Modulation of Electrogenic Sodium Bicarbonate Transport

描述（由申请人提供）：碳酸氢钠共转运蛋白参与细胞内pH （pHi）调节以及钠和碳酸氢钠在几种组织中的经上皮转运。最近电致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。电致碳酸氢钠共转运蛋白kNBC1是近端小管基底外侧碳酸氢钠流出的主要途径。NBC1基因的功能突变缺失导致严重的常染色体隐性近端肾小管酸中毒。目前，关于kNBC 1的电原性的结构基序和调节其功能的重要的生物学蛋白相互作用的信息都很缺乏。在最近的研究中，我们已经证明pka依赖的c端Ser982残基磷酸化通过将其HCO3-:Na +化学计量从3:1改变为2:1，改变了kNBC1的电性。在Ser982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，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端这个带负电荷的区域是否会与转运体中的一个碳酸氢盐结合位点（2:1模式）或一个假定的结合蛋白（3:1模式）发生静电相互作用。在此基础上，我们以knbc1的c端为诱饵，用酵母双杂交法筛选了人肾脏cDNA文库，并分离出了天冬氨酸酰化酶。天冬氨酸酰化酶具有若干n端碱性残基，可介导其与kNBC1的c端静电相互作用。天冬氨酸酰化酶定位于近端小管细胞的基底外膜，并与肾knbc1共免疫沉淀。pka依赖的kNBC1-Ser982磷酸化阻止了蛋白之间的相互作用。此外，在n -乙酰天冬氨酸存在的情况下，kNBC1和天冬氨酸酰化酶共转染的细胞中，kNBC1的功能显著增强。我们将使用mPCT细胞系作为模型系统来实现本提案的目标。这个项目的成功完成将增强我们对H+/碱转运体调节机制的理解。

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.