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

描述(申请人提供)：调节酸碱平衡(血液pH~7.4)和钠离子平衡对动物生命至关重要。在包括哺乳动物在内的所有脊椎动物中，动态平衡是通过严格调节NaHCO3水平来实现的。电生Na+碳酸氢盐共转运体NBCel通过其在肾近端小管基底外侧膜上的转运活性对NaHCO3水平起主要调节作用。这种转运蛋白的重要性体现在人类肾脏NBCel(HkNBCel)的自然发生的隐性点突变(R298S，R510H，S427L)上，这些突变会导致严重的近端肾小管酸中毒(PRTA)、青光眼和白内障。血pH 7.1和[HCO3-]11 mm提示hkNBCel是肾脏吸收HC03的主要途径。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，离子亲和力局限于NBCel蛋白的离散区域。生物物理分析和未补偿的PRTA(突变的NBCel表型)表明，kNBCel在呼吸补偿和肾脏运输中具有重要作用。由于kNBCel单一氨基酸突变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，以揭示负责其功能组件的蛋白质区域。来自人类突变的证据明确地表明，hkNBCel中单一氨基酸的变化会极大地改变其活性。我们假设，对带有额外序列修改的hkNBCel功能的检查将确定负责其功能的关键亚域。这些信息可以用来设计针对这些亚区的治疗剂，以改变这种关键转运蛋白的活性，以治疗代谢性酸中毒、青光眼和白内障。为了研究这一假设，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、新的人类NBCel突变和NBCel二聚体的作用来从功能上测试我们的结构模型。其次，我们将确定NBCEL的异构体特异性N末端的功能作用。第三，我们将使用人kNBCel与FUGU-NBCel的嵌合体来界定通过蛋白质的NBCel跨膜域的离子结合和/或渗透路径。Lay公共健康声明：NBCel是肾脏中负责吸收小苏打(小苏打)的蛋白质。人类NBCel突变会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有一些戏剧性的功能差异，尽管其蛋白质与人类的NBCel只有轻微的不同。我们将利用这些NBCel突变和人/鱼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来预防或治疗疾病。

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.