Molecular mechanisms of the electrogenic Na+ Bicarbonate Cotransporter (NBCe1)

产电碳酸氢钠协同转运蛋白 (NBCe1) 的分子机制

• 批准号: 7335536
• 负责人: MICHAEL F. ROMERO
• 金额: $ 33.89万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7335536
• 关键词: Molecular; mechanisms; electrogenic; Na+; Bicarbonate

DESCRIPTION (provided by applicant): Regulation of acid-base homeostasis (blood pH ~7.4) and Na+ homeostasis is critical for animal life. In all vertebrates, including mammals, homeostasis is achieved through strict regulation of levels of NaHCO3. The electrogenic Na+ bicarbonate cotransporter, NBCel, is a major regulator of NaHCO3 levels through its transport activity at the basolateral membrane of the renal proximal tubule. The importance of this transporter is shown by naturally occurring, recessive, point mutations (R298S , R510H, S427L) in human kidney NBCel (hkNBCel), which cause profound proximal renal tubular acidosis (pRTA), glaucoma and cataracts. Blood pH < 7.1 and [HCO3-] < 11 mM in these patients indicate that hkNBCel is THE major HC03 absorption path of the kidney. However, the mechanism by which NBCel inactivation leads to pRTA and ocular pathologies is unclear. Our preliminary experiments with hkNBCel mutations show that ion affinities are localized to discrete areas of the NBCel protein. Biophysical analysis and uncompensated pRTA (the mutant NBCel phenotype), indicate that kNBCel has a major role in respiratory compensation as well as renal transport. Because of the compelling phenotype of patients with single amino acid mutations in kNBCel, we propose using kNBCel for biophysical experiments designed to reveal regions of the protein responsible for components of its function. Evidence from human mutations shows definitively that single amino acid changes in hkNBCel drastically alter its activity. We hypothesize that examination of the function of hkNBCel bearing additional sequence modifications will identify critical subdomains responsible for its function. This information can be used to design therapeutic agents targeted to those subdomains to modify the activity of this critical transporter to treat metabolic acidosis, glaucoma and cataracts. To investigate this hypothesis we will pursue 3 aims. First, we will functionally test our structural model by evaluating biophysical properties of mutations in the N-terminus, new human NBCel mutations and the role of NBCel dimers. Second, we will determine the functional roles of the isoform specific N-termini of NBCel. Third, we will use chimeras of human kNBCel with fugu-NBCel to delimit ion binding and/or permeation paths via the NBCel transmembrane domain of the protein. Lay Public Health statement: NBCel is the protein in the kidney responsible for absorbing sodium bicarbonate (baking soda). Human NBCel mutations cause severe kidney disease (metabolic acidosis) and eye disease (glaucoma and cataracts). NBCel from a salt-water puffer fish has some dramatic functional differences though the protein is only slightly different from human NBCel. We will use these NBCel mutations and human/fish differences to determine how this protein causes kidney and eye disease and how to modify its activity to prevent or treat disease.

描述（由申请方提供）：酸碱平衡（血液pH值约为7.4）和Na+平衡的调节对动物生命至关重要。在所有脊椎动物中，包括哺乳动物，体内平衡是通过严格调节NaHCO 3的水平来实现的。产电Na+碳酸氢盐协同转运蛋白NBCel是通过其在肾近端小管基底外侧膜的转运活性来调节NaHCO 3水平的主要调节剂。这种转运蛋白的重要性通过人肾NBCel（hkNBCel）中天然存在的隐性点突变（R298 S、R510 H、S427 L）显示，其引起深度近端肾小管酸中毒（pRTA）、青光眼和白内障。在这些患者中，血液pH < 7.1和[HC 0 3-] < 11 mM表明hkNBCel是肾脏的主要HC 0 3吸收途径。然而，NBCel失活导致pRTA和眼部病理的机制尚不清楚。我们对hkNBCel突变的初步实验表明，离子亲和力定位于NBCel蛋白的离散区域。生物物理分析和未补偿pRTA（突变NBCel表型）表明，kNBCel在呼吸补偿以及肾转运中具有主要作用。由于kNBCel中具有单个氨基酸突变的患者的引人注目的表型，我们建议使用kNBCel进行生物物理实验，旨在揭示负责其功能组分的蛋白质区域。来自人类突变的证据明确地表明，hkNBCel中的单个氨基酸变化极大地改变了其活性。我们假设检查带有额外序列修饰的hkNBCel的功能将鉴定负责其功能的关键亚结构域。这些信息可用于设计靶向这些亚结构域的治疗剂，以改变这种关键转运蛋白的活性，从而治疗代谢性酸中毒、青光眼和白内障。为了研究这个假设，我们将追求三个目标。首先，我们将通过评估N-末端突变的生物物理性质、新的人NBCel突变和NBCel二聚体的作用来功能性地测试我们的结构模型。其次，我们将确定NBCel亚型特异性N末端的功能作用。第三，我们将使用人kNBCel与fugu-NBCel的嵌合体来界定经由蛋白质的NBCel跨膜结构域的离子结合和/或渗透路径。公众健康声明：NBCel是肾脏中负责吸收碳酸氢钠（小苏打）的蛋白质。人类NBCel突变导致严重的肾脏疾病（代谢性酸中毒）和眼部疾病（青光眼和白内障）。来自咸水河豚的NBCel具有一些显著的功能差异，尽管该蛋白质与人类NBCel仅略有不同。我们将利用这些NBCel突变和人类/鱼类的差异来确定这种蛋白质如何导致肾脏和眼睛疾病，以及如何改变其活性以预防或治疗疾病。