Structural Aspects of Bicarbonate Transport Metabolons

描述(由申请人提供)：负责上皮HCO3转运的机制对肾脏生理学非常重要。肾小管重新吸收过滤后的HCO3，生成“新的HCO3”，以维持全身酸碱平衡。HCO3-转运体NBCe1和AE1以及碳酸氢酶CA II和CAIV是在肾小管重吸收HCO3过程中起重要作用的蛋白质。最近对AE1的研究和NBCel的初步数据表明，这些HCO3-转运体与CA II和CA IV结合，形成HCO3-转运体代谢物。该项目的具体目的是用X射线结晶学来描述1)CA II和HCO3-转运体之间的细胞内相互作用，以及2)CA IV和HCO3-转运体之间的细胞外相互作用的结构基础。该方法将产生与AE1和NBCe1的CA-II和CA-IV结合域相对应的重组多肽，并将它们与各自的CA共结晶。此外，还将使用固相结合和下拉分析等技术来表征蛋白质-蛋白质相互作用的结合特性。了解CaS和HCO3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，并可能揭示它们之间的相互作用如何影响AE1和NBCE1的功能。拟议的研究将提供关于肾脏HCO3转运更精细机制的关键细节，这将有助于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.