NA/ION REABSORPTION AND H/ION SECRETION RENAL MECHANISMS

NA/离子重吸收和 H/离子分泌肾脏机制

基本信息

批准号：
3226361
负责人：
DAVID Gene WARNOCK
金额：
$ 16.83万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-09-01 至 1995-07-31
项目状态：
已结题

项目摘要

The long term objective of this proposal is to characterize the ion transport processes in apical and basolateral membranes of rental epithelial cells, with a specific focus on the factors which account for long term regulation of these transporters at the cellular and subcellular level. A specific hypothesis to be tested in the present application is that two related forms of the Na/H antiporter are expressed in the apical and basolateral membranes of renal epithelial cells. While similar in structure, and perhaps even the produce of the same gene, the two forms have district functional differences which may be related to distinct structural domains of the protein. In addition, we hypothesize that there are critical groups (e.., histidine, lysine glutamate) in a hydrophobic region of the amiloride-binding site which regulate the maximal transport rate of the Na/H antiporter and interact specifically with 5-N substituted amiloride analogues. The specific aims for the current proposal are: 1) To define the functional and physical distinctions between the apical and basolateral forms of the human Na/H antiporter. 2) To clone, sequence and express the apical and basolateral forms of the human NA/H antiporter. 3)To examine the role of transcriptional regulation, antiporter to acid-base perturbations and other long term "regulators" of antiporter activity. 4) To define the functional domains of the apical and basolateral form of the a/H antiporter, and relate these findings to the functional characteristics of the two forms as determined under specific aim #1. 5) To determine if a "charge relay" mechanism is involved in the transepithelial transport of sodium an protons by the apical and basolateral forms of the Na/H antiporter. These studies will provide basic insights into the function and regulation of the apical and basolateral forms of the Na/H antiporter, their roles in the maintenance of intracellular pH and, in polarized epithelial systems, in the vectorial transport of protons and sodium. These experiments will use affinity-purifed antibodies to the Na/H antiporter to immunopurify the apical form of the exchanger for human renal BBMV, and the basolateral form from human placental membrane vesicles. The specific amino acid sequences obtained from the purified proteins and peptide will be compared to sequences obtained for cDNA libraries constructed from human renal cortex, and human placenta. Transcriptional regulation of the Na/H antiporter(s) activity will be studied in cultured cells which express apical and/or basolateral exchangers. Site specific mutagenesis will be undertake to examine the molecular details of the active site of the various forms of the Na/H antiporter. In vitro expression systems frog oocytes, transection assays) will be used to assess the structure-function aspects of the Na/H antiporter and its polarized expression.

该提议的长期目标是表征离子租赁的顶端和基底外侧膜中的运输过程上皮细胞，具体侧重于考虑的因素这些转运蛋白在细胞和亚细胞上的长期调节等级。在本应用中要测试的特定假设是 Na/h抗胞剂的两种相关形式在顶端表达和肾上皮细胞的基底外侧膜。同时相似结构，甚至相同基因的产物，两种形式具有区域功能差异，可能与不同蛋白质的结构结构域。此外，我们假设那里是疏水性的关键组（E ..，组氨酸，赖氨酸谷氨酸）阿米洛里德结合部位的区域，该区域调节最大运输 Na/h抗胞药物的速率，并与5-N取代特别相互作用 Amiloride类似物。当前建议的具体目的是：1）定义功能以及顶端和基底外侧形式的物理区分人NA/H抗毒剂。 2）克隆，序列并表达顶端和人Na/H抗植物的基底外侧形式。 3）检查转录调节，抗酸碱扰动和其他长期的抗植物活性的“调节剂”。 4）定义功能 A/H抗植物的顶端和基底外侧形式的域，以及将这些发现与两种形式的功能特征相关联根据特定目标＃1确定。 5）确定是否“电荷继电器” 机理参与了质子钠的旋转运输由Na/H抗胞菌的顶端和基底外侧形式。这些研究将提供有关功能和调节的基本见解 Na/h抗胞剂的顶端和基底外侧形式，它们在维持细胞内pH，在极化上皮系统中，在质子和钠的矢量运输中。这些实验将使用与Na/H的亲和力抗体抗胞膜剂，以使人肾脏的交换器的顶端形式 BBMV和人胎盘膜囊泡的基底外侧形式。这从纯化的蛋白质获得的特定氨基酸序列和将肽与获得cDNA文库获得的序列进行比较由人类肾皮质和人胎盘建造。转录 Na/h抗植物活性的调节将在培养中研究表达顶端和/或基底外侧交换器的细胞。特定地点将进行诱变，以检查 Na/h抗胞菌的各种形式的活性位点。体外表达系统青蛙卵母细胞，横断测定）将用于评估 Na/h抗胞菌及其极化的结构功能方面表达。