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

基本信息

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
项目类别：
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

项目摘要

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心脏中最重要的通道之一是 IKr (Kv11.1) 电压门控。钾通道，由人类 ether-à-go-go 相关基因 (hERG) 编码，控制心室这些通道是由 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，异聚体通道的组装和功能异常。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，我已经使用异聚体组装测试了 1a 和 1b mRNA 直接相互作用的假设。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，我发现它们在该提案的重点是 mRNA 结合蛋白可能含有 1a 和 1b 的假设。这一假设得到了相关 RNA 结合蛋白 (RBP) 初步数据的支持。通过液相色谱串联质谱法（LC- MS/MS）。这些研究将得到扩展，目标 RBP 将在活细胞中得到验证。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，看看是否可以重构这些复合物的结果。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，并通过我还将学习使用人类细胞培养物以及收集和分析。在本次奖学金培训期间，我将积极使用 smFISH 和 IF 进行 mRNA-RBP 共定位数据。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，以及我的培训的目标是成为一名灵活、协作且受人尊敬的科学家。