CHARACTERIZATION OF NA-DEPENDENT HCO3-TRANSPORTERS

NA 依赖性 HCO3 转运蛋白的表征

• 批准号: 6178160
• 负责人: MANOOCHER SOLEIMANI
• 金额: $ 23.52万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6178160
• 关键词: acidity /alkalinity; bicarbonates; cell line; electrophysiology; ion transport; laboratory rat; membrane transport proteins; molecular cloning; nucleic acid sequence; phosphoprotein phosphatase; protein binding; protein isoforms; protein kinase; protein structure function; sodium ion; stoichiometry; transfection

DESCRIPTION (Adapted from the Applicant's Abstract): Na-coupled HCO3 transporters share similarities with AE isoforms (i.e. transport of HCO3 and sensitivity to inhibition by DIDS) indicating structural homology with each other. They have cloned the human kidney Na:HCO3 cotransporter (NBC-1) based on homology with the AE family. NBC-1 is 7.6 kb, encodes a protein with an apparent MW of 116 kD, and contains consensus binding sites for PKA, PKC, and Casein kinase II. Functional data support the presence of more than one isoform of NBC (e.e. kidney versus liver) and this is confirmed by the abundance of NBC-1 mRNA in the kidney and its absence in the liver and stomach. The purpose of the current proposal is to clone and characterize the Na-coupled HCO3 transporters (NBC isoforms and Na-dependent Cl/HCO3 exchange) in mammalian tissues. The Specific Aims of this proposal include: 1. Cloning and functional expression of Na-coupled HCO3 transporters; 2. Characterization and regulation of Na-coupled HCO3 transporters in acute and chronic states; and 3. Examining structure-function relationship of NBC-1. Aim 1) They plan to clone the Na-dependent Cl/HCO3 exchanger and NBC isoforms and express them in cultured HEK293 cells. The work on these areas is in progress. Aim 2) They will characterize NBC-1 (and NBC isoforms) with respect to stoichiometry, inhibitory profile, ion specificity and examine its acute regulation by phosphatase and kinase pathways in transfected cells. Chronic regulation of NBC-1 will be examined by mRNA and protein abundance in acid-base disorders. Aim 3) They will examine the effect of progressive DNA truncation (on the C-terminal) region on pHi sensitivity and regulation of NBC-1 in cells transfected with mutant cDNAs. Insight into regulation and gene structure of Na-coupled HCO3 transporters should enhance our knowledge on cell regulation and acid-base homeostasis.

描述（改编自申请人的摘要）：Na-偶联的 HCO3 转运蛋白与 AE 亚型有相似之处（即 HCO3 和 对 DIDS 抑制的敏感性）表明与每个的结构同源性 其他。 他们克隆了人类肾脏 Na:HCO3 协同转运蛋白 (NBC-1) 基于与AE家族的同源性。 NBC-1 为 7.6 kb，编码蛋白质 具有 116 kD 的表观分子量，并包含 PKA 的共有结合位点， PKC 和酪蛋白激酶 II。 功能数据支持更多的存在 比 NBC 的一种同种型（即肾脏与肝脏）不同，这一点得到了证实 肾脏中 NBC-1 mRNA 的丰度以及肝脏中的缺失，以及 胃。 当前提案的目的是克隆和表征 Na 偶联 HCO3 转运蛋白（NBC 异构体​​和 Na 依赖性 Cl/HCO3 交换）在哺乳动物组织中。 该提案的具体目标包括： 1. Na偶联HCO3转运蛋白的克隆及功能表达； 2. 急性和急性期 Na 偶联 HCO3 转运蛋白的表征和调节 慢性状态； 3.检查NBC-1的结构-功能关系。 目标 1) 他们计划克隆 Na 依赖性 Cl/HCO3 交换器和 NBC 同种型并在培养的 HEK293 细胞中表达它们。 这些领域的工作 正在进行中。 目标 2) 他们将描述 NBC-1（和 NBC 同工型） 关于化学计量、抑制谱、离子特异性并检查 其在转染过程中受磷酸酶和激酶途径的急性调节 细胞。 NBC-1的慢性调节将通过mRNA和蛋白质进行检查 丰富的酸碱紊乱。 目标 3) 他们将检查以下效果 渐进式 DNA 截断（C 端）区域对 pHi 敏感性的影响 NBC-1 在用突变 cDNA 转染的细胞中的调节。 洞察 Na 偶联 HCO3 转运蛋白的调控和基因结构应增强 我们关于细胞调节和酸碱稳态的知识。