The Biology of NBCe1 in Health and Disease

项目总结 肾脏中的SLC4蛋白在调节偶联转运(符号转运体， Na+、HCO3-⎯、CO32-⎯和Cl-⎯在近端小管和集合管中的交换者)。婴幼儿 随着Na+-CO32⎯同向转运体NbCe1和氯离子⎯/HCO3⎯交换器Ae1的突变，形成了 重度近端肾小管酸中毒和远端肾小管酸中毒。疾病如何导致突变损害它们的 分子水平上的转运机制尚不清楚。为了实现这一目标，我们最近通过 低温电子显微镜(CryoEM)NBCe1的外向结构，内向和外向 得到了Na~+-CO_3~(2+)⎯/Cl⎯交换剂NDCBE的2D类平均值 代表着该领域的重大进步。这些重大进展与我们初步的功能 诱变和分子动力学计算分析使其首次成为可能 实现交通领域的长期目标，了解详细的结构--功能 SLC4转运蛋白的特性及其致病突变所致的损伤。要做到这一点 根据目标，该项目涉及以下具体目标：目标1：第一阶段的结构决定因素； NBCe1和NDCBE离子配位位置、传输模式和离子特性：在这一目标中，使用 低温电子显微镜、功能突变和分子动力学计算分析，假设 这些转运体中的离子配位位点编码它们各自的运输方式和独特的离子 将审查运输的具体情况。目的2：鉴定AE1、NBCe1和NbCe1的结构成分 NDCBE渗透孔、离子选择性和能量学：在这个目标中，使用低温EM、功能突变 和分子动力学计算分析，假设结构和分子间的差异 离子在这些转运体之间渗透的能量学在决定选择性方面起着重要作用。 在向外和向内的构象中到达其配位位置的离子的数量将被检测。 目的3：AE1、NBCe1和NDCBE的运输模型，以及运输异常的特征 肾小管酸中毒致突变：AE1、NBCe1和NbCe1的动态转运模型 NDCBE将基于初步数据和在目标1和2中获得的结果生成。 导致突变损害这些机械转运蛋白的分子模型将被确定。这项建议 鉴于SLC4转运蛋白的重要性，对肾脏病和医学做出了重大贡献 调节肾脏离子平衡、全身酸碱化学、血压和细胞维持 功能和成长。

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.