CHARACTERIZATION OF NA-DEPENDENT HCO3-TRANSPORTERS

描述(改编自申请人的摘要)：NA偶联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)表示与各自的结构同源 其他的。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全长7.6kb，编码一种蛋白质 表观分子量为116kD，并包含与PKA的共识结合位点， PKC和酪蛋白激酶II。功能数据支持更多 NbC的一种异构体(e.E.肾与肝)，这一点得到了 NBC-1mRNA在肾脏中的丰度及其在肝脏和肝脏中的缺失 胃。当前提案的目的是克隆和描述 钠偶联HCO3转运体(NBC异构体和钠依赖的Cl/HCO3 交换)在哺乳动物组织中。这项建议的具体目标包括： 1.钠偶联HCO3转运蛋白的克隆及功能表达； 急性和慢性阻塞性肺疾病中Na-偶联HCO3转运体的特性和调控 慢性状态；3.检测NBC-1的结构与功能关系。 目的1)他们计划克隆钠依赖的Cl/HCO3交换器和NBC 并在培养的HEK293细胞中表达。这些领域的工作 正在进行中。目标2)他们将以NBC-1(和NBC亚型)为特征 关于化学计量学、抑制谱、离子特异性和检查 磷酸酶和激酶途径对其的急性调节作用 细胞。NBC-1的慢性调节将通过mRNA和蛋白质来检验 在酸碱紊乱中含量丰富。目标3)他们将检查 递进DNA截断(在C-末端)区域对phi敏感性和 突变型cDNA对NBC-1细胞的调控洞察 钠偶联HCO3转运体的调控和基因结构应加强 我们对细胞调节和酸碱平衡的知识。

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.