Regulatory and Functional Mechanisms in hERG Ion Channels

人类etherago-go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动作复极化的电流在心脏中发挥重要作用 潜力hERG基因突变和处方脱靶作用对hERG通道的抑制 药物导致hERG电流减少，这是遗传性和获得性长QT的原因 综合征（LQTS），易患心律失常。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 优化它们在心脏和专门亚单位中的细胞作用的特性（打开和关闭） 也控制通道门控的程序集属性。hERG（也称为主要亚型，hERG 1a） 与另一种α亚基亚型hERG 1b结合。亚基缔合的机制是一个主要的 了解hERG 1a/hERG 1b通道是如何调控和门控的。的 提出的实验的目标是了解这些基础的分子机制， 以及它们如何控制同源hERG 1a和异源hERG 1a/hERG 1b通道。我们将 使用新的荧光方法检查hERG 1a和hERG 1b亚基蛋白-蛋白相互作用， 蛋白质生物化学测定。我们将测试静态N-和C-末端结构域相互作用的最新结构 在hERG 1a中，测试这些域相互作用如何控制组装并在组装过程中动态重排。 通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调查 通道构象变化和荧光显微镜研究结构相互作用如何控制 通道门控和调节。我们将利用非经典氨基酸生物学来工程化小分子 探针hERG 1a，并在最近的结构和测试的指导下，在位置引入金属结合位点， 运动与过渡金属FRET和电压。我们还将使用方法的功能工具箱， 研究hERG 1a和hERG 1b亚基的结构和功能相互作用以及hERG 1a和hERG 1b亚基的细胞作用。 人诱导多能干细胞衍生的心肌细胞中的致病突变， 由hERG 1a和hERG 1b通道亚基形成的强心脏IKr电流。 完成这些研究将有助于更好地了解 同源hERG 1a和异源hERG Ga 1a/hERG 1b通道门控，深入了解细胞内结构域如何 这些通道调节hERG 1a和hERG 1b亚基的组装，以及突变如何干扰这些亚基 交互.基于我们对机制的深刻理解，我们已经开发并将测试hERG 1a 用于拯救hERG 1a和hERG 1b LQTS突变体的编码hERG 1a功能结构域的多肽 渠道我们的研究结果预计将导致合理的生物医学战略，以抵消或加强 hERG 1a和hERG 1b亚基的功能缺失突变导致心律失常。

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.