Structure/function analysis of understudied pH-sensitive Kir4.2 channels in vitro with conformation specific nanobodies

使用构象特异性纳米体对正在研究的 pH 敏感 Kir4.2 通道进行体外结构/功能分析

• 批准号: 10217510
• 负责人: Sun Joo Lee
• 金额: $ 15.75万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2022-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217510
• 关键词: Arrhythmia; Atrial Heart Septal Defects; Biochemical; Biochemistry; Biological Assay; C-terminal; Cells; Cholesterol; Complex; Congenital diaphragmatic hernia; Cryoelectron Microscopy; Defect; Dependence; Detergents; Development; Diabetes Mellitus; Disease; Docking; Down Syndrome; Drug Screening; Drug or chemical Tissue Distribution; Electrophysiology (science); Epilepsy; Epitopes; Family; Functional disorder; Generations; Genes; Goals; Homeostasis; Homo; Human; Immunoglobulin G; In Vitro; Individual; Inflammation; Ion Channel; Lead; Libraries; Lipids; Liposomes; Membrane Lipids; Membrane Potentials; Membrane Proteins; Methods; Molecular; Molecular Conformation; Mutation; Non-Insulin-Dependent Diabetes Mellitus; Pathologic; Patients; Pharmaceutical Preparations; Phosphatidylinositol 4,5-Diphosphate; Physiological; Polyunsaturated Fatty Acids; Positioning Attribute; Potassium; Property; Protein Chemistry; Proteins; Protocols documentation; Proximal Renal Tubular Acidosis; Publishing; Regulation; Resolution; Rest; Structure; Surface; System; Testing; Therapeutic; Time; Tissues; Tracer; Vascular Diseases; Work; X-Ray Crystallography; base; extracellular; gene product; human disease; improved; in silico; in vitro testing; insight; inward rectifier potassium channel; large scale production; mutant; nanobodies; novel; novel strategies; novel therapeutics; particle; programs; protein purification; reconstitution; screening; simulation; small molecule; stable cell line; systemic juvenile idiopathic arthritis; therapeutic target; tool; web site

Project Summary Inward rectifying potassium (Kir) channels regulate excitability in many tissues, and multiple diseases result from mutations of Kir channel genes. The long-term goal of this project is to understand the molecular details of understudied pH-sensitive Kir4 sub-family channels. Previously, I defined molecular mechanisms of bulk anionic lipid regulation of Kir2 channels. These previous studies were made possible by my establishing a new expression system and purification protocols for eukaryotic Kir2 channels, as well as uniquely combining the multiple methods of (1) computational docking simulations, (2) functional assays of purified proteins reconstituted in synthetic liposomes, and (3) high resolution structure determination. These studies provide a template for gaining new understanding of understudied pH-sensitive Kir4 channels at the molecular level. We will establish an efficient expression system and a purification protocol for large scale purifications of Kir4.2 homo- and Kir4.2/Kir5.1 hetero-tetramer proteins that are understudied as well as develop nanobodies specific to functional states that will not only facilitate structure determinations in certain functional states but also have therapeutic potentials. Once established, we will utilize the same `triple combination' of approaches to determine molecular details of these understudied channel regulation and function.

项目摘要 内向整流钾（KIR）通道调节许多组织的兴奋性，并且 多种疾病是由KIR通道基因突变引起的。长期目标 该项目是为了了解研究不足的pH敏感KIR4的分子细节 亚家族通道。以前，我定义了散装阴离子的分子机制 KIR2通道的脂质调节。这些先前的研究使我成为可能 建立真核KIR2的新表达系统和纯化协议 通道，以及唯一组合（1）计算的多种方法 对接模拟，（2）在重构中重构的纯化蛋白的功能测定 合成脂质体和（3）高分辨率结构测定。这些研究 提供了一个模板，以获得对研究不足的pH敏感KIR4的新理解 在分子水平上的通道。我们将建立一个有效的表达系统 以及针对Kir4.2均质和纯化纯化的纯化协议 Kir4.2/kir5.1杂种蛋白被研究和发展 特定于功能状态的纳米体不仅会促进结构 在某些功能状态的确定，但也具有治疗潜力。 建立后，我们将利用相同的方法的“三重组合” 确定这些研究的通道调节和功能的分子细节。