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

K 通道门控的高分辨率晶体学和功能研究

• 批准号: 8290873
• 负责人: Luis Gonzalo Cuello
• 金额: $ 28.12万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8290873
• 关键词: Address; Affect; Alkali Metals; Amino Acids; Aromatic Amino Acids; Biological Process; Cell membrane; Cells; Cellular Membrane; Cessation of life; Characteristics; Chemicals; Communication; Complex; Coupled; Coupling; Cysteine; Data; Disease; Drug Design; Elements; Engineering; Functional disorder; Goals; Human body; Immune system; Ions; Kinetics; Knowledge; Lead; Life; Mechanics; Mediating; Membrane; Metabolic; Methods; Molecular; Molecular Conformation; NK Cell Activation; Nature; Pancreas; Pharmaceutical Preparations; Phenylalanine; Physiological Processes; Potassium Channel; Property; Proteins; Resolution; Role; Series; Side; Structure; Therapeutic; X-Ray Crystallography; base; blood glucose regulation; design; disulfide bond; driving force; electrical potential; extracellular; improved; information model; mutant; solute; voltage

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.

描述（由申请人提供）：该项目的长期目标是确定 K+ 通道功能背后的结构变化。这些跨膜蛋白对于控制跨膜电位差至关重要，而跨膜电位差又构成了溶质交换和细胞兴奋性的基础。由此可见，充分认识 K+通道结构和功能之间的关系是一个高度优先的问题。实现这一目标需要了解介导离子渗透和门控这两个关键特性的构象变化。在本提案中，我们将通过将通道复合物捕获在不同的动力学状态来在原子水平上表征这两个特性。后续的结构分析将解决以下基本但尚未解答的问题：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 激活门和选择性过滤器之间变构通讯的氨基酸网络是什么？ 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了解动态背景下 K+ 通道的离子选择性、渗透和门控所需的结构背景，并最终有助于合理设计治疗 K+ 通道相关疾病的药物。我们将重点研究原型原核通道 KcsA，它包含 K+ 通道功能特征的所有结构元素（即离子渗透、激活和 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 该模型通道处于各种动力学状态。 公共健康相关性：K+ 通道是位于所有活细胞质膜中的分子，可有效控制 K+ 离子流出细胞的流动，这对于大量生理过程（例如自然杀伤细胞的激活）至关重要免疫系统中的细胞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，它们的功能障碍会产生灾难性的代谢后果，常常导致死亡，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至关重要，这反过来将有助于我们设计更安全的治疗药物治疗多种 K+ 通道相关疾病。