Modulation of Electrogenic Sodium Bicarbonate Transport

碳酸氢钠协同转运蛋白有助于细胞内pH（pHi）调节和跨上皮转运 钠和碳酸氢盐的含量克隆、功能表达和免疫定位研究进展 产电和电中性碳酸氢钠共转运（NBC）蛋白提供了一个机会， 研究负责调节其功能的分子机制。产电钠 碳酸氢盐协同转运体kNBC 1是近端小管基底外侧碳酸氢盐流出的主要途径。损失 NBC 1基因的功能突变导致严重形式的常染色体隐性近端肾小管酸中毒。 目前缺乏关于负责产电性的结构基序的信息 以及调节其功能的生物学上重要的蛋白质相互作用。在最近的研究中，我们 已经证明C-末端Ser 982残基的PKA依赖性磷酸化改变了 通过将其HCO 3-：Na +化学计量从3：1改变到2：1来改变kNBC 1的产电性。在邻近的地区， Ser 982，结构分析揭示了一个带有天冬氨酸残基的带电区域，该区域可能潜在地发挥作用。 在这方面的重要作用。我们假设Ser 982的磷酸化状态决定了这是否是一种蛋白质。 在kNBC 1 C-末端的带负电荷的区域将与一个碳酸氢盐静电相互作用 转运蛋白中的结合位点（2：1模式）或推定的结合蛋白（3：1模式）。在此基础上，我们筛选了 使用kNBC 1的C末端作为诱饵，在酵母双杂交试验中构建人肾脏cDNA文库，并分离出 β-酰化酶天冬氨酸酰化酶的N端有几个碱性残基，它们可以介导天冬氨酸酰化酶的相互作用 与kNBC 1的C末端静电连接。天冬氨酸酰化酶定位于基底外侧膜， 近曲小管细胞，并与来自肾脏的kNBC 1共免疫沉淀。PKA依赖性磷酸化 kNBC 1-Ser 982阻止了蛋白质之间的相互作用。此外，kNBC 1的功能是 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。成功完成 本项目的研究将增强我们对H+/碱基转运蛋白调控机制的理解。

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.