Mechanism of Ligand Gating in Potassium Channels

描述（由申请人提供）：钾通道控制K+进出细胞的流动，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，即通道在响应外部刺激时的打开和关闭。K+通道门控在神经和肌肉细胞的兴奋等许多重要的生物过程中起着至关重要的作用。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本知识。目前，有大量关于K+通道门控活动的功能数据，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+通道的结构和力学。更具体地说，我们的实验室将专注于研究门控机制，这个门控机制已经被证明是一个很好的模型系统，一个叫做MthK的Ca2+激活的K+通道，来自热自养甲烷细菌。我们的方法将是多学科的，同时利用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封闭构象中的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，将提供离子通道在开放和封闭构象中详细的原子分辨率图像的第一个例子。第二个具体目标是借助单克隆抗体获得MthK的高分辨率x射线结构。高分辨率结构将阐明配体结合位点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。第三个具体目标是研究Ca2+结合和通道门控之间耦合的功能机制。我们将使用基于结构的诱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.