Biogenesis of hERG1a/1b ion channels in health and disease model cardiomyocytes

项目概要/摘要 心脏IKr是一种重要的复极化钾电流，塑造了人类心室动作电位。是 通过人类ether-à-go-go相关基因（hERG 1）1a和1b亚基的异聚体组装进行。 这些亚基由hERG/KCNH 2基因的交替转录物编码，仅在其氨基上不同。 终端区域。hERG 1a/1b异聚化对正常CM功能至关重要，因为亚基的失衡， 表达和/或功能导致细胞促凋亡行为。hERG 1a/1b组装是由 HEK 293细胞、来源于人的心肌细胞、来源于人的心肌细胞和来源于人的心肌细胞中编码mRNA的共翻译关联 诱导多能干细胞（hiPSC-CM）和人心肌。有证据表明， 新生蛋白质之间的相互作用对于共翻译复合物组装不是必需的。这个格兰特 初步的研究结果表明，这种复杂的组装发生在转录后，并由直接 hERG 1a和1b mRNA之间的相互作用由其二级结构控制。在初步研究中， RNA结合蛋白DDX 3X和DDX 5被鉴定为复合物的一部分，并且纯化的DDX 3X促进了RNA结合蛋白DDX 3X和DDX 5的表达。 hERG 1a/1b mRNA的体外结合。在K99阶段，我将定义mRNA的结构特征 促进共翻译结合并决定RNA/RNA相互作用的亲和力和能量 使用体外系统、等温量热法（ITC）、诱变、杂合蛋白-RNA免疫沉淀 (RIP)和活细胞成像。我还将确定DDX 3X和DDX 5是否影响hERG 1a和1b mRNA 使用qPCR、电生理学、蛋白质印迹、核糖体在hiPSC-CM中的稳定性、翻译和缔合 分析、RIP和单分子荧光原位杂交（smFISH）。我将使用定量ITC， 体外重建方法用于确定之间相互作用的特异性、亲和力和能量 用hERG 1a和1b mRNA纯化DDX 3X和DDX 5。我还将评估DDX 3X和DDX 5是否能促进 在体外系统中的mRNA的关联。在R 00阶段，我将确定是否稳定， hERG 1a和1b mRNA的翻译和结合在2型长时相心律失常中受损 QT综合征（LQT 2）。我将使用hiPSC-CM疾病模型来评估半衰期、转化率和 mRNA与qPCR、核糖体分析、RIP和smFISH的关联。这些实验将有助于 了解离子通道的生物起源和阐明LQT 2相关的分子机制 心律不齐这个建议是为了实现我的短期目标，扩大我的技能，在心血管 研究和生物物理学，并过渡到我职业生涯的独立阶段。这将最终使 我实现了我的长期目标，将RNA和离子通道生物物理学与转译心血管疾病联系起来， research.

PROJECT SUMMARY/ABSTRACT Cardiac IKr is a critical repolarizing potassium current shaping the human ventricular action potential. It is conducted by heteromeric assemblies of the human ether-à-go-go-related gene (hERG1) 1a and 1b subunits. These subunits are encoded by alternate transcripts of the hERG/KCNH2 gene and differ only in their amino- terminal regions. hERG1a/1b heteromerization is vital for normal CM function, as the imbalance of subunit expression and/or function results in cellular pro-arrhythmic behaviors. hERG1a/1b assembly is mediated by the co-translational association of the encoding mRNAs in HEK293 cells, cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), and human myocardium. Evidence suggests that interaction between the nascent proteins is not required for the co-translational complex assembly. This grant's preliminary findings indicate that this complex assembly occurs post-transcriptionally and is promoted by direct interactions between hERG1a and 1b mRNAs governed by their secondary structures. In preliminary studies, RNA binding proteins DDX3X and DDX5 were identified as part of the complex, and purified DDX3X promoted hERG1a/1b mRNAs' association in vitro. In the K99 phase, I will define the mRNA structural features promoting the co-translational association and determine the affinity and energies of the RNA/RNA interaction using in vitro systems, isothermal calorimetry (ITC), mutagenesis, hybrid protein-RNA immunoprecipitation (RIP), and live-cell imaging. I will also determine whether DDX3X and DDX5 affect hERG1a and 1b mRNAs stability, translation, and association in hiPSC-CMs using qPCR, electrophysiology, Western Blot, ribosome profiling, RIP, and single molecule fluorescent in situ hybridization (smFISH). I will use quantitative ITC and in vitro reconstitution approaches to determine the specificity, affinity, and energies of the interaction between purified DDX3X and DDX5 with hERG1a and 1b mRNAs. I will also evaluate if DDX3X and DDX5 promote the association of the mRNAs in in vitro systems. In the R00 phase, I will determine whether the stability, translation, and association of hERG1a and 1b mRNAs are impaired in arrhythmias associated with type 2 long QT syndrome (LQT2). I will use hiPSC-CM disease models to evaluate half-life, translation rate, and association of the mRNAs with qPCR, ribosome profiling, RIP, and smFISH. These experiments will contribute to understanding ion channel biogenesis and elucidate molecular mechanisms underlying LQT2 related arrhythmias. This proposal is designed to fulfill my short-term goals of expanding my skills in cardiovascular research and biophysics and transitioning into the independent phase of my career. This will ultimately allow me to obtain my long-term purpose of linking RNA and ion channel biophysics to translational cardiovascular research.