Structure/function analysis of the na/bicarbonate cotransporters

摘要肾脏的任务之一是通过回收过滤后的碳酸氢盐并制造和储存新的碳酸氢盐来维持细胞和全身酸碱平衡。电致钠/碳酸氢盐共转运体（NBCe1）在近端小管重吸收碳酸氢盐中起重要作用，其中80%以上的过滤碳酸氢盐被回收。NBCe1的电致功能对这种碳酸氢盐重吸收至关重要，它为钠和碳酸氢盐穿过近端小管细胞的基底外膜产生驱动力。NBCe活性的改变导致严重的近端肾小管酸中毒以及青光眼和白内障。本项目的长期目标是阐明NBCe1电致钠/碳酸氢盐转运的分子机制。我们建议对NBCe1进行综合结构/功能分析，并确定对电原性至关重要的结构域和氨基酸残基。实验设计是：1)系统地构建一系列由NBCe1和电子中性的钠/碳酸氢盐转运体NBCn1组成的嵌合转运体，将钠和碳酸氢盐转移到细胞中；2)通过与NBCn1的序列比较构建NBCe1的点突变体。有三个具体目标。在目的1中，我们将鉴定影响电原性的NBCe1的跨膜结构域。嵌合转运体将通过与NBCn1的同源区域交换NBCe1的单个跨膜结构域来构建。这些嵌合转运体的功能将通过测量表达蛋白质的爪蟾卵母细胞的pH回收率和碳酸氢盐依赖电流来分析。在目标2中，我们将确定影响电原性的氨基酸残基。我们将在确定的结构域内选择可能严重改变电致功能并使其发生突变的残基。在目标3中，我们将区分功能必需的跨膜结构域和氨基酸与结构支持的氨基酸。这将通过将NBCe1的已识别结构域/氨基酸重组为电中性转运体来完成。这项工作将有助于通过NBCe1建立电致钠/碳酸氢盐运动的分子模型

DESCRIPTION (provided by applicant): ABSTRACT One of the tasks of the kidney is to maintain cellular and total body acid-base homeostasis by reclaiming filtered bicarbonate and making and saving new bicarbonate. The electrogenic sodium/bicarbonate cotransporter (NBCe1) plays a significant role in reabsorbing bicarbonate in the proximal tubules, where more than 80% of filtered bicarbonates are reclaimed. Electrogenic function of NBCe1 is essential for this bicarbonate reabsorption, producing a driving force for sodium and bicarbonate exit across the basolateral membranes of the proximal tubule cells. Altered activities of NBCe cause severe proximal renal tubule acidosis as well as glaucoma and cataracts. The long-term goal of this project is to elucidate molecular mechanisms for electrogenic sodium/bicarbonate transport of NBCe1. We propose to perform an integrated structure/function analysis of NBCe1 and identify the structural domains and amino acid residues that are essential for electrogenicity. The experimental designs are i) to systematically construct a series of chimeric transporters from NBCe1 and the electroneutral sodium/bicarbonate transporter NBCn1 that moves sodium and bicarbonate into the cell, and ii) to construct point mutants of NBCe1 by sequence comparison with NBCn1. There are three specific aims. In Aim 1, we will identify the transmembrane domains of NBCe1 that affect electrogenicity. Chimeric transporters will be constructed by swapping individual transmembrane domains of NBCe1 with the homologous regions of NBCn1. The function of those chimeric transporters will be analyzed by measuring the pH recovery rate and bicarbonate-dependent currents in Xenopus oocytes expressing the proteins. In Aim 2, we will determine amino acid residues affecting electrogenicity. We will select the residues, within identified domains, that might severely alter electrogenic function and mutate them. In Aim 3, we will distinguish functionally essential transmembrane domains and amino acids from structurally supportive ones. This will be done by reconstituting identified domains/amino acids of NBCe1 into the electroneutral transporter. The proposed work will help develop molecular models of electrogenic sodium/bicarbonate movement via NBCe1