The Biology of NBCe1 in Health and Disease

NBCe1 在健康和疾病中的生物学

• 批准号: 10379238
• 负责人: IRA KURTZ
• 金额: $ 62.8万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-19 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10379238
• 关键词: Acids; Address; Ammonia; Bicarbonates; Biology; Blood; Blood Chemical Analysis; Blood Pressure; Bone Growth; Carbonates; Carrier Proteins; Cattle; Cell Maintenance; Cell physiology; Cells; Chemistry; Child; Chronic Kidney Failure; Computer Analysis; Coupled; Coupling; Cryoelectron Microscopy; Data; Development; Disease; Distal renal tubular acidosis Type 1; Duct (organ) structure; Electrolytes; Equilibrium; Erythrocyte Anion Exchange Protein 1; Erythrocytes; Eye; Family; Genetic Anticipation; Goals; Grant; Health; Human; Hypokalemia; Impairment; Infant; Intercalated Cell; Ion Transport; Ions; Kidney; Kidney Calculi; Kidney Diseases; Mammals; Mediating; Medicine; Modeling; Molecular; Molecular Conformation; Mutagenesis; Mutation; Nephrocalcinosis; Nephrology; Newborn Infant; Physiology; Play; Prevalence; Process; Production; Property; Proteins; Public Health; Publications; Rattus; Renal function; Renal tubular acidosis; Research Personnel; Resolution; Role; Seminal; Site; Sodium Bicarbonate; Source; Specificity; Structure; Thermodynamics; Time; Tooth structure; Urine; absorption; base; basolateral membrane; blood pressure regulation; body system; cell growth; clinically relevant; disease-causing mutation; experimental study; human disease; hypercalciuria; member; molecular dynamics; molecular modeling; molecular transporter; symporter

PROJECT SUMMARY SLC4 proteins in the kidney play an essential role in mediating the coupled transport (symporters, exchangers) of Na+, HCO3⎯, CO32⎯, and Cl⎯ in the proximal tubule and collecting duct. Infants and children with mutations in the electrogenic Na+-CO32⎯ symporter NBCe1 and the Cl⎯/HCO3⎯ exchanger AE1 develop severe proximal and distal renal tubular acidosis respectively. How disease causing mutations impair their transport mechanisms at the molecular level is unknown. Towards this goal, we have recently solved by cryoelectron microscopy (cryoEM) the outward facing structure of NBCe1, the inward and outward facing structures of AE1, and have obtained 2D class averages of the Na+-CO32⎯/Cl⎯ exchanger NDCBE representing major advances in the field. These significant advances coupled with our preliminary functional mutagenesis and Molecular Dynamics computational analyses make it possible for the first time in the transport field to achieve the long-term objective of understanding the detailed structure-functional properties of SLC4 transporters and their impairment by disease causing mutations. To accomplish this objective, the project addresses the following specific aims: Aim 1: Structural Determinants of the AE1, NBCe1, and NDCBE Ion Coordination Sites, Transport Modes and Ion Specificities: In this aim, using cryoEM, functional mutagenesis and Molecular Dynamics computational analyses, the hypothesis that the ion coordination sites in these transporters encode both their respective transport modes and unique ion transport specificities will be examined. Aim 2: Characterize the Structural Components of AE1, NBCe1 and NDCBE Permeation Pores, Ion Selectivity and Energetics: In this aim, using cryoEM, functional mutagenesis and Molecular Dynamics computational analyses, the hypothesis that differences in the structure and energetics of ion permeation among these transporters plays an important role in determining the selectivity of ions reaching their coordination sites in both outward and inward facing conformations will be examined. Aim 3: Transport Models for AE1, NBCe1 and NDCBE, and Characterization of the Transport Abnormalities Induced by Renal Tubular Acidosis Causing Mutations: The dynamic transport models of AE1, NBCe1 and NDCBE will be generated based on preliminary data, and the results obtained in Aims 1 and 2. How disease causing mutations impair these mechanistic transporter molecular models will be determined. This proposal represents a significant contribution to Nephrology and Medicine given the importance of SLC4 transporters in regulating kidney ion balance, systemic acid-base chemistry, blood pressure, and the maintenance of cell function and growth.

项目概要 肾脏中的 SLC4 蛋白在介导偶联转运（同向转运蛋白、 近曲小管和集合管中的 Na+、HCO3⎯、CO32⎯ 和 Cl⎯ 交换器）。婴儿和儿童 随着产电 Na+-CO32⎯ 同向转运体 NBCe1 和 Cl⎯/HCO3⎯ 交换器 AE1 的突变而发展 分别为严重的近端肾小管和远端肾小管酸中毒。引起突变的疾病如何损害它们 分子水平的运输机制尚不清楚。为了实现这一目标，我们最近解决了 冷冻电子显微镜 (cryoEM) NBCe1 的朝外结构、朝内和朝外 AE1的结构，并获得了Na+-CO32⎯/Cl⎯交换剂NDCBE的二维类平均值 代表了该领域的重大进展。这些重大进步加上我们初步的功能 诱变和分子动力学计算分析首次使之成为可能 运输领域以实现了解详细结构-功能的长期目标 SLC4 转运蛋白的特性及其因致病突变而受到的损害。为了实现这一点 目标，该项目致力于以下具体目标： 目标 1：AE1 的结构决定因素， NBCe1 和 NDCBE 离子配位位点、传输模式和离子特异性：为此目的，使用 冷冻电镜、功能诱变和分子动力学计算分析，假设 这些转运蛋白中的离子配位位点编码它们各自的转运模式和独特的离子 将审查运输的特殊性。目标 2：表征 AE1、NBCe1 和 NDCBE 渗透孔、离子选择性和能量学：为此，使用冷冻电镜、功能诱变 和分子动力学计算分析，假设结构和 这些转运蛋白之间的离子渗透能量在确定选择性方面起着重要作用 将检查在向外和向内构象中到达其配位点的离子的数量。 目标 3：AE1、NBCe1 和 NDCBE 的传输模型以及传输异常的表征 肾小管酸中毒引起的突变：AE1、NBCe1和 NDCBE 将根据初步数据以及目标 1 和 2 中获得的结果生成。 引起突变损害这些机械转运蛋白的分子模型将被确定。这个提议 鉴于 SLC4 转运蛋白的重要性，代表着对肾病学和医学的重大贡献 调节肾脏离子平衡、全身酸碱化学、血压和细胞维护 功能和生长。