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

描述(申请人提供)：这项研究的目标是了解电压门控钠通道(VGSCs)失活的机制，以及这一调节功能的故障如何导致疾病。这种失活形成了可兴奋细胞动作电位启动的关键部分。VGSCs广泛存在于组织中，包括心肌细胞、神经、骨骼肌，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，其重要性很容易得到强调。本文提出的实验重点在于了解细胞外钙离子如何通过与通道C末端结构域(CTD)相互作用来调节VGSC的两种异构体。有人假设，钙离子以一种异构体特异性的方式调节通道失活，可能是通过辅助蛋白，如钙调素(CaM)的参与，或通过间接方法，涉及钙/钙调蛋白激酶II(Ca2+/CaMKII)，而扰乱通道门控的突变改变了与调节相关的构象动力学。提出的具体目标是：(1)确定导致VGSC调控的钙离子诱导的无序到有序转变的机制；(2)确定VGSC是否可以通过间接机制发生调控，如钙/CaMKII与CTD结合；(3)确定已知的导致VGSC调控失调的CTD突变的分子基础。为了验证这些假设，提出了一系列的核磁共振实验，包括自旋松弛、化学位移微扰和结构确定。核磁共振非常适合于解决这些实验问题，因为它提供了关于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可兴奋细胞的功能至关重要，如心肌细胞、神经和骨骼肌。这项拟议的研究直接关系到了解调控发生的机制，以及这一调控的故障如何导致与这些渠道相关的一系列疾病。

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.