NBCe1 and n1: Crystal Structure Determination

描述(由申请人提供)：碳酸氢盐(HCO3-)通过肾脏上皮细胞的转运对维持全身酸碱平衡非常重要。HCO3-转运蛋白NBCe1和NBCn1是完整的膜蛋白，它们分别在近端小管的HCO3重吸收和粗大上肢的NH4+重吸收中发挥重要作用。本项目的具体目标是：(1)利用X射线结晶学来阐明NBCe1和N1的N-末端结构域的结构；(2)探索唯一的124个残基外显子7在NBCn1中的生理作用；(3)建立NBCe1或NBCn1膜结构域的大规模真核表达系统，用于以后的结晶实验。我在NBCe1 N末端的纯化和结晶方面的初步工作正在为研究HCO3转运调控所需的重要结构信息铺平道路，而我的新的NBCn1剪接变体拓宽了我们目前对NBCn1在各种细胞类型中作用的理解。这项拟议的研究将为肾脏HCO3转运的分子机制提供关键的见解，并将增强我们对肾脏疾病基本过程的理解。

DESCRIPTION (provided by applicant): Bicarbonate (HCO3-) transport across epithelial cells of the kidney is of great importance to maintain systemic acid-base balance. The HCO3- transporters NBCe1 & NBCn1 are integral, membrane-proteins that, respectively, play important roles in HCO3- reabsorption in the proximal tubule and NH4+ reabsorption in the thick ascending limb. The specific aims of this project are: (1) To use X-ray crystallography to illuminate the structures of the N-terminal domains of NBCe1 and n1; (2) To explore the physiological roles in NBCn1 of the unique 124-residue exon 7, which may be spliced in or out of the protein; (3) To develop a large-scale, eukaryotic expression system for the membrane domain of NBCe1 or NBCn1, to be used later for crystallization trials. My preliminary work on the purification and crystallization of the N-terminus of NBCe1 is paving the way for vital structural information needed to study the regulation of HCO3- transport, and my new splice variants of NBCn1 broadens our current understanding of the role of NBCn1 in various cell types. The proposed study will provide key insights on the molecular mechanisms of renal HCO3- transport and will enhance our understanding of processes fundamental to renal disease.