RNF207: A Potential Modifier of HERG in Long QT Syndrome

RNF207：HERG 在长 QT 综合征中的潜在修饰剂

• 批准号: 9257218
• 负责人: Hannah Aizad Ledford
• 金额: $ 3.6万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9257218
• 关键词: Action Potentials; Affect; Arrhythmia; Biochemistry; Biological Assay; Calmodulin; Cardiac; Cardiac Myocytes; Cells; Chromosomes, Human, Pair 1; Co-Immunoprecipitations; Coiled-Coil Domain; Defect; Degradation Pathway; Disease; Dominant-Negative Mutation; Drug Targeting; Electrophysiology (science); Ethers; Female; Genes; Health; Human; Immunofluorescence Immunologic; Inborn Genetic Diseases; Individual; Infant; Inherited; Ion Channel; Kinetics; Knowledge; Lead; Life; Long QT Syndrome; Methods; Microscopy; Missense Mutation; Molecular; Multiprotein Complexes; Mutation; Nature; Parents; Patch-Clamp Techniques; Pathogenicity; Pathway interactions; Patients; Perinatal; Pharmaceutical Preparations; Potassium Channel; Proteins; Recurrence; Research; Resolution; Role; Severities; Techniques; Terminator Codon; Testing; Time; Torsades de Pointes; Variant; Ventricular; Ventricular Arrhythmia; Ventricular Fibrillation; Ventricular Tachycardia; Zinc Fingers; density; design; experimental study; genome sequencing; induced pluripotent stem cell; innovation; insight; interest; microscopic imaging; multidisciplinary; mutant; new therapeutic target; novel; protein protein interaction; sudden cardiac death; trafficking; ubiquitin-protein ligase; variant of unknown significance; voltage; whole genome

PROJECT SUMMARY/ ABSTRACT Background: Long QT Syndrome Type 2 (LQT2) is characterized by mutations in the cardiac K+ channel known as hERG (human ether-a-go-go related gene) channel or Kv11.1, encoded by the KCNH2 gene. Mutations in the hERG channel result in a decrease in an important cardiac, repolarizing current: the rapidly activating delayed rectifier K+ current (IKr). The fundamental defect in LQTS is prolonged ventricular repolarization, caused by the imbalance of inward and outward currents, and an increase in occurrences of life-threatening arrhythmias— such as torsades de pointes polymorphic ventricular tachycardia, which can lead to ventricular fibrillation and sudden cardiac death. LQTS manifests not only from mutations in cardiac ion channels (as in LQT1, LQT2, and LQT3), but also mutations in ion channel interacting proteins (such as calmodulin in LQT14-16). A recent study revealed RNF207 as a potential modifier of the hERG channel. The hypothesis is that RNF207 alters cardiac repolarization through its interaction with the hERG K+ channel. Methods and Objectives: This interaction will be examined by looking at wild-type and mutant variants of hERG and RNF207. Mutant variants of interest will be hERG T613M and RNF207 G603fs. Feasibility for this project has been verified by positive co-localization near cardiac t-tubules seen in preliminary immunofluorescent experiments and multi- protein complex formation in co-immunoprecipitation experiments. The extent and nature of this interaction will be determined using a combination of biochemistry and electrophysiology techniques. Aim 1 will verify the protein-protein interaction using immunofluorescence (visualized with both confocal and standard emission depletion microscopy), co-immunoprecipitation, and GST pull-down assays. Aim 2 will analyze the functional interaction between hERG and RNF207 in human induced pluripotent stem cell- derived cardiomyocytes (hiPSC- CMs) and HEK 293 cells using whole-cell patch-clamp techniques. Conclusion: Our study will further reveal the cellular and molecular effects of RNF207 on hERG channels and ventricular repolarization. Furthermore, we will test the mechanisms for the underlying hypothesis that polygenic variability influences LQTS severity. The value of the proposed research lies in its ability to provide insights into novel mechanisms for LQTS and elucidate possible targets for new therapeutic treatments.

项目总结/摘要 背景资料： 长QT综合征2型（LQT 2）的特征是心脏K+通道突变， hERG（人类ether-a-go-go相关基因）通道或Kv11.1，由KCNH 2基因编码。突变 hERG通道导致重要的心脏复极化电流减少：快速激活的延迟电流 整流器K+电流（IKr）。LQTS的基本缺陷是心室复极延长，由心室肌的收缩和舒张功能引起。 内向电流和外向电流的不平衡，以及危及生命的心律失常的发生率增加- 例如尖端扭转型多形室性心动过速，其可导致心室纤维性颤动， 心源性猝死LQTS不仅表现为心脏离子通道的突变（如LQT 1，LQT 2， 和LQT 3），而且还包括离子通道相互作用蛋白（例如LQT 14 -16中的钙调蛋白）中的突变。最近的一 研究揭示RNF 207作为hERG通道的潜在修饰剂。假设RNF 207改变了 通过与hERG K+通道的相互作用实现心脏复极化。 方法和目的： 将通过观察hERG和RNF 207的野生型和突变变体来检查这种相互作用。突变体 感兴趣的变体是hERG T613 M和RNF 207 G603 fs。该项目的可行性已通过 在初步的免疫荧光实验和多重免疫荧光实验中， 免疫共沉淀实验中的蛋白质复合物形成。这种相互作用的程度和性质将 使用生物化学和电生理学技术的组合来确定。目标1将验证 使用免疫荧光的蛋白质-蛋白质相互作用（用共聚焦和标准发射可视化 耗尽显微术）、免疫共沉淀和GST下拉测定。目标2将分析功能 hERG与RNF 207在人诱导多能干细胞心肌细胞中的相互作用 使用全细胞膜片钳技术对hiPSC-CM细胞和HEK 293细胞进行了细胞周期分析。 结论： 我们的研究将进一步揭示RNF 207对hERG通道的细胞和分子作用， 心室复极此外，我们将测试潜在假设的机制， 多基因变异影响LQTS的严重程度。拟议研究的价值在于它能够提供 深入了解LQTS的新机制，并阐明新治疗方法的可能靶点。