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

项目摘要 本基金申请的中心原则包含两个相互依存的组成部分：1）结构如何 确定K+通道的功能？以及细胞膜磷脂成分 调节它们的结构-功能相关性我们的工作涉及这两个基本问题， 包括离子通道生理学的三个方面：1）K+-K 通道门控、渗透和选择性？2)如何使一个双向变构耦合之间的 激活门（AG）和选择性过滤器（SF）控制K+通道功能？以及3）细胞如何 膜脂组成调节K+通道行为？在原子水平上理解K+- 渠道的工作将有助于发现新的治疗药物。我们将使用几个 我们在过去10年中从NIH持续资助中开发的方法学进步。 这些成果是：1）阐明了钾离子通道的原子分辨率门控周期; 2） 二硫键桥联锁定开放KcsA支架的工程化，其产生原子分辨率衍射晶体 并允许我们通过电生理学在pH 7.0（生理相关pH）下表征其功能。 用等温滴定量热法测定整个选择性过滤器的碱金属离子结合亲和力 K+通道的开放构象和/或单个离子结合位点4）发现了一种新的 通过减少细胞膜的厚度和5）KcsA激活的机制， 一种新的方法，用于过度表达正确折叠和功能性的人K+通道， 生物医学的重要性在E。coli细胞。我们使用KcsA作为结构替代物的工作是我们的基础， 目前对K+通道功能的理解。现在，我们正在通过开发一种 一种突破性的新方法，用于过度表达正确折叠和功能性的人类Kv通道 在大肠大肠杆菌细胞，这消除了其他耗时和昂贵的使用真核生物， 细胞我们将对离子通道的结构如何改变其构象有一个完整的了解 在由细胞膜的脂质组成决定的能量环境中调节它们的功能。 我们的目的是确定潜在的离子渗透，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双层组成的相互依赖性 以及两个与生物医学相关的人体KV通道。最后，我们将提出一个概念框架， K+通道选择性过滤器和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协同性和细胞膜的磷脂组成来调节。 完成这项拨款申请将产生宝贵的信息，以协助智能设计的安全 治疗药物

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.