Modulation of Electrogenic Sodium Bicarbonate Transport

描述(由申请人提供)：碳酸氢钠共转运体有助于调节细胞内pH(Phi)和几种组织中的钠和碳酸氢盐的跨上皮运输。最近电生和电中和碳酸氢钠共转运蛋白(NBC)的克隆、功能表达和免疫定位为研究其功能调控的分子机制提供了机会。电生碳酸氢钠共转运体kNBC1是近端小管基底外侧碳酸氢盐外流的主要途径。NBC1基因功能突变的丧失会导致一种严重的常染色体隐性遗传性近端肾小管酸中毒。目前，关于KNBC-1致电活性的结构基序和调节其功能的生物重要蛋白质相互作用的信息很少。在最近的研究中，我们已经证明了依赖于PKA的C-末端Ser982残基的磷酸化通过改变其HCO3-：Na+的化学计量比从3：1改变到2：1来改变kNBC1的生电活性。在Ser982的邻近区域，结构分析揭示了一个含有天冬氨酸残基的带电区域，这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kNBC1C末端的这个负电区是与转运蛋白中的一个碳酸氢盐结合位点(2：1模式)，还是与假定的结合蛋白(3：1模式)相互作用。在此基础上，利用酵母双杂交技术，以KNBC-1的C末端为诱饵，筛选出人肾cDNA文库，并分离出天冬氨酸氨基转移酶。天冬氨酸氨基转移酶有几个N-端的碱性残基，这些残基可以通过静电方式介导其与kNBC1的C-端的相互作用。天冬氨酸氨基转移酶定位于近端小管细胞的基侧膜上，并与来自肾脏的KNBC-1免疫共沉淀。依赖于PKA的kNBC1-Ser982的磷酸化阻止了蛋白质之间的相互作用。此外，在有N-乙酰天冬氨酸存在的情况下，KNBC-1和天冬氨酸氨基转移酶共转染的细胞中，KNBC1的功能显著增强。我们将使用MPCT细胞系作为实现本提案目标的模型系统。这个项目的成功完成将加深我们对调节H+/碱基转运蛋白的机制的了解。

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.