A comprehensive thermodynamic and structural characterization of ion channel function and its regulation by the lipid bilayer composition

离子通道功能的综合热力学和结构表征及其由脂质双层组成的调节

• 批准号: 10623911
• 负责人: Luis Gonzalo Cuello
• 金额: $ 59.4万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623911
• 关键词: Achievement; Affinity; Alkali Metals; Applications Grants; Arrhythmia; Autoimmune Diabetes; Autoimmune Diseases; Behavior; Binding Sites; Calorimetry; Cell membrane; Cells; Consumption; Coupling; Development; Disease; Disulfides; Drug Targeting; Electrophysiology (science); Engineering; Epilepsy; Escherichia coli; Eukaryotic Cell; Functional disorder; Funding; Human; Individual; Ion Channel; Ions; Lipid Bilayers; Lipids; Measures; Membrane Lipids; Metal Ion Binding; Methodology; Molecular Conformation; Pathologic; Pharmaceutical Preparations; Phospholipids; Physiological; Physiology; Potassium Channel; Regulation; Resolution; Structure; Syndrome; Therapeutic; Thermodynamics; Thick; Time; Titrations; United States National Institutes of Health; Work; design; novel; novel therapeutics; overexpression; scaffold

Project summary The central tenet of this grant application contains two interdependent components 1) how does the structure determine the function of K+-channels? and how does the cell membrane phospholipid composition regulate their structure-function correlations? Our work deal with these two fundamental questions, which encompasses three aspects of ion channel physiology: 1) Which are the structural changes underlying K+- channel gating, permeation, and selectivity? 2) How does a bidirectional allosteric coupling between the activation gate (AG) and the selectivity filter (SF) control K+-channels function? and 3) How does the cell membrane lipid composition regulate K+-channels behavior? Understanding at the atomic level how K+- channels work will assist during the discovery of novel therapeutic drugs. We will use several methodological advancements developed by us during the last 10 years of continuous funding from the NIH. These achievements are: 1) the elucidation of an atomic resolution gating cycle of a K+-channel 2) the engineering of a disulfide bridged locked open KcsA scaffold that produces atomic resolution diffracting crystals and allow us to characterize its function by electrophysiology at pH 7.0 (a physiologically relevant pH) 3) the measuring of the alkali metal ions binding affinity by Isothermal Titration Calorimetry of the whole selectivity filter of a K+-channel in the open conformation and/or of individual ion binding sites 4) the discovery of a novel mechanism of KcsA activation by reducing the thickness of the cell membrane and 5) the development of a new methodology for the overexpression of properly folded and functional Human K+-channels of Biomedical Importance in E. coli cells. Our work using KcsA as a structural surrogate is foundational of our current understanding of K+-channel function. Now we are expanding into human K+-channels by developing a groundbreaking new methodology for the overexpression of properly folded and functional human Kv-channels in E. coli cells, which eliminates the otherwise time consuming and outrageously expensive use of eukaryotic cells. We will develop an integrative understanding of how the structure of ion channels change its conformation to regulate their function within an energetic landscape determined by the lipid composition of the cell membrane. We aim to determine the structural changes underlying ion permeation, ion selectivity and C-type inactivation gating and their interdependence with the lipid bilayer composition of the cell membrane in a bacterial channel and in two human Kv-channels of Biomedical Relevance. Finally, we will produce a conceptual framework about how the allosteric coupling between a K+-channel’s selectivity filter and its activation gate define ion channel function and how is modulated by subunit cooperativity and the phospholipid composition of the cell membrane. The completion of this grant application will produce invaluable information to assist in the smart design of safer therapeutic drugs.

项目摘要 该赠款应用程序的中心宗旨包含两个相互依存的组件1）结构如何 确定K+通道的功能？细胞膜磷脂组成如何 调节其结构 - 功能相关性？我们的工作处理了这两个基本问题，这是 包括离子通道生理学的三个方面：1）是K+ - 的结构变化 通道门控，渗透和选择性？ 2）双向变构耦合如何 激活门（AG）和选择性滤波器（SF）控制K+通道功能？ 3）细胞如何 膜脂质组成调节K+通道行为？在原子层中了解K+如何 渠道工作将在发现新型治疗药物期间有助于。我们将使用几个 我们在NIH持续资金的最后十年中发展的方法学进步。 这些成就是：1）阐明A+通道的原子分辨率门控循环2） 二硫键架锁定的开放式kcsa脚手架的工程，产生原子分辨率衍射晶体 并允许我们通过pH 7.0的电生理学来表征其功能（一个物理相关的pH）3） 通过等温滴定量热法对碱金属离子结合亲和力的测量整个选择性过滤器 在开放构象和/或单个离子结合位点中的K+ - 通道的发现4）发现小说 通过减少细胞膜的厚度，KCSA激活的机制和5） 过表达正确折叠和功能性的人K+通道的新方法 大肠杆菌细胞中的生物医学重要性。我们使用KCSA作为结构代理的工作是我们的基础 当前对K+通道功能的理解。现在，我们通过开发一个 开创性的新方法，用于过表达正确折叠和功能性的人类KV通道 在大肠杆菌细胞中，消除了原本耗时和非常昂贵的真核的使用 细胞。我们将对离子渠道结构如何改变其构象的结构有综合的理解 在通过细胞膜的脂质组成确定的能量景观中调节其功能。 我们旨在确定离子渗透，离子选择性和C型失活的结构变化 门控及其与细菌通道中细胞膜的脂质双层组成的相互依存关系 以及在生物医学相关性的两种人类KV通道中。最后，我们将建立一个概念框架 K+通道的选择性滤波器及其激活门之间的变构耦合如何定义离子通道 功能以及如何通过亚基配位和细胞膜的磷脂组成调节。 该赠款申请的完成将产生宝贵的信息，以协助安全设计 治疗药物。