NA+ REABSORPTION AND H+ ION SECRETION RENAL MECHANISMS

总体目标是提供详细的了解， 阿米洛利耐药Na+/H+亚型的表征， 在肾上皮组织中的功能重要性。具体目标 包括：A.确定阿米洛利耐药的关键功能域 NHE亚型。cDNA构建体将被稳定表达，并且定点 诱变、截短、缺失沿着交联实验， 有限的胰蛋白酶溶解和蛋白质印迹定量将 用于确定阿米洛利相互作用的位点，阳离子（底物） 结合位点，关键官能团，包括组氨酸和 谷氨酸和关键的细胞质和跨膜结构域。B。到 确定阿米洛利耐药的表达和活性的调节 NHE亚型。明确的饮食和激素控制（高与 低盐饮食，甲状腺激素给药5天，慢性 代谢性酸中毒）将用于长期调节NHE水平 同种型体内表达。蛋白质印迹、北方印迹、RNA酶 将使用保护试验、免疫沉淀和表面标记 以确定稳态mRNA水平和特定亚型的变化 蛋白质表达对NHE的这些体内扰动的响应 表情C.鉴定、克隆、稳定表达和表征 阿米洛利耐药NHE亚型。商业化cDNA文库将 筛选并对克隆进行测序和分析。PCR方法将 用于染色体定位，以确定这些是否是唯一的 同种型。部分序列信息将用于比对 比较以描述关于井的定义的感兴趣的域 特征异构体。然后可以获得全长构建体， 在小鼠胰岛β细胞中稳定表达。动力学研究（阳离子， 胞浆pH敏感性、协同性）和抑制剂敏感性 （阿米洛利类似物，西咪替丁）将进行。D.来定义 阿米洛利耐药NHE同种型的组织特异性表达。多 组织北方和西方印迹将允许比较相对的 在各种大鼠和人体组织之间的丰度使用同种型- 特异性cDNA探针和亲和纯化的抗体。 免疫组织化学将确定特定的细胞膜分布。 使用亲和纯化的大鼠皮质小管制备物中的同种型 抗体的这些研究将提供对结构的独特见解- NHE亚型的功能关系，并确定其慢性调节 在特定的肾单位节段中。

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.