CHARACTERIZATION OF NA-DEPENDENT HCO3-TRANSPORTERS

描述（改编自申请人的摘要）：Na-偶联的HCO 3 转运蛋白与AE亚型有相似之处（即HCO 3和 对DIDS抑制的敏感性），表明与每个 其他. 他们克隆了人类肾脏Na：HCO 3协同转运蛋白（NBC-1） 基于与AE家族的同源性。 NBC-1全长7.6kb，编码一种蛋白质， 表观分子量为116 kD，含有PKA的共有结合位点， PKC和酪蛋白激酶II。 功能数据支持更多 NBC的一种以上同种型（e. e.肾脏与肝脏），这一点得到了证实， 肾脏中NBC-1 mRNA的丰度和肝脏中的缺失， 胃 当前提案的目的是克隆和表征 Na偶联HCO 3转运蛋白（NBC亚型和Na依赖性Cl/HCO 3 交换）。 该提案的具体目标包括： 1. 钠偶联HCO 3转运蛋白的克隆和功能表达; 2. 急性心肌梗死患者Na-偶联HCO 3转运蛋白的特征和调控 慢性状态;和3. 研究NBC-1的结构与功能关系。 目的1）他们计划克隆Na依赖的Cl/HCO 3交换器和NBC 同种型并在培养的HEK 293细胞中表达它们。 这些领域的工作 正在进行 目的2）他们将用以下方法表征NBC-1（和NBC同种型）： 化学计量、抑制特性、离子特异性和检查 磷酸酶和激酶途径对转染细胞急性调节作用 细胞 将通过mRNA和蛋白质水平检测NBC-1的慢性调节 在酸碱失衡中大量存在。 （3）将研究 进行性DNA截短（在C末端）区域对pHi敏感性的影响， 用突变cDNA转染的细胞中的NBC-1的调节。 洞察 钠偶联HCO 3转运蛋白的调控和基因结构应增强 我们对细胞调节和酸碱平衡的了解。

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.