will use electrophysiological recordings to investigate channel conformational changes and fluorescence microscopy to study how structural interactions control channel gating and regulation. We will take advantage of non-canonical amino acid biology to engineer small probes to hERG1a and introduce metal binding sites at locations guided by recent structures and test for movements with transition metal FRET and voltage. We will also use a functional toolbox of approaches to examine the structural and functional interactions of hERG1a and hERG1b subunits and the cellular role of disease-causing mutations in human induced pluripotent stem cell-derived cardiomyocytes, which have a robust cardiac IKr current formed by hERG1a and hERG1b channel subunits. Completion of these studies will lead to a greater understanding of the basic mechanisms for homomeric hERG1a and heteromeric hERGa1a/hERG1b channel gating, insight into how intracellular domains of the channels regulate the assembly of hERG1a and hERG1b subunits and how mutations perturb these interactions. Based on our deep understanding of mechanism, we have developed and will test hERG1a polypeptides that encode hERG1a functional domains for rescue of hERG1a and hERG1b LQTS mutant channels. Our outcomes are anticipated to lead to rational biomedical strategies to counteract or enhance the loss-of-function mutations in hERG1a and hERG1b subunits that cause arrhythmias.