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

 描述(申请人提供)：遗传性心律失常综合征(IAS)的遗传学基础的发现对患者的诊断和临床治疗产生了重大影响。作为自然界的实验，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本的见解。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，它是由&gt；29钠通道相互作用蛋白(SCIP)组成的钠通道复合体(SCC)的一部分。在已确定的IAS基因中，40%存在于影响INA的SCIP中；许多发现有待于进一步加深我们对此类IAS的理解，如长QT综合征(LQTS)、Brugada综合征(BRS)和婴儿猝死综合征(SID)。这一新的申请要求部分资金用于一个基因发现项目，该项目在过去曾为建立10种心律失常综合征的遗传学基础做出了贡献。另外两个SCIP基因(SAP97和Nedd4.2L)的突变，以前不是已知的心律失常基因，将通过INA功能障碍来研究BRS的可能致病性。将继续对遗传性心律失常队列进行额外的筛查，并调查新的发现。除了发现之外，该项目还解决了SCIP突变导致INA功能障碍的机制。这一应用建立在先前发表的工作的基础上，该工作表明，NaV1.5的增强的直接S-亚硝化(SNO)分别通过丧失小窝蛋白-3和α1-合营养素/质膜钙泵复合体的SNO抑制作用，导致LQT9和LQT12的晚期INA增加。目的1将探讨NaV1.5半胱氨酸残基的直接SNO导致晚期INA增加的重要原因，并将包括关于NaV1.5上特定SNO位点的新假说，INA的反硝化调节作用，以及SNO机制是否导致8种典型的遗传性和获得性疾病的晚期INA。目的2研究E3泛素连接酶Nedd4.2L的SNO调节异丙肾上腺素浓度的机制。一些LQT9和LQT12突变也影响Ina峰，这一目的将探讨Nedd4.2L在特定半胱氨酸位点被SNO失活从而降低NaV1.5泛素化从而增加Ina的假设。Nedd4.2L在短剪接变异体和长剪接变异体中都存在，长剪接变异体受钙离子调控。我们的初步数据显示，这两种变体都存在于人类心脏中，以长型为主。在确定了SCC中Nedd4.2L变异体的组成后，我们将确定Ca~(2+)和SNO对Ina浓度的调节。对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，揭示NaV1.5和SCIPs的SNO调节INA的机制，通过建立关键膜蛋白和底物共定位的局部调控原理，对于理解细胞信号特异性的基本机制具有特殊的意义。SNO对INA的调节可能适用于获得性心脏病的心律失常发生，如心力衰竭和缺血，已知SNO信号会受到影响。

 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.