High-resolution crystallographic and functional studies of K+ channel gating

描述（由申请人提供）：本项目的长期目标是确定K+通道功能的结构变化。这些跨膜蛋白在控制跨膜电位差方面至关重要，而跨膜电位差又形成了溶质交换和细胞兴奋性的基础。因此，充分理解 钾离子通道的结构和功能之间的关系是一个高度优先事项。实现这一目标将需要了解介导两个关键特性（离子渗透和门控）的构象变化。在这个提议中，我们将在原子水平上通过捕获不同动力学状态的通道复合物来表征这两种性质。随后的结构分析将解决以下基本但未回答的问题：1）处于开放和C型失活状态的KcsA的高分辨率结构是什么？2)KcsA的激活门和选择性过滤器之间的变构通讯背后的氨基酸网络是什么？和3）KcsA选择性过滤器的哪些结构变化导致C型失活？这些问题的答案将为我们提供在动态背景下理解K+通道的离子选择性、渗透性和门控所需的结构背景，并最终将有助于合理设计用于治疗K+通道相关疾病的药物。我们将集中研究原型原核通道KcsA，它包含K+通道功能的所有结构元件（即，离子渗透、活化和C型失活门控），但具有简单的结构 足以方便分析。结合晶体学和电生理学方法的双重方法将为以下方面提供高分辨率的功能和结构信息： 这个模型在各种动力学状态下的通道。 公共卫生相关性：K+通道是便利地位于所有活细胞的质膜中的分子，它们从质膜有效地控制从细胞出来的K+离子的流动，这对于大量的生理过程是至关重要的，即免疫系统中自然杀伤细胞的激活或胰腺细胞对血糖含量的调节。鉴于K+通道在正常功能中的关键作用， 对于人体来说，它们的功能障碍具有灾难性的代谢后果，往往导致死亡，这并不奇怪。因此，确定K+通道在发挥其生物学功能时必须承受的原子水平上的结构变化是非常重要的，这反过来将有助于我们设计更安全的治疗药物来治疗许多K+通道相关疾病。

DESCRIPTION (provided by applicant): The long-term goal of this project is to determine the structural changes that underlie K+ channel function. These membrane-spanning proteins are critical in controlling the electrical potential difference across the membrane, which, in turn, forms the basis for solute exchange and cellular excitability. It follows that a full understanding of the relation between structure and function in K+ channels is a high priority. Achieving this goal will require knowledge of the conformational changes that mediate two key properties, ion permeation and gating. In this proposal, we will characterize at the atomic level these two properties by trapping the channel complex in different kinetic states. Subsequent structural analysis will address the following fundamental but unanswered questions: 1) what is the high-resolution structure of KcsA trapped in the open and C-type inactivated state? 2) what is the amino-acid network underlying allosteric communication between the activation gate and the selectivity filter of KcsA? and 3) which are the structural changes of the KcsA selectivity filter that lead to C-type inactivation? The answers to these questions will provide us with the structural background needed to understand ion selectivity, permeation, and gating of K+ channels in a dynamic context and eventually will assist in the rational design of drugs for the treatment of K+ channel- related diseases. We will focus our study on an archetypal prokaryotic channel, KcsA, that contains all the structural elements characteristic of K+ channel function (i.e., ion permeation, activation, and C-type inactivation gating) but possesses a structure simple enough to facilitate analysis. A dual approach combining crystallographic and electrophysiological methods will provide high-resolution functional and structural information for this model channel in various kinetic states. PUBLIC HEALTH RELEVANCE: K+ channels are molecules conveniently located in the plasma membrane of all living cells, from which they effectively control the flow of K+ ions coming out of the cell, which is crucial for a large number of physiological processes i.e. activation of natural killer cells in the immune system or the regulation of the blood glucose content by the pancreatic cells. Given the critical role of K+ channel in the normal functioning of the human body, it is not surprising that their dysfunction has catastrophic metabolic consequences that very often lead to death. For this reason, it is of surmount importance to determine what are the structural changes at the atomic level that a K+ channel has to endure when it is doing its biological function, which in turn it will assist us in the design of safer therapeutic drugs to treat many K+ channel related diseases.