Structure/function analysis of the na/bicarbonate cotransporters

描述（由申请人提供）：摘要肾脏的任务之一是通过回收过滤的碳酸氢盐并产生和储存新的碳酸氢盐来维持细胞和全身酸碱平衡。产电钠/碳酸氢盐协同转运蛋白（NBCe 1）在近端小管重吸收碳酸氢盐中起着重要作用，其中超过80%的过滤的碳酸氢盐被回收。NBCe 1的产电功能对于这种重吸收是必不可少的，产生驱动力，使钠和重碳酸盐穿过近端小管细胞的基底外侧膜离开。NBCe的活性改变会导致严重的近端肾小管酸中毒以及青光眼和白内障。该项目的长期目标是阐明NBCe 1的产电钠/碳酸氢盐转运的分子机制。我们建议进行NBCe 1的结构/功能的综合分析，并确定结构域和氨基酸残基，是必不可少的产电。实验设计是i）系统地构建来自NBCe 1和将钠和碳酸氢盐移动到细胞中的电中性钠/碳酸氢盐转运体NBCn 1的一系列嵌合转运体，以及ii）通过与NBCn 1的序列比较构建NBCe 1的点突变体。有三个具体目标。在目标1中，我们将确定NBCe 1的跨膜结构域，影响产电性。嵌合转运蛋白将通过将NBCe 1的单个跨膜结构域与NBCn 1的同源区域交换来构建。这些嵌合转运蛋白的功能将通过测量表达蛋白的爪蟾卵母细胞中的pH恢复速率和碳酸氢盐依赖性电流来分析。在目标2中，我们将确定影响产电性的氨基酸残基。我们将选择的残基，在确定的结构域，可能会严重改变产电功能和突变。在目标3中，我们将区分功能上必需的跨膜结构域和氨基酸结构上的支持。这将通过将NBCe 1的鉴定结构域/氨基酸重组到电中性转运蛋白中来完成。拟议的工作将有助于开发通过NBCe 1的产电钠/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