Structural studies of NaV1.5 and functional implications

描述(申请人提供)：电压门控钠通道(NAV)在神经元和心脏启动电脉冲。分别编码神经元通道Nav1.1和NaV1.2的SCN1A和SCN2A突变会导致遗传性癫痫综合征。SCN5A编码心脏NAV通道的主要亚型(NaV1.5)，其突变与许多致心律失常有关，如长QT综合征(LQTS)和Brugada综合征(BRS)。NAV通道的主要结构成分是四个同源重复序列，它们在膜内以四聚体的形式组装，控制电压依赖性的钠传导，以及细胞质的C-末端结构域(CTD)，它是许多辅助调节蛋白的结合部位，是致病突变的热点。在这些与CTD相互作用的辅助蛋白中，普遍存在的钙传感器钙调蛋白(CaM)和成纤维细胞生长因子同源因子(FHFs；FGF11-FGF14)尤其令人感兴趣：许多致病突变定位于特定NAV通道CTD内的推测相互作用区域；FGF14本身是神经退行性疾病脊髓小脑性共济失调27的基因座；而CaM对NAV通道的钙调节知之甚少。然而，缺乏这些与CTD相互作用的结构信息，阻碍了人们对CTD的作用、这些辅助蛋白在通道调节中的作用以及导致疾病的NAV突变效应的分子基础的了解。在最近成功地确定了NAV CTD与CaM和FHF的复合体的晶体结构，以及FHFs在调节NAV通道门控和运输中的新角色的基础上，我们建议定义钙/CaM和FHFs调节NAV通道的机制，并揭示导致通道病变的NAV CTD及其相关蛋白突变的分子基础。为了实现这些目标，我们提出了以下具体目标：1.结构-功能研究，以确定钙/钙调素是如何调节NAV通道功能的，使用电生理学和生物化学方法，以探索从结构测定中获得的见解；2.结构-功能研究，以确定FHF如何调节NAV通道功能，以及FHF如何与CaM协同进行通道调节；3.生理学研究FHF调节NAV1.5的后果。由于NAV通道病和FHFs突变与神经退行性疾病、癫痫综合征和心律失常相关，该项目具有阐明NAV CTD突变引起的多器官系统多通道病的分子基础以及更广泛地揭示NAV通道结构和功能的基本方面的巨大潜力。

DESCRIPTION (provided by applicant): Voltage-gated Na channels (NaV) initiate electrical impulses in neurons and the heart. Mutations in SCN1A and SCN2A, which encode the neuronal channels NaV1.1 and NaV1.2, respectively, cause inherited epilepsy syndromes. Mutations in SCN5A, which encodes the major subtype of NaV channel in heart (NaV1.5), are linked to many cardiac arrhythmogenic disorders, such as Long QT syndrome (LQTS) and Brugada syndrome (BrS). The main structural components of NaV channels are four homologous repeats, which assemble in a tetrameric configuration within the membrane to control voltage-dependent Na+ conduction, and a cytosolic C-terminal domain (CTD) that serves as a binding site for many auxiliary regulatory proteins and is a hotspot for disease-causing mutations. Among these auxiliary CTD-interacting proteins, the ubiquitous Ca2+ sensor calmodulin (CaM) and fibroblast growth factor homologous factors (FHFs; FGF11-FGF14) are of particular interest: many disease- causing mutations are localized to their putative interaction domains within the CTDs of specific NaV channels; FGF14 is itself a locus for the neurodegenerative disorder spinocerebellar ataxia 27; and Ca2+ regulation of NaV channels by CaM is poorly understood. A lack of structural information for these interactions with the CTD, however, has prevented an understanding of the roles of the CTDs, these auxiliary proteins in channel regulation, and the molecular basis of mutational effects on NaV that lead to disease. Building on recent success in determining the crystal structure of a NaV CTD in complex with CaM and an FHF; and in defining new roles for FHFs in regulation of NaV channel gating and trafficking, we propose to define the mechanisms of NaV channel regulation by Ca2+/CaM, and FHFs and to uncover the molecular basis of the mutational effects on NaV CTDs and their associated proteins that lead to channelopathies. To achieve these goals, we propose the following specific aims: 1. Structure-function studies to determine how Ca2+/CaM regulate NaV channel function, using electrophysiology and biochemistry, to probe insights gained from structural determination; 2. Structure-function studies to determine how FHFs regulate NaV channel function and how FHFs co-operate with CaM for channel regulation; 3. Physiological investigations of the consequences of FHF regulation of NaV1.5. Because NaV channelopathies and mutations in FHFs are associated with neurodegenerative diseases, epilepsy syndromes, and cardiac arrhythmias, the proposed project has great potential to illuminate the molecular basis of multiple channelopathies in multiple organ systems due to NaV CTD mutations and, more generally, to uncover fundamental aspects of NaV channel structure-function.