NA/ION REABSORPTION AND H/ION SECRETION RENAL MECHANISMS

这项提议的长期目标是描述离子的特性 月牙根尖膜和基底外侧膜的转运过程 上皮细胞，特别关注占主导地位的因素 这些转运蛋白在细胞和亚细胞中的长期调节 水平。在本申请中要检验的特定假设是 Na/H逆向转运蛋白的两种相关形式在顶端表达 和肾上皮细胞的基侧膜。虽然相似的是 结构，甚至可能是同一基因的产物，这两种形式 有地区功能差异，可能与不同的 蛋白质的结构域。此外，我们假设在那里 是疏水分子中的关键基团(如组氨酸、赖氨酸、谷氨酸) 调节最大转运的阿米洛利结合部位区域 Na/H逆向转运体的速率及其与5-N取代物的特异性相互作用 阿米洛利类似物。 当前提案的具体目标是：1)定义功能 以及根尖形态和基底外侧形态之间的物理区别 人类Na/H逆向转运蛋白。2)克隆、测序和表达顶端和 人类NA/H逆向转运蛋白的基侧形式。3)研究 转录调控、酸碱干扰的逆向转运蛋白和其他 反转运蛋白活动的长期“监管者”。4)定义功能 A/H反向转运蛋白顶端和基侧形式的结构域，以及 将这些发现与这两种形式的功能特征联系起来 在特定目标1.5下确定)，以确定是否有“充电继电器” 钠和质子跨上皮转运的机制 通过Na/H逆向转运蛋白的顶端和基底侧向形式。 这些研究将提供对功能和调节的基本见解 在Na/H逆向转运蛋白的顶端和基底外侧形式中的作用 维持细胞内的pH，在极化的上皮系统中， 在质子和钠的矢量运输中。 这些实验将使用针对Na/H的亲和纯化抗体 反转运蛋白免疫纯化人肾交换器的顶端形式 BBMV，以及来自人胎盘膜囊泡的基侧形态。这个 从纯化的蛋白质中获得特定的氨基酸序列和 多肽将与从cDNA文库中获得的序列进行比较 由人类肾皮质和人类胎盘构建而成。转录的 Na/H逆向转运蛋白(S)活性的调节将在培养的 表达顶端和/或基底外侧交换器的细胞。站点特定 将进行诱变以检查基因的分子细节 各种形式的Na/H逆向转运蛋白的活性部位。离体 表达系统，青蛙卵母细胞，横切试验)将被用于评估 Na/H逆向转运蛋白及其极化的结构与功能 表情。

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.