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

项目总结 这项赠款申请的中心原则包含两个相互依赖的组成部分1)结构如何 确定K+通道的功能？以及细胞膜磷脂是如何组成的？ 调控它们的结构-功能相关性？我们的工作涉及到这两个基本问题，即 包括离子通道生理学的三个方面：1)K+-的结构变化 通道门控、渗透和选择性？2)通道之间的双向变构耦合是如何 激活门(AG)和选择性过滤器(SF)控制K+通道的功能？以及3)细胞是如何 膜脂成分调节K+通道的行为？在原子水平上理解K+是如何 在发现新的治疗药物过程中，通道工作将起到帮助作用。我们将使用几个 在美国国立卫生研究院持续资助的过去10年里，我们在方法上取得的进步。 这些成果是：1)阐明了K+通道的原子分辨率选通周期2) 产生原子分辨衍射晶体的二硫代桥联KCSA开放支架的工程设计 并允许我们在pH 7.0(生理上相关的pH)3下通过电生理学来表征其功能 全选择性滤池等温滴定量热法测定碱金属离子结合亲和力 在开放构象和/或单个离子结合位点中的K+通道4)一种新的 KCsA通过减少细胞膜厚度而激活的机制和5)A的形成 人类正常折叠和功能K+通道过表达的新方法 在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 目前对K+通道功能的认识。现在我们正在扩展到人类的K+通道，通过开发一种 突破性的人类Kv通道正确折叠和功能过表达的新方法 在大肠杆菌细胞中，这消除了原本耗时和昂贵的真核细胞的使用 细胞。我们将对离子通道的结构如何改变其构象有一个综合的理解 在由细胞膜的脂类成分决定的能量环境中调节它们的功能。 我们的目标是确定离子渗透、离子选择性和C型失活背后的结构变化 门控及其与细菌通道细胞膜脂质双层组成的相互依赖 以及在两个与生物医学相关的人类Kv通道中。最后，我们将提出一个概念性框架 K+通道的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 功能和方式受亚单位协作性和细胞膜磷脂组成的调节。 这项拨款申请的完成将产生宝贵的信息，以协助SAFER的智能设计 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.