Structural Aspects of Bicarbonate Transport Metabolons

描述（由申请人提供）：负责上皮HCO 3转运的机制对肾脏生理学非常重要。肾小管重吸收过滤的HCO 3并产生“新的HCO 3”，以维持全身酸碱平衡。HCO 3转运蛋白NBCe 1和AE 1以及碳酸酐酶CA II和CAIV是在肾小管重吸收HCO 3中发挥重要作用的蛋白质。最近对AE 1的研究和NBCel的初步数据表明，这些HCO 3转运蛋白与CA II和CA IV结合，形成HCO 3转运代谢子。本项目的具体目标是使用X射线晶体学来描述1）CA II和HCO 3-转运蛋白之间的细胞内相互作用和2）CA IV和HCO 3-转运蛋白之间的细胞外相互作用的结构基础。该方法将产生对应于AE 1和NBCe 1的CA-II和CA-IV结合结构域的重组肽，并将其与各自的CA共结晶。此外，蛋白质-蛋白质相互作用的结合特性将使用固相结合和下拉测定等技术进行表征。了解CA和HCO 3-转运蛋白之间的相互作用将揭示这些蛋白如何相互作用的重要特征，并可能揭示它们的相互作用如何影响AE 1和NBCe 1的功能。拟议的研究将提供关于肾脏HCO 3转运的更精细机制的关键细节，这将提供更好地理解所研究的蛋白质对肾脏功能的生理学和重要性。

DESCRIPTION (provided by applicant): Mechanisms responsible for epithelial HCO3-transport are of great importance to renal physiology. The renal tubule reabsorbs filtered HCO3 and generates "new HCO3" in order to maintain systemic acid-base homeostasis. The HCO3-transporters NBCe1& AE1, and carbonic anhydrases CA II & CAIV are proteins that play important roles in HCO3-reabsorption by the renal tubule. Recent studies on AE1, and preliminary data on NBCel, suggest that these HCO3-transporters bind to both CA II and CA IV and form HCO3- transport metabolons. The specific aims of this project are to use X-ray crystallography to describe the structural basis of 1) the intracellular interaction between CA II and the HCO3-transporters, and 2) the extracellular interaction between CA IV and the HCO3-transporters. The approach will be to generate recombinant peptides that correspond to the CA-II and CA-IV binding domains of AE1 and NBCe1 and cocrystallize them with their respective CA. In addition, binding properties of the protein-protein interactions will be characterized using such techniques as solid-phase binding and pull-down assays. Understanding the interactions between CAs and HCO3-transporters will reveal important features of how these proteins interact with one another and may reveal how their interactions influence the function of AE1 and NBCe1.The proposed study will provide key details on the finer mechanisms of renal HCO3 transport, which will provide a better understanding of the physiology and importance of the studied proteins to kidney function.