Uncovering the Mechanism of Heteromeric hERG Channel Biosynthesis Through mRNA Association

通过 mRNA 关联揭示异聚 hERG 通道生物合成机制

• 批准号: 10066401
• 负责人: Jennifer Knickelbine
• 金额: $ 6.74万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10066401
• 关键词: Action Potentials; Affect; Amino Acids; Anabolism; Antibodies; Arrhythmia; Binding; Binding Proteins; Biogenesis; Cardiac; Cardiac Myocytes; Cardiovascular system; Cell Culture Techniques; Cells; Chemicals; Co-Immunoprecipitations; Collaborations; Complex; Data; Digestion; Disease; Dot Immunoblotting; Electrophoretic Mobility Shift Assay; Equilibrium; Ethers; Fellowship; Fluorescent in Situ Hybridization; Future; Genes; Goals; Heart; Human; Immunofluorescence Immunologic; Immunoprecipitation; Impairment; In Vitro; Industry; Inosine Diphosphate; Ion Channel; Journals; Label; Laboratories; Lead; Learning; Link; Liquid Chromatography; Long QT Syndrome; Medical Research; Mentors; Messenger RNA; Myocardium; Neurosciences; Patients; Pharmacy Schools; Post-Translational Protein Processing; Property; Proteins; Proteomics; Protocols documentation; RNA; RNA-Binding Proteins; Research; Safety; Science; Scientist; System; Testing; Training; Transcript; Translations; Trypsin; Update; Ventricular; Voltage-Gated Potassium Channel; Woman; Work; base; career; career networking; cohesion; experimental study; flexibility; genome wide association study; induced pluripotent stem cell; insight; knock-down; meetings; new therapeutic target; novel; novel therapeutic intervention; protein protein interaction; reconstitution; single molecule; small hairpin RNA; sudden cardiac death; tandem mass spectrometry

PROJECT SUMMARY/ABSTRACT The expression of cardiac ion channels must be precisely controlled to produce the regular beating of the heart and protect from arrhythmia. One of the most important channels in the heart is the IKr (Kv11.1) voltage-gated potassium channel, encoded by the human ether-à-go-go-related gene (hERG), which controls ventricular repolarization. These channels are tetramers composed of 2 types of subunits, termed 1a and 1b, and heteromeric channels conduct much more current than homomers. Thus, aberrant assembly and function of the channel can give rise to impaired repolarization, Long QT syndrome, and sudden cardiac death. My project aims to determine the mechanism by which hERG mRNAs associate to facilitate co-translational, heteromeric assembly. I have already tested the hypothesis that 1a and 1b mRNAs directly interact using an electrophoretic mobility shift assay, a dot blot to screen for binding of 1a and 1b fluorescently labelled probes, and mixing cell lysates from separately transfected HEK cells, and I found that they do not associate under these conditions. This proposal focuses on the hypothesis that mRNA binding proteins may hold 1a and 1b together. This hypothesis is supported by preliminary data identifying RNA binding proteins (RBPs) associated with the mRNAs in complexes as determined by liquid chromatography tandem mass spectrometry (LC- MS/MS). These studies will be extended and the target RBPs will be validated in living cells using immunofluorescence (IF) and single molecule fluorescence in situ hybridization (smFISH), shRNA knockdown of candidate RBPs to see if the 1a/1b mRNA complex is disrupted, and by adding the purified RBP into an in vitro translation system with 1a and 1b to see if the complex can be reconstituted. The results of these experiments will establish a mechanism by which 1a and 1b mRNAs come together to produce heteromeric hERG channels and could lead to new therapeutic approaches for patients with cardiac arrhythmias. This fellowship training plan is intended to prepare me for a career as an independent scientist in medical research, and will include weekly mentoring sessions with Dr. Gail Robertson to discuss progress toward IDP goals, participation in weekly lab meetings, regular participation and presentation in 3 journal clubs, serving as the lab's Chemical Safety Officer and updating our biosafety and chemical safety protocols, managing a collaboration with Dr. Lingjun Li's lab in the School of Pharmacy, and participation in industry tours through the Graduate Women in Science. I will also be learning to work with human cell cultures and to collect and analyze mRNA-RBP co-localization data using smFISH and IF. During this fellowship training period I will actively broaden my professional network in translational cardiovascular research, neuroscience, and industry, and my goal is to come out of this training as a flexible, collaborative, and respected scientist.

项目概要/摘要 必须精确控制心脏离子通道的表达才能产生心脏的规律跳动 并防止心律失常。心脏中最重要的通道之一是 IKr (Kv11.1) 电压门控通道 钾通道，由人类 ether-à-go-go 相关基因 (hERG) 编码，控制心室 复极化。这些通道是由 2 种亚基（称为 1a 和 1b）组成的四聚体， 异聚体通道比同聚体通道传导更多的电流。因此，异常的组装和功能 该通道可导致复极受损、长 QT 综合征和心源性猝死。 我的项目旨在确定 hERG mRNA 关联以促进共翻译的机制， 异聚组装。我已经测试了 1a 和 1b mRNA 使用 电泳迁移率变动测定，一种用于筛选 1a 和 1b 荧光标记探针结合的点印迹， 和混合来自单独转染的 HEK 细胞的细胞裂解物，我发现它们在 这些条件。该提案重点关注 mRNA 结合蛋白可能持有 1a 和 1b 的假设 一起。这一假设得到了初步数据的支持，该数据确定了与RNA结合蛋白（RBP）相关的RNA结合蛋白（RBP）。 通过液相色谱串联质谱法（LC- 质谱/质谱）。这些研究将得到扩展，并且目标 RBP 将在活细胞中使用进行验证 免疫荧光 (IF) 和单分子荧光原位杂交 (smFISH)、shRNA 敲低 候选 RBP 以确定 1a/1b mRNA 复合物是否被破坏，并将纯化的 RBP 添加到 in 用 1a 和 1b 进行体外翻译系统，看看复合物是否可以重构。这些结果 实验将建立一种机制，通过该机制 1a 和 1b mRNA 结合在一起产生异聚体 hERG 通道可能会为心律失常患者带来新的治疗方法。 该奖学金培训计划旨在帮助我为医学领域独立科学家的职业生涯做好准备 研究，并将包括每周与盖尔·罗伯逊博士进行指导会议，讨论 IDP 的进展 目标、参加每周实验室会议、定期参加 3 个期刊俱乐部并进行演讲，担任 实验室的化学安全官员并更新我们的生物安全和化学安全协议，管理 与药学院李令军博士实验室合作，并通过 科学领域的女研究生。我还将学习使用人类细胞培养物并收集和分析 使用 smFISH 和 IF 的 mRNA-RBP 共定位数据。在这次联谊培训期间我会积极 拓宽我在转化心血管研究、神经科学和工业方面的专业网络，以及我的 目标是通过培训成为一名灵活、协作且受人尊敬的科学家。