Mechanism of Ligand Gating in Potassium Channels

描述(申请人提供)：钾通道控制钾离子流入和流出细胞，在从最简单的细菌到人类的几乎所有生物体中都普遍表达。K+通道最重要的特性之一是门控，即通道对外界刺激的开放和关闭。钾通道门控在神经和肌肉细胞的兴奋等许多重要的生物学过程中起着至关重要的作用。了解K+通道门控将提供与K+通道相关的生物学活动和疾病的基本基础知识。目前，关于K+通道门控活动的功能数据很多，但对门控过程背后的结构知之甚少。我研究的主要目标是了解K+通道的结构和机制。更具体地说，我们的实验室将专注于研究门控机制，这个系统已经被证明是一个很好的模型系统，来自古细菌甲烷热自养杆菌的一种名为MthK的钙激活K+通道。我们的方法将是多学科的，利用X射线结晶学和电生理学。这项拟议的研究有三个具体目标。第一个具体目标是确定MthK通道在封闭构象中的X射线结构。这与开放形式的MthK的已知结构相结合，将提供开放构象和闭合构象的离子通道的详细原子分辨率图像的第一个例子。第二个具体目的是在单抗的帮助下获得MthK的高分辨率X射线结构。高分辨结构将阐明配体结合部位的特定相互作用的原子细节，以及构象变化背后的蛋白质-蛋白质接触。第三个具体目的是研究钙离子结合和通道门控之间耦合的作用机制。我们将使用基于结构的突变结合单通道电生理记录来分析配体门控的能量过程。

DESCRIPTION (provided by applicant): Potassium channels control the flow of K+ into and out of the cell, and are ubiquitously expressed in nearly all organisms ranging from the simplest bacterium to humans. One of the most important properties of K+ channels is gating, that is, the opening and closing of the channel in response to external stimuli. K+ channel gating plays a vital role in many important biological processes such as the excitation of nerve and muscle cells. Understanding K+ channel gating will provide basic, fundamental knowledge about K+ channel-related biological activities and diseases. Currently, there is a large body of functional data on K+ channel gating activity, but little is known about the structure that underlies the gating process. The broad goal of my research is to understand the structure and mechanics of the K+ channel. More specifically, our lab will focus on studying the gating mechanism in what has already proven to be an excellent model system, a Ca2+-activated K+ channel called MthK, from the archaebacterium Methanobacterium thermoautotrophicum. Our approach will be multi-disciplinary, utilizing both X-ray crystallography and electrophysiology. The proposed research has three specific aims. The first specific aim is to determine the X-ray structure of the MthK channel in a closed conformation. This combined with the known structure of MthK in the open form will provide the first example of detailed, atomic resolution pictures of an ion channel in both the opened and closed conformations. The second specific aim is to obtain high resolution X-ray structures of MthK with the help of monoclonal antibodies. The high-resolution structures will elucidate atomic details of the specific interactions at the ligand binding site and the protein-protein contacts that underlie conformational changes. The third specific aim is to study the functional mechanics of the coupling between Ca2+ binding and channel gating. We will use structure-based mutagenesis combined with single channel electrophysiological recordings to analyze the energetic process of ligand gating.