Molecular Mechanism of Renal Na+ Dependent Cotransport

描述（由申请人提供）：低亲和力Na+/二羧酸盐共转运蛋白NaDC1存在于肾近端小管上皮的顶膜上，它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）的重吸收中起重要作用。NaDC1的转运活性影响着柠檬酸盐稳态（以及肾结石的发生）、血压的调节、酸碱平衡的维持以及机体的整体代谢状态等重要生理功能。在我们上一个项目期间，我们鉴定了NaDC1的细菌同源物，称为sdc，这为我们研究SLC13家族提供了一个独特的工具。sdc是一种来自金黄色葡萄球菌的Na+依赖性二羧酸转运蛋白，与NaDC1有35%的同源性。我们研究的长期目标是确定Na+/二羧酸盐共转运体中底物和阳离子结合和转运的结构基础，并更好地了解离子耦合转运的机制。我们下一个项目期的研究计划是在四个具体目标中阐明NaDC1和sdc的结构和功能。Aim 1将使用半胱氨酸扫描诱变技术来鉴定NaDC1中的底物和阳离子渗透途径，重点关注跨膜螺旋（TM） 7和8，预计它们将构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新的13-TM NaDCt结构模型。目的3将使用sdc作为模型转运体来表征在运输周期中可从细胞内部获得的氨基酸。最后，Aim 4将使用诱变选择来确定sdc中运输柠檬酸盐所需的残基。本项目的实验将为SLC13家族成员和离子耦合输运机制提供重要信息。我们的研究结果将为开发涉及NaDC1的疾病的未来治疗奠定基础。与人类健康的相关性：NaDC1是代谢中间体（包括琥珀酸盐和柠檬酸盐）的重要转运体，介导许多生理过程；因此，NaDC1可能与肾结石、高血压和肥胖等疾病有关。细菌同源物sdc与NaDC1密切相关，使我们能够进行哺乳动物转运蛋白无法进行的实验。了解NaDC1和sdc如何起作用将有助于我们开发针对NaDC1介导疾病的治疗方法。

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.