Structure/function analysis of the na/bicarbonate cotransporters

na/碳酸氢盐协同转运蛋白的结构/功能分析

• 批准号: 7931611
• 负责人: INYEONG CHOI
• 金额: $ 24.75万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931611
• 关键词: Acid-Base Equilibrium; Acids; Address; Affect; Amino Acids; Attention; Bicarbonates; Buffers; C-terminal; Cataract; Cells; Chimera organism; Chronic; Classification; Data; Dependence; Electrodes; Elements; Epithelial Cells; Experimental Designs; Glaucoma; Goals; Hand; Homeostasis; Individual; Kidney; Limb structure; Measures; Mediating; Metabolic acidosis; Molecular; Molecular Models; Movement; Mutate; Mutation; Oocytes; Pathology; Pathway interactions; Play; Positioning Attribute; Property; Proteins; Recovery; Regulation; Renal tubular acidosis; Research Personnel; Role; Series; Side; Sodium Bicarbonate; Sodium-Bicarbonate Symporters; Structure; Testing; Thick; Transmembrane Domain; Work; Xenopus oocyte; abstracting; base; basolateral membrane; driving force; extracellular; immunocytochemistry; insight; molecular modeling; mutant; protein expression; reconstitution; stoichiometry; voltage clamp

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

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制造和保存新的碳酸氢盐来维持细胞和全身酸碱稳态。生电钠/碳酸氢盐协同转运蛋白 (NBCe1) 在近端肾小管重吸收碳酸氢盐方面发挥着重要作用，其中超过 80% 的过滤碳酸氢盐被回收。 NBCe1 的生电功能对于碳酸氢盐重吸收至关重要，为钠和碳酸氢盐穿过近端小管细胞的基底外侧膜排出提供驱动力。 NBCe 活性的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 NBCe1 电化学钠/碳酸氢盐运输的分子机制。我们建议对 NBCe1 进行综合结构/功能分析，并确定对生电性至关重要的结构域和氨基酸残基。实验设计是i)系统地构建一系列由NBCe1和电中性钠/碳酸氢盐转运蛋白NBCn1组成的嵌合转运蛋白，将钠和碳酸氢盐转移到细胞中，ii)通过与NBCn1进行序列比较来构建NBCe1的点突变体。具体目标有三个。在目标 1 中，我们将识别影响生电性的 NBCe1 跨膜结构域。通过将 NBCe1 的各个跨膜结构域与 NBCn1 的同源区域交换来构建嵌合转运蛋白。这些嵌合转运蛋白的功能将通过测量表达蛋白质的非洲爪蟾卵母细胞中的 pH 恢复率和碳酸氢盐依赖性电流来分析。在目标 2 中，我们将确定影响生电性的氨基酸残基。我们将在已识别的结构域内选择可能严重改变产电功能并使其突变的残基。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性的跨膜结构域和氨基酸。这将通过将 NBCe1 的已识别结构域/氨基酸重建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 NBCe1 产生电的钠/碳酸氢盐运动的分子模型