Molecular Mechanism of Renal Na+ Dependent Cotransport

肾Na依赖性协同转运的分子机制

• 批准号: 7921094
• 负责人: Ana M Pajor
• 金额: $ 16.68万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-29 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7921094
• 关键词: Acid-Base Equilibrium; Affect; Affinity; Amino Acids; Anions; Binding; Biotinylation; Carbon; Carrier Proteins; Cations; Cells; Citrates; Citric Acid Cycle; Complement; Coupled; Cysteine; Development; Disease; Epithelium; Epitopes; Escherichia coli; Family; Foundations; Fumarates; Future; Grant; Health; Homeostasis; Homologous Gene; Human; Hypertension; Ions; Kidney; Kidney Calculi; Label; Laboratories; Longevity; Maintenance; Maps; Mediating; Membrane; Metabolic; Modeling; Molecular; Molecular Conformation; Mutagenesis; Mutation; N-biotinylaminoethyl methanethiosulfonate; Obesity; Pathway interactions; Physiological; Physiological Processes; Play; Property; Proteins; Reagent; Research; Research Personnel; Role; Scanning; Side; Site-Directed Mutagenesis; Source; Specificity; Staphylococcus aureus; Structural Models; Structure; Succinates; Tertiary Protein Structure; Testing; Tubular formation; Vesicle; Work; apical membrane; base; blood pressure regulation; design; dicarboxylate; dicarboxylate-binding protein; directed evolution; interest; member; mutant; novel strategies; protein structure; research study; tool

DESCRIPTION (provided by applicant): The low-affinity Na+/dicarboxylate cotransporter NaDC1 is found on the apical membrane of the renal proximal tubular epithelium, where it plays an important role in the reabsorption of citric acid cycle intermediates, including citrate and succinate, from the tubular lumen. The transport activity of NaDC1 affects such important physiological functions as citrate homeostasis (and the development of kidney stones), the regulation of blood pressure, maintenance of acid-base balance, and the overall metabolic status of the body. In our last project period we identified a bacterial homolog of NaDC1, called SdcS, which provides a unique tool in our studies of the SLC13 family. SdcS is a Na+dependent dicarboxylate transporter from Staphylococcus aureus, with 35% identity to NaDC1. The long-term objective of our research is to determine the structural basis of substrate and cation binding and translocation in the Na+/dicarboxylate cotransporters, and to better understand the mechanism of ion-coupled transport. The research plan for our next project period is designed to elucidate the structure and function of NaDC1 and SdcS in four Specific Aims. Aim 1 will use cysteine-scanning mutagenesis to identify the substrate and cation permeation pathway in NaDC1, focusing on transmembrane helices (TM) 7 and 8, predicted to form part of the substrate access channel. Aim 2 will use epitope insertions to test our new 13-TM structural model of NaDCt. Aim 3 will use SdcS as a model transporter to characterize amino acids that are accessible from the inside of the cell during the transport cycle. Finally, Aim 4 will use mutagenic selection to identify the residues required to transport citrate in SdcS. The experiments in this project should provide important information on the members of the SLC13 family and on the ion-coupled mechanism of transport. Our results should lay the foundations to develop future treatments of disorders involving NaDC1. Relevance to human health: NaDC1 is an important transporter for metabolic intermediates (including succinate and citrate) that mediate many physiological processes; thus, NaDC1 may be involved in diseases such as kidney stones, high blood pressure, and obesity. The bacterial homolog, SdcS, is closely related to NaDC1 and allows us to perform experiments that are not possible with the mammalian transporters. Understanding how NaDC1 and SdcS work will help us to develop treatments for NaDC1-mediated diseases.

描述（由申请方提供）：低亲和力Na+/二羧酸协同转运蛋白NaDC 1存在于肾近端肾小管上皮细胞的顶膜上，在肾小管腔中柠檬酸循环中间体（包括柠檬酸盐和琥珀酸盐）的重吸收中发挥重要作用。NaDC 1的转运活性影响重要的生理功能，如柠檬酸盐稳态（和肾结石的发展），血压调节，酸碱平衡的维持和身体的整体代谢状态。在我们的上一个项目期间，我们确定了NaDC 1的细菌同系物，称为SdcS，它为我们研究SLC 13家族提供了一个独特的工具。SdcS是来自金黄色葡萄球菌的Na+依赖性二羧酸转运蛋白，与NaDC 1具有35%的同一性。本研究的长期目标是确定Na+/二羧酸共转运蛋白中底物和阳离子结合和转运的结构基础，并更好地理解离子耦合转运的机制。我们下一个项目期的研究计划旨在阐明NaDC 1和SdcS在四个特定目的中的结构和功能。目的1将使用半胱氨酸扫描诱变来确定NaDC 1中的底物和阳离子渗透途径，重点是跨膜螺旋（TM）7和8，预测形成底物进入通道的一部分。目的2将使用表位插入来测试我们的新的NaDCt的13-TM结构模型。目标3将使用SdcS作为模型转运蛋白，以表征在转运周期中可从细胞内部进入的氨基酸。最后，目标4将使用诱变选择来鉴定在SdcS中转运柠檬酸盐所需的残留物。该项目的实验将提供有关SLC 13家族成员和离子耦合运输机制的重要信息。我们的研究结果应为开发未来涉及NaDC 1的疾病的治疗方法奠定基础。与人类健康的相关性：NaDC 1是代谢中间体（包括琥珀酸和柠檬酸）的重要转运蛋白，介导许多生理过程;因此，NaDC 1可能与肾结石、高血压和肥胖等疾病有关。细菌同源物SdcS与NaDC 1密切相关，使我们能够进行哺乳动物转运蛋白不可能进行的实验。了解NaDC 1和SdcS的工作原理将有助于我们开发NaDC 1介导的疾病的治疗方法。