BIOLOGY OF SODIUM BICARBONATE TRANSPORT

说明（改编自申请人的摘要）：碳酸氢钠 肾脏中的协同转运蛋白有助于维持整个身体 酸碱平衡这些整合的膜蛋白发挥着重要的作用 在调节细胞内pH和介导跨上皮钠和 重碳酸盐转运我们对功能特性的理解 这些转运蛋白在最近克隆了几个成员后得到了改善 碳酸氢钠cotranport（NBC）系列。我们最近克隆了 NBC 3是NBC家族的一个独特成员， 二苯乙烯不敏感，电中性，和EIPA可降解。在肾脏中，NBC 3是 在A型闰细胞中分选到顶膜， B型闰细胞的基底侧膜。此外，NBC 3 与液泡H+-ATP酶共定位和共免疫沉淀， 共有PDZ相互作用结构域，-埃塔尔和-DTAL，它们可以 在膜分选中起重要作用。在此基础上，我们筛选了一名 使用NBC 3的C-末端作为 诱饵，并分离出一种新的蛋白质，NBC 3BP，它具有三个潜力， PDZ结合结构域。NBC 3BP定位于A型细胞的顶膜 插入细胞并与来自肾脏的NBC 3共免疫沉淀。我们 假设NBC 3和液泡H+-ATP酶58 kD亚基被分类为 A型闰细胞的顶膜，通过它们之间的相互作用， 与PDZ结合蛋白NBC 3BP的C-末端PDZ相互作用结构域， 在该细胞类型的顶膜上表达，而在B型中不表达 闰细胞我们将使用MDCK I细胞系，其分选NBC 3BP和NBC 3BP。 NBC 3以顶膜类似A型闰细胞为模型 为实现本提案的目标。成功完成本 该项目将提高我们对负责的分子机制的理解 用于确定酸碱转运蛋白的极性。

DESCRIPTION (Adapted from the Applicant's Abstract): Sodium bicarbonate cotransporters in the kidney contribute to the maintenance of whole body acid-base homeostasis. These integral membrane proteins play an important role in regulating intracellular pH and in mediating transepithelial sodium and bicarbonate tranport. Our understanding of the functional characteristics of these transporters has improved following the recent cloning of several members of the sodium bicarbonate cotranport (NBC) family. We have recently cloned and functionally characterized NBC3, a unique member of the NBC family which is stilbene-insensitive, electroneutral, and EIPA-inhibitable. In kidney, NBC3 is sorted to the apical membrane in type A intercalated cells and to the basolateral membrane in type B intercalated cells. Furthermore, NBC3 co-localized and co-immunoprecipitated with the vacuolar H+-ATPase have consensus PDZ-interacting domain, -ETAL and -DTAL respectively, which could play an important role in membrane sorting. On this basis, we screened a human kidney cDNA library in a yeast two-hybrid assay using the C-terminus of NBC3 as bait, and isolated a novel protein, NBC3BP, which has three potential PDZ-binding domains. NBC3BP was localized to the apical membrane of type A intercalated cells and was co-immunoprecipitated with NBC3 from kidney. We hypothesize that NBC3 and 58 kD subunit of the vacuolar H+-ATPase are sorted to the apical membrane of type A intercalated cells, via interaction of their C-terminal PDZ-interacting domains with the PDZ-binding protein, NBC3BP, which is expressed on the apical membrane of this cell type and not in type B intercalated cells. We will use the MDCK I cell line which sorts NBC3BP and NBC3 to the apical membrane similar to the type A intercalated cell as a model system for achieving the goals of this proposal. Successful completion of this project will enhance our understanding of the molecular mechanisms responsible for determining the polarity of acid-base transporters.