Mechanisms for arrhythmia: Nitrosylation and mutations in the Na current complex

心律失常的机制：Na电流复合体中的亚硝基化和突变

• 批准号: 9119036
• 负责人: JONATHAN C MAKIELSKI
• 金额: $ 38.25万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9119036
• 关键词: ATP phosphohydrolase; Action Potentials; Address; Affect; Arrhythmia; Brugada syndrome; Calcium; Candidate Disease Gene; Cardiac; Cardiac Myocytes; Cell membrane; Cell surface; Cells; Clinical Treatment; Complex; Cysteine; DLG1 gene; Data; Diagnosis; Disease; Electrocardiogram; Electrophysiology (science); Family; Functional disorder; Funding; Gene Mutation; Genes; Genetic; Goals; Health; Heart; Heart Atrium; Heart Diseases; Heart failure; Homeostasis; Human; Inherited; Ion Channel; Ischemia; Lead; Long QT Syndrome; Membrane Proteins; Modeling; Molecular; Morbidity - disease rate; Muscle Cells; Mutation; Myocardial Ischemia; Nature; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Pathogenicity; Pathology; Pathway interactions; Patients; Phase; Play; Pluripotent Stem Cells; Protein Subunits; Proteins; Proteomics; Publishing; Pump; RNA Splicing; Regulation; Request for Applications; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Site; Sodium; Sodium Channel; Specificity; Sudden infant death syndrome; Syndrome; System; Techniques; Tissues; Transgenic Mice; Ubiquitination; Variant; Ventricular; Work; base; caveolin-3; clinical phenotype; clinically significant; cohort; cytochemistry; density; gain of function; gene discovery; genetic regulatory protein; heart rhythm; improved; inhibitor/antagonist; insight; loss of function; mortality; neuron loss; novel; protein complex; research study; screening; sodium channel proteins; syntrophin alpha1; ubiquitin-protein ligase

 DESCRIPTION (provided by applicant): Discovery of the genetic basis of inherited arrhythmia syndromes (IAS) has had significant impact on the diagnosis and clinical treatment of patients. As "experiments of nature" it has also contributed basic insights into the normal and abnormal function of ion channels, signaling pathways and mechanisms of arrhythmia. The cardiac Na current INa underlies excitability in heart; it is carried by the cardiac sodium channel NaV1.5, which is part of a sodium channel complex (SCC) composed of >29 Na channel interacting proteins (SCIPs). Of the established IAS genes 40% are in SCIPs that affect INa; much discovery remains to further our understanding of such IAS as long QT syndrome (LQTS), Brugada Syndrome (BrS), and Sudden Infant Death Syndrome (SIDS). This new application requests funds in part for a gene discovery project that in the past has contributed to establishing the genetic basis of ten arrhythmia syndromes. Mutations in two more SCIPs (SAP97 and Nedd4.2L), not previously known to be arrhythmia genes, will be investigated for plausible pathogenicity of BrS through INa dysfunction. Additional screening of inherited arrhythmia cohorts will continue and new discoveries investigated. Beyond discovery, this project addresses mechanisms by which mutations in SCIPs cause dysfunction of INa. This application builds on previously published work showing that enhanced direct S-Nitrosylation (SNO) of NaV1.5 underlies increased late INa for both LQT9 and LQT12 through loss of function of the SNO inhibitory actions of caveolin-3 and the alpha1-syntrophin/plasma-membrane Ca2+ pump complexes, respectively. Aim 1 will address important questions about causes of increased late INa by direct SNO of NaV1.5 cysteine residues and will include novel hypotheses about specific SNO sites on NaV1.5, roles for denitrosylation regulation of INa, and if SNO mechanisms cause late INa in eight representatives inherited and acquired disorders. Aim 2 will investigate mechanisms of SNO of the E3 ubiquitin Ligase Nedd4.2L in regulating INa density. Some LQT9 and LQT12 mutations also affected peak INa, this aim will investigate the hypothesis that Nedd4.2L is inactivated by SNO at a specific cysteine site decreasing ubiquitination of NaV1.5 and thereby increasing INa. Nedd4.2L exists in both short and long splice variants with the long variant regulated by Ca2+. Our preliminary data show both variants are present in human heart with the long form dominant. After determining the composition of Nedd4.2L variants in the SCC we will determine Ca2+ and SNO regulation of INa density. The appreciation of the role of SNO of the SCC in regulating excitability is just beginning. SNO is a general cell phenomenon and unraveling the mechanisms by which SNO of NaV1.5 and SCIPs regulate INa has particular significance for understanding basic mechanisms of specificity of cell signaling by establishing the principle of local control by co-localization of key membrane proteins and substrates. SNO regulation of INa will likely have applicability to arrhythmogenesis in acquired heart disease, such as heart failure and ischemia, where SNO signaling is known to be affected.

 描述（由申请人提供）：遗传性心律失常综合征（IAS）遗传基础的发现对患者的诊断和临床治疗产生了重大影响。作为“自然实验”，它也为了解离子通道的正常和异常功能、信号通路和心律失常的机制提供了基础知识。心脏钠电流INa是心脏兴奋性的基础;它由心脏钠通道NaV1.5携带，NaV1.5是由>29个钠通道相互作用蛋白（SCIP）组成的钠通道复合物（SCC）的一部分。在已确定的IAS基因中，40%在影响INa的SCIP中;仍有许多发现需要进一步了解长QT综合征（LQTS），Brugada综合征（BrS）和婴儿猝死综合征（SIDS）等IAS。这项新的申请要求部分资金用于基因发现项目，该项目过去曾为建立十种心律失常综合征的遗传基础做出贡献。另外两个SCIP（SAP 97和Nedd4.2L）中的突变，以前不知道是心律失常基因，将通过INa功能障碍研究BrS的合理致病性。将继续对遗传性心律失常队列进行额外筛查，并研究新发现。除了发现，该项目还解决了SCIP突变导致INa功能障碍的机制。本申请建立在先前发表的工作的基础上，该工作表明NaV1.5的直接S-亚硝基化（SNO）增强是LQT 9和LQT 12的晚期INa增加的基础，这分别是通过小窝蛋白-3和α 1-促突触蛋白/质膜Ca 2+泵复合物的SNO抑制作用的功能丧失。目的1将解决有关NaV1.5半胱氨酸残基的直接SNO导致晚期INa增加的原因的重要问题，并将包括关于NaV1.5上的特定SNO位点的新假设，INa的脱亚硝基化调节的作用，以及SNO机制是否导致8种代表性遗传性和获得性疾病的晚期INa。目的2探讨E3泛素连接酶Nedd4.2L的SNO调控INa浓度的机制。一些LQT 9和LQT 12突变也影响INa峰，这一目的将研究Nedd4.2L在特定的半胱氨酸位点被SNO灭活，从而降低NaV1.5的泛素化，从而增加INa的假设。Nedd4.2L存在于短剪接体和长剪接体中，长剪接体受Ca 2+调控。我们的初步数据显示，这两种变体都存在于人类心脏中，长型占主导地位。在确定SCC中Nedd4.2L变体的组成后，我们将确定Ca 2+和SNO对INa密度的调节。对SCC的SNO在调节兴奋性中的作用的认识才刚刚开始。SNO是一种普遍的细胞现象，阐明NaV1.5和SCIPs的SNO调节INa的机制对于通过建立关键膜蛋白和底物的共定位的局部控制原理来理解细胞信号传导特异性的基本机制具有特别重要的意义。SNO对INa的调节可能适用于获得性心脏病如心力衰竭和缺血中的血管生成，其中已知SNO信号传导受到影响。