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.

项目总结/文摘