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

描述(申请人提供)：罕见遗传综合征(特发性室颤和长QT综合征)的潜在致命性心律失常与去极化钠电流缺陷有关。因此，描述心脏钠通道的分子基础和机制对于准确理解心脏去极化是至关重要的。细菌内向整流钾通道晶体结构的确定是一项重大进展，它为检验有关通道结构的假说提供了一个框架。然而，晶体结构有一个局限性，即运动是快速和缓慢失活的一个主要特征，是不可察觉的。因此，这项提议将强调对心脏钠通道快速和缓慢失活的结构-功能分析的重要方法，并强调了解这些门控机制在遗传性心律失常中的作用。这项建议的主要重点是开发一个包含两个荧光蛋白的人心脏钠通道构建库，以便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描述心脏钠通道的分子机制和运动。通过结合膜片钳和FRET，我们将能够在正常和病理条件下测量不同门控状态下钠通道不同区域之间的距离。因此，这项提议将验证以下假设：1.可以构建一个同时包含CFP和YFP的功能性NaV1.5构建库，用于使用FRET研究心脏钠通道的运动。2.在这一门控机制中，心脏钠通道的C-末端区域参与了快速失活和移动。3.外孔口中的运动是通道缓慢失活的基础。4.导致长QT综合征的心脏钠通道突变会影响与失活有关的通道区域。这个项目将产生对渠道结构的动态洞察，这是迄今为止包括结晶在内的任何其他方法都无法产生的。这些关于通道结构的信息将极大地帮助理解该通道的突变是如何导致心律失常的。

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.