Elucidating the Mechanisms Involved in (DYS)regulation of Sodium Channels

描述（由申请人提供）：本研究的目的是了解电压门控钠通道（VGSC）失活的机制以及这种调节功能障碍如何导致疾病。这种失活形成可兴奋细胞中动作电位起始的关键部分。VGSC在组织（包括心肌细胞、神经、骨骼肌）中无处不在的性质以及与其调节失调相关的疾病（包括心律失常、癫痫发作和肌强直）很容易强调VGSC的重要性。本文提出的实验集中在理解细胞外Ca 2+如何通过与通道C-末端结构域（CTD）相互作用来促进两种VGSC亚型的调节。据推测，Ca2+调节通道失活的亚型特异性的方式，可能通过参与的辅助蛋白，如钙调蛋白（CaM），或通过间接的方法，涉及Ca2 +/钙调蛋白激酶II（Ca2 +/CaMKII），和突变，破坏通道门控改变构象动力学与监管。提出的具体目标是：（1）确定负责VGSC调节的Ca 2+诱导的无序到有序转变的机制;（2）确定VGSC调节是否可以通过间接机制发生，例如Ca 2 +/CaMKII与CTD结合;（3）并确定已知导致VGSC失调的CTD突变的分子基础。为了验证这些假设，提出了一系列的NMR实验，包括自旋弛豫，化学位移扰动，和结构测定。NMR非常适合解决这些实验问题，因为它提供了有关系统构象和动力学变化的原子分辨率信息。公共卫生相关性：电压门控钠通道的适当调节对于可兴奋细胞（例如心肌细胞、神经和骨骼肌）的功能至关重要。拟议的研究与理解调节发生的机制以及这种调节的故障如何导致与这些通道相关的许多疾病直接相关。

DESCRIPTION (provided by applicant): The goal of this research is to understand the mechanism by which voltage gated sodium channels (VGSCs) are inactivated and how the malfunction of this regulation leads to disease. This inactivation forms a critical part of action potential initiation in excitable cells. The importance of VGSCs is readily underscored by their ubiquitous nature in tissues, including cardiac myocytes, nerves, skeletal muscle, and by diseases associated with their dysregulation, including cardiac arrhythmias, epileptic seizures, and myotonia. The experiments proposed herein are focused on understanding how extracellular Ca2+ contributes to the regulation of two VGSC isoforms by interacting with the channel C-terminal domain (CTD). It is hypothesized that Ca2+ regulates channel inactivation in an isoform specific manner, possibly via the involvement of accessory proteins, such as calmodulin (CaM), or by indirect methods, involving Ca2 +/calmodulin kinase II (Ca2+/CaMKII), and that mutations which disrupt channel gating alter the conformational dynamics associated with regulation. The specific aims proposed are: (1) to ascertain the mechanism of Ca2+-induced disordered-to-ordered transitions responsible for the regulation of VGSCs; (2) to determine if VGSC regulation can occur by an indirect mechanism, such as Ca2+/CaMKII binding to the CTD; (3) and to determine the molecular basis of CTD mutations known to cause VGSC dysregulation. To test these hypotheses, a series of NMR experiments, including spin-relaxation, chemical shift perturbation, and structure determination, are proposed. NMR is well-suited to address these experimental questions as it provides atomic resolution information about changes in the conformation and dynamics of a system. PUBLIC HEALTH RELEVANCE: The proper regulation of voltage gated sodium channels is critical to the function of excitable cells, such as cardiac myocytes, nerves, and skeletal muscle. The proposed research is directly relevant to understanding the mechanism by which regulation occurs and how the malfunction of this regulation results in a host of diseases associated with these channels.