Mechanism of Ligand Gating in Potassium Channels

描述（由申请人提供）：钾通道控制K+流入和流出细胞，并且在从最简单的细菌到人类的几乎所有生物体中普遍表达。K+通道最重要的特性之一是门控，即通道响应外部刺激的打开和关闭。钾通道门控在许多重要的生物学过程中起着重要的作用，如神经和肌肉细胞的兴奋。了解K+通道门控将提供有关K+通道相关生物活动和疾病的基础知识。目前，有大量关于K+通道门控活性的功能数据，但对门控过程的基础结构知之甚少。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已经被证明是一个很好的模型系统的门控机制，一个被称为MthK的Ca 2+激活的K+通道，来自古细菌嗜热甲烷杆菌。我们的方法将是多学科的，利用X射线晶体学和电生理学。拟议的研究有三个具体目标。第一个具体目标是确定闭合构象中MthK通道的X射线结构。这与已知的开放形式的MthK结构相结合，将提供第一个详细的例子，在开放和封闭构象的离子通道的原子分辨率图片。第二个具体目标是在单克隆抗体的帮助下获得MthK的高分辨率X射线结构。高分辨率的结构将阐明在配体结合位点和蛋白质-蛋白质接触，基础构象变化的具体相互作用的原子细节。第三个具体目标是研究钙离子结合和通道门控之间的耦合的功能机制。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.