CHARACTERIZATION OF NA-DEPENDENT HCO3-TRANSPORTERS

NA 依赖性 HCO3 转运蛋白的表征

• 批准号: 2906284
• 负责人: MANOOCHER SOLEIMANI
• 金额: $ 22.83万
• 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/2906284
• 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耦合HCO3 转运蛋白与AE同工型共享相似之处（即HCO3和HCO3的运输 对dids抑制的敏感性）指示每个人的结构同源性 其他。 他们克隆了人类肾脏NA：HCO3共转运蛋白（NBC-1） 基于与AE家族的同源性。 NBC-1为7.6 kb，编码蛋白质 明显的MW为116 kD，并包含PKA共识结合位点， PKC和酪蛋白激酶II。 功能数据支持更多 NBC的一种同工型（E.E.肾脏和肝脏），这是由 NBC-1 mRNA在肾脏中及其在肝脏中的缺失和 胃。 当前建议的目的是克隆和表征 Na耦合的HCO3转运蛋白（NBC同工型和Na依赖性CL/HCO3 交换）在哺乳动物组织中。 该提案的具体目的包括： 1。Na耦合HCO3转运蛋白的克隆和功能表达； 2。 Na耦合HCO3转运蛋白在急性和调节中的表征和调节 慢性国家；和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 同工型并在培养的HEK293细胞中表达它们。 这些领域的工作 正在进行中。 目标2）他们将以NBC-1（和NBC同工型）来表征 尊重化学计量，抑制谱，离子特异性并检查 它在转染中对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和蛋白质检查 酸碱疾病的丰度。 目标3）他们将检查 PHI敏感性和 用突变cDNA转染的细胞中NBC-1的调节。 深入了解 NA耦合HCO3转运蛋白的调节和基因结构应增强 我们对细胞调节和酸碱稳态的了解。