Molecular Mechanism of Renal Na+ Dependent Cotransport

本研究的重点是肾近端小管的Na/二羧酸盐共转运蛋白，特别是肾顶膜的低亲和力转运蛋白NaDC-1。NaDC-1在克雷布斯循环中间体如柠檬酸盐和琥珀酸盐的吸收中起重要作用。NaDC-1的转运活性影响生理功能，如尿中柠檬酸的稳态（影响肾结石的发生）、有机阴离子的分泌、酸碱平衡的维持和身体的整体代谢状态。本研究的长期目标是确定Na+/二羧酸盐共转运体中离子与底物结合和转运的结构基础，从而更好地了解离子耦合转运的机制。在当前的资助期内产生的结果已经确定了几个可能参与底物和阳离子结合的关键氨基酸，并可能形成通过蛋白质的渗透途径的一部分。在本提案的第一个具体目标中，我们计划确定NaDC-1转运体中决定特定功能特性的其他残基和结构域，例如底物和阳离子的选择性和亲和力，以及对抑制剂的敏感性。在第二个具体目标中，我们计划使用取代半胱氨酸可及性方法来鉴定NaDC-1中的离子和底物渗透途径。在第三个目标中，我们计划完善NaDC-1的二级结构模型。本提案中概述的研究应提供nadc相关钠偶联转运蛋白家族的功能特性以及这些转运蛋白在肾脏中的生理作用的基本信息。

The focus of the current proposal is the Na/dicarboxylate cotransporters of the renal proximal tubule, particularly NaDC-1, the low affinity transporter of the apical membrane. NaDC-1 plays an important role in the absorption of Krebs cycle intermediates, such as citrate and succinate. The transport activity of NaDC-1 affects physiological functions such as citrate homeostasis in the urine (which influences the development of kidney stones), organic anion secretion, maintenance of acid-base balance, and overall metabolic status of the body. The long-term objective of this research is to determine the structural basis of ion and substrate binding and translocation in the Na+/dicarboxylate cotransporters to better understand the mechanism of ion-coupled transport. The results generated in the current grant period have identified several key amino acids that may participate in substrate and cation binding and may form part of the permeation pathway through the protein. In the first specific aim of this proposal, we plan to identify additional residues and domains in the NaDC-1 transporters that determine specific functional properties, such as substrate and cation selectivity and affinity, and sensitivity to inhibitors. In the second specific aim, we plan to use the substituted cysteine-accessibility method to identify the ion and substrate permeation pathways in NaDC-1. In the third aim, we plan to refine the secondary structure model of NaDC-1. The studies outlined in this proposal should provide fundamental information on the functional properties of the NaDC-related family of sodium-coupled transporters and on the physiological role of these transporters in the kidney.