Tertiary Structures of Potassium Channel Domains

描述(由申请人提供)：这是一个竞争性的继续提案，以支持该项目的三个新目标，名为“钾通道结构域的三级结构”。这项更新建议通过关注6个跨膜(TM)-螺旋电压门控K通道的分子工作，将范围和方向扩展到一个新的水平。在第一个目标中，我们建议确定细菌6 TM K通道KvPae与标志导向的Fab复合体。这项工作将解决电压介导的通道激活的结构基础及其机制，以及通道中赋予的构象灵活性。在第二个目标中，我们建议完成真核细胞Kv通道的整个N-末端结构域aKv1.1的结构，并结合功能表征用核磁共振波谱分析构象变化。这项工作将阐述通道失活机制的结构基础，该机制通过蛋白质-蛋白质相互作用的自身失活亚域来实现。在第三个目标中，我们建议研究钙结合的细胞质KChIP蛋白与真核细胞Kv通道的完整N-末端结构域Kv4.2的复合体中发生的结构变化。钙诱导的KChIP/通道复合体的构象变化将用X射线结晶学和核磁共振波谱方法相结合的方法来探索。这项研究将扩大我们对真核细胞钾通道不同调控机制的分子理解。我们将学习许多关于电压激活(目标1)、其亚区介导的失活(目标2)和钙介导的调节(目标3)的结构机制。

DESCRIPTION (provided by applicant): This is a competitive continuation proposal to support three new aims of the project entitled "Tertiary structures of potassium channel domains." This renewal proposal expands the scope and direction to a newer level by focusing on molecular working of 6 transmembrane (TM)-helix voltage-gated K channels. In the first aim, we propose to determine a bacterial 6 TM K channel, KvPae in complex with an FLAG-directed Fab complex. This work will address the structural basis of voltage-mediated channel activation and its mechanism, and conformational flexibility endowed in the channel. In the second aim, we propose to complete the structure of the entire N-terminal domain of eukaryotic Kv channel, aKv1.1, and analyze the conformational change by NMR spectroscopy combined with functional characterization. This work will address the structural basis of the mechanism of channel inactivation by its own inactivation subdomain by protein-protein interaction. In the third aim, we propose to study structural changes occurring in the complex of calcium-binding cytoplasmic KChIP protein in complex with the "entire" N-terminal domain of eukaryotic Kv channel, Kv4.2. Calcium-induced conformational change in KChIP/channel complex will be probed by a combination of x-ray crystallography and NMR spectroscopy methods. This study will expand our molecular understanding of diverse regulatory mechanisms of eukaryotic potassium channels. We will learn a great deal about structural mechanisms underlying voltage-activation (Aim 1), inactivation mediated by its subdomain (Aim 2), and calcium-mediated regulation (Aim 3).