NA+ REABSORPTION AND H+ ION SECRETION RENAL MECHANISMS

总体目标是提供详细的理解和 抗阿米洛利钠氢异构体的特性 肾上皮组织的功能重要性。具体目标 包括：A.确定阿米洛利耐药的关键功能结构域 他是一种异构体。构建的cdna将稳定表达，并且是定点定向的。 突变、截断、缺失以及交联性实验， 限制性胰酶降解和蛋白质定量的蛋白质印迹分析 用于确定阿米洛利相互作用、阳离子(底物)的位置 结合部位，关键官能团，包括组氨酸和 谷氨酸，以及关键的细胞质和细胞膜跨域。B.到 确定阿米洛利耐药基因的表达和活性调节 他是一种异构体。明确的饮食和正常操作(高与 低盐饮食，甲状腺激素治疗5天，慢性 代谢性酸中毒)将用于慢性调节NHE水平 体内异构体表达。蛋白质印迹、蛋白质印迹、核糖核酸酶 将使用保护分析、免疫沉淀和表面标记 定义稳态信使核糖核酸水平和特定亚型的变化 NHE在体内对这些扰动的蛋白表达 表情。C.鉴定、克隆、稳定表达和鉴定 耐阿米洛利的NHE亚型。商业cDNA文库将被 将对筛选和克隆进行测序和分析。聚合酶链式反应方法将 用于染色体定位，以确定它们是否是唯一的 异构体。部分序列信息将用于比对 描述与油井相关的已定义的感兴趣领域的比较 特征化的异构体。然后可以获得全长的构建体，并且 在小鼠LAP细胞中稳定表达。动力学研究(阳离子、 胞液pH敏感性、协作性)和抑制剂敏感性 (阿米洛利类似物，西咪替丁)将进行。D.定义 耐阿米洛利NHE异构体的组织特异性表达。多重 组织Northern和Western杂交将允许比较亲缘关系 大鼠和人的各种组织之间的丰度使用亚型- 特异的cDNA探针和亲和纯化的抗体。 免疫组织化学将定义特定的细胞膜分布 亲和纯化的大鼠皮质小管制剂中的异构体 抗体。这些研究将提供对该结构的独特见解- NHE异构体的功能关系及其慢性调节 在特定的肾单位节段。

The overall goal is to provide a detailed understanding and characterization of the amiloride-resistant Na+/H+ isoforms which are of functional importance in renal epithelial tissues. The SPECIFIC AIMS include: A. To define critical functional domains of amiloride-resistant NHE isoforms. cDNA constructs will be stably expressed, and site-directed mutagenesis, truncations, deletions along with cross linking experiments, limited trypsinolysis and protein quantification with Western blots will be used to define the sites of amiloride interation, cation (substrate) binding sites, critical functional groups including histidine and glutamate, and critical cytoplasmic and membrane spanning domains. B. To define the regulation of expression and activity of amiloride-resistant NHE isoforms. Well defined dietary and hormal manipulations (high versus low salt diet, thyroid hormone administration for 5 days, chronic metabolic acidosis) will be used to chronically modulate the level of NHE isoform expression in vivo. Western blots, Northern blots, RNase protection assays, immunoprecipitation and surface labeling will be used to define changes in steady-state mRNA levels and in specific isoform protein expression in response to these in vivo pertubations of NHE expression. C. To identify, clone, stably express and characterize amiloride-resistant NHE isoforms. Commercial cDNA libraries will be screened and clones will be sequenced and analyzed. PCR methods will be used for chromosomal locaiizaton to determine if these are unique isoforms. Partial sequence information will be used for alignment comparisons to describe defined domains of interest with respect to well characterized isoforms. Full length constructs can then be obtained and stably expressed in mouse LAP- cells. Studies of the kinetics (cations, cytosolic pH sensitivity, cooperativity), and inhibitor sensitivity (amiloride analogues, cimetidine) will be carried out. D. To define the tissue specific expression of amiloride-resistant NHE isoforms. Multiple Tissue Northern and Western blots will permit comparisons of relative abundance between a variety of rat and human tissues using isoform- specific cDNA probes and affinity purified antibodies. Immunohistochemistry will define the membrane distribution of specific isoforms in rat cortical tubule preparations using affinity purified antibodies. The studies will provide unique insights into the structure- function relationships of NHE isoforms and define their chronic regulation in specific nephron segments.