Modulation of Electrogenic Sodium Bicarbonate Transport

电化学碳酸氢钠传输的调节

• 批准号: 7000379
• 负责人: IRA KURTZ
• 金额: $ 35万
• 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/7000379
• 关键词: Modulation; Electrogenic; Sodium; Bicarbonate; Transport

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（由申请人提供）：碳酸氢钠协同转运蛋白有助于细胞内 pH (pHi) 调节以及钠和碳酸氢盐在多种组织中的跨上皮转运。最近的生电和电中性碳酸氢钠共转运（NBC）蛋白的克隆、功能表达和免疫定位为研究调节其功能的分子机制提供了机会。生电碳酸氢钠协同转运蛋白 kNBC1 是近曲小管基底外侧碳酸氢盐流出的主要途径。 NBC1 基因的功能缺失突变会导致严重的常染色体隐性遗传性近端肾小管酸中毒。 目前，有关 kNBC 1 产生电的结构基序以及调节其功能的生物学上重要的蛋白质相互作用的信息很少。在最近的研究中，我们证明 C 端 Ser982 残基的 PKA 依赖性磷酸化通过将 HCO3-:Na + 化学计量从 3:1 改变为 2:1 改变了 kNBC1 的电原性。在 Ser982 附近的区域中，结构分析揭示了一个带有天冬氨酸残基的带电区域，可能在这方面发挥重要作用。我们假设 Ser982 的磷酸化状态决定了 kNBC1 C 末端的带负电荷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）或假定的结合蛋白（3:1 模式）发生静电相互作用。在此基础上，我们以kNBC 1的C端为诱饵，通过酵母双杂交实验筛选了人肾cDNA文库，并分离出了天冬氨酸酰化酶。天冬氨酸酰化酶具有多个 N 端碱性残基，可以介导其与 kNBC1 C 端的静电相互作用。天冬氨酸酰化酶定位于近端小管细胞的基底外侧膜，并与来自肾脏的 kNBC 1 进行共免疫沉淀。 kNBC1-Ser982 的 PKA 依赖性磷酸化阻止了蛋白质之间的相互作用。此外，在 N-乙酰天冬氨酸存在的情况下，kNBC1 的功能在 kNBC 1 和天冬氨酸酰化酶共转染的细胞中显着增强。 我们将使用 mPCT 细胞系作为模型系统来实现本提案的目标。该项目的成功完成将增强我们对负责调节 H+/碱转运蛋白的机制的理解。