Modulation of Electrogenic Sodium Bicarbonate Transport

电化学碳酸氢钠传输的调节

• 批准号: 6562362
• 负责人: IRA KURTZ
• 金额: $ 35.84万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6562362
• 关键词: acidity /alkalinity; amidohydrolases; aspartate; binding proteins; genetic library; ion transport; ionic bond; membrane transport proteins; phosphorylation; protein kinase A; protein protein interaction; protein structure function; renal tubular transport; sodium bicarbonate

DESCRIPTION (provided by applicant): Sodium bicarbonate cotransporters contribute to intracellular pH (pHi) regulation and the transepithelial transport of sodium and bicarbonate in several tissues. The recent cloning, functional expression, and immunolocalization of electrogenic and electroneutral sodium bicarbonate cotransport (NBC) proteins provides an opportunity to investigate the molecular mechanisms responsible for modulating their function. The electrogenic sodium bicarbonate cotransporter kNBC1 is the main pathway for proximal tubule basolateral bicarbonate effiux. Loss of function mutations in the NBC1 gene cause a severe form of autosomal recessive proximal renal tubular acidosis. There is currently a paucity of information regarding both the structural motifs responsible for the electrogenicity of kNBC 1, and the biologically important protein interactions that modulate its function. In recent studies, we have demonstrated that PKA-dependent phosphorylation of the C-terminal Ser982 residue altered the electrogenicity of kNBC1 by shifting its HCO3-:Na + stoichiometry from 3:1 to 2:1. In the region adjacent to Ser982, structural analysis reveals a charged region with aspartic acid residues that could potentially play an important role in this regard. We hypothesized that the phosphorylation state of Ser982 determines whether this negatively charged region in the kNBC1 C-terminus will interact electrostatically either with one bicarbonate binding site in the transporter (2:1 mode), or a putative binding protein (3:1 mode). On this basis we screened a human kidney cDNA library in a yeast two-hybrid assay using the C-terminus of kNBC 1 as bait, and isolated the enzyme aspartoacylase. Aspartoacylase has several N-terminal basic residues which could mediate its interaction electrostatically with the C-terminus of kNBC1. Aspartoacylase was localized to the basolateral membrane of proximal tubule cells, and co-immunoprecipitated with kNBC 1 from kidney. PKA-dependent phosphorylation of kNBC1-Ser982 prevented the interaction between the proteins. Furthermore, the function of kNBC1 was significantly greater in cells co-transfected with kNBC 1 and aspartoacylase in the presence of N-acetylaspartate. We will use the mPCT cell line as a model system for achieving the goals of this proposal. Successful completion of this project will enhance our understanding of the mechanisms responsible for regulating H+/base transporters.

描述（由申请方提供）：碳酸氢钠协同转运蛋白有助于细胞内pH（pHi）调节以及几种组织中钠和碳酸氢盐的跨上皮转运。最近的克隆，功能表达和免疫定位的产电和电中性碳酸氢钠共转运（NBC）蛋白提供了一个机会，调查负责调节其功能的分子机制。产电性碳酸氢钠协同转运体kNBC 1是近曲小管基底外侧碳酸氢盐流出的主要途径。NBC 1基因功能缺失突变导致严重的常染色体隐性近端肾小管酸中毒。 目前有一个缺乏的信息负责kNBC 1的致电性的结构基序，和生物学上重要的蛋白质相互作用，调节其功能。在最近的研究中，我们已经证明，PKA依赖的磷酸化的C-末端Ser 982残基改变了kNBC 1的致电性，通过改变其HCO 3-：Na +的化学计量比从3：1到2：1。在Ser 982附近的区域，结构分析揭示了一个带有天冬氨酸残基的带电区域，该区域可能在这方面发挥重要作用。我们假设Ser 982的磷酸化状态决定了kNBC 1 C-末端带负电荷的区域是否会与转运蛋白中的一个碳酸氢盐结合位点（2：1模式）或推定的结合蛋白（3：1模式）静电相互作用。在此基础上，我们使用kNBC 1的C-末端作为诱饵，在酵母双杂交分析中筛选人肾cDNA文库，并分离出酶的乙酰化酶。天冬氨酸酰化酶的N端有几个碱性残基，这些残基可以介导天冬氨酸酰化酶与kNBC 1的C端的静电相互作用。天冬氨酸酰化酶定位于近曲小管细胞的基底外侧膜，并与肾脏的kNBC 1免疫共沉淀。kNBC 1-Ser 982的PKA依赖性磷酸化阻止了蛋白质之间的相互作用。此外，在存在N-乙酰天冬氨酸的情况下，在用kNBC 1和乙酰化酰化酶共转染的细胞中，kNBC 1的功能显著更大。 我们将使用mPCT细胞系作为实现本提案目标的模型系统。该项目的成功完成将增强我们对负责调节H+/碱基转运蛋白的机制的理解。