Structural studies of NaV1.5 and functional implications

描述（由申请人提供）：电压门控Na通道（NaV）在神经元和心脏中启动电脉冲。 SCN1A 和 SCN2A（分别编码神经元通道 NaV1.1 和 NaV1.2）的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)，其突变与许多心律失常疾病有关，例如长 QT 综合征 (LQTS) 和布鲁格达综合征 (BrS)。 NaV 通道的主要结构成分是四个同源重复序列，它们在膜内组装成四聚体结构，以控制电压依赖性 Na 传导，以及一个胞质 C 端结构域 (CTD)，它作为许多辅助调节蛋白的结合位点，也是致病突变的热点。 在这些辅助 CTD 相互作用蛋白中，普遍存在的 Ca2 传感器钙调蛋白 (CaM) 和成纤维细胞生长因子同源因子 (FHF；FGF11-FGF14) 特别令人感兴趣：许多致病突变位于特定 NaV 通道 CTD 内的假定相互作用域； FGF14 本身是神经退行性疾病脊髓小脑共济失调 27 的基因座； CaM 对 NaV 通道的 Ca2 调节知之甚少。然而，由于缺乏这些与 CTD 相互作用的结构信息，阻碍了对 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理解。 基于最近成功确定 NaV CTD 与 CaM 和 FHF 复合物的晶体结构；在确定 FHF 在 NaV 通道门控和运输调节中的新作用时，我们建议定义 Ca2 /CaM 和 FHF 调节 NaV 通道的机制，并揭示 NaV CTD 及其相关蛋白导致通道病的突变效应的分子基础。 为了实现这些目标，我们提出以下具体目标： 1. 结构功能研究，确定 Ca2/CaM 如何调节 NaV 通道功能，利用电生理学和生物化学，探讨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配合进行通道调节； 3. FHF 调节 NaV1.5 后果的生理学调查。 由于 NaV 通道病和 FHF 突变与神经退行性疾病、癫痫综合征和心律失常相关，因此拟议的项目具有巨大的潜力，可以阐明 NaV CTD 突变导致的多个器官系统中多种通道病的分子基础，更广泛地说，揭示 NaV 通道结构功能的基本方面。

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.