Development of a new approach to study 3D structure of the sodium channel

描述（由申请人提供）：罕见遗传性综合征（特发性室颤和长QT综合征）中的潜在致死性心律失常与去极化钠电流缺陷相关。因此，阐明心脏钠通道的分子基础和机制对于准确理解心室去极化至关重要。细菌内向整流钾通道晶体结构的确定是一个重大进展，为检验有关相关通道结构的假设提供了框架。然而，晶体结构具有运动的局限性，运动是快速和缓慢失活的主要特征，是不可感知的。因此，这项建议将强调重要的方法来结构功能分析的快速和缓慢失活的心脏钠通道，重点是了解这些门控机制在遗传性心律失常的作用。该建议的主要重点是开发一个包含两种荧光蛋白的人心脏钠通道构建体库，用于以后用荧光共振能量转移（FRET）进行分析。FRET和生物物理分析将结合起来，以表征心脏钠通道的分子机制和运动。将膜片钳技术与FRET技术相结合，我们将能够在正常和病理条件下测量钠通道不同门控状态下不同区域之间的距离。因此，本提案将测试以下假设：1。可以产生含有CFP和YFP的功能性Nav1.5构建体的文库，以使用FRET研究心脏钠通道中的运动。2.心脏钠通道的C-末端区域参与快速失活，并在此门控机制期间移动。3.外孔口的运动是通道缓慢失活的基础。4.导致长QT综合征的心脏钠离子通道突变会影响通道失活区域。该项目将产生对渠道结构的动态见解，这是迄今为止包括结晶化在内的任何其他方法都无法产生的。关于该通道结构的信息将对理解该通道中的突变如何导致心律失常有巨大帮助。

DESCRIPTION (provided by applicant): Potentially lethal arrhythmias in rare inherited syndromes (idiopathic ventricular fibrillation and Long QT syndrome) have been associated with defects in depolarizing sodium currents. Delineation of the molecular basis and mechanism of the cardiac sodium channel are therefore essential for an accurate understanding of cardiac ventricular depolarization. The determination of the crystal structure of a bacterial inwardly rectifying K channel was a major advance that provided a framework for testing hypotheses concerning the structure of related channels. Nevertheless, crystal structures have the limitation that movement, a major feature of both fast and slow inactivation, is imperceptible. Thus, this proposal will underline vital approaches to structure-function analysis of both fast and slow inactivation of the cardiac sodium channel with an emphasis on understanding the role of these gating mechanisms in inherited arrhythmias. The main focus of this proposal is to develop a library of human cardiac sodium channel constructs containing two fluorescent proteins for later analysis with Fluorescence Resonance Energy Transfer (FRET). FRET and biophysical analysis will be combined to characterize the molecular mechanism and movements of the cardiac sodium channel. By combining patch clamping with FRET, we will be able to measure distance between different regions of the sodium channel in its different gating states under normal a pathological conditions. Therefore this proposal will test the hypotheses that: 1. a library of functional Nav1.5 constructs containing both CFP and YFP can be generated to study motion in cardiac sodium channel using FRET. 2. The C- terminal region of the cardiac sodium channel is involved in fast inactivation and moves during this gating mechanism. 3. Motion in the outer pore mouth underlies slow forms of inactivation of the channel. 4. Mutations in the cardiac sodium channel that cause Long QT syndrome affect regions of the channel involved in inactivation. This project will generate dynamic insights on the structure of the channel that no other approaches, including crystallization have been able to engender thus far. This information on the structure of the channel will be of tremendous help to understand how mutations in this channel are responsible for arrhythmias.