NA/ION REABSORPTION AND H/ION SECRETION RENAL MECHANISMS

本提案的长期目标是表征离子 视网膜顶膜和基底膜的转运过程 上皮细胞，特别关注的因素，占 这些转运蛋白在细胞和亚细胞的长期调节 水平在本申请中待测试的具体假设是 Na/H反向转运蛋白的两种相关形式在顶膜表达， 和肾上皮细胞的基底外侧膜。虽然类似于 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，这可能与不同的 蛋白质的结构域。此外，我们假设存在 是关键组（例如，组氨酸、赖氨酸谷氨酸盐）， 调节最大转运的阿米洛利结合位点区域 Na/H反向转运蛋白的速率，并与5-N取代的 阿米洛利类似物。 本建议的具体目标是：1）界定职能 和身体之间的区别顶端和基底外侧形式的 人Na/H反向转运蛋白。2)克隆、测序并表达其顶端和 人NA/H反向转运蛋白的基底外侧形式。（3）研究 转录调节、酸碱扰动反向转运蛋白和其他 反转运蛋白活动的长期“调节者”。4)定义函数 的顶侧和基底侧形式的域， a/H反向转运蛋白，和 将这些发现与两种形式的功能特征联系起来， 具体目标#1。5)为了确定“充电继电器” 机制涉及钠和质子的跨上皮转运 由Na/H反向转运蛋白的顶端和基底侧形式所决定。 这些研究将提供基本的了解功能和调节 Na/H反向转运蛋白的顶端和基底侧形式，它们在 维持细胞内pH值，在极化上皮系统中， 质子和钠的矢量传输。 这些实验将使用亲和纯化的Na/H抗体， 反向转运蛋白免疫纯化人肾交换器的顶端形式 BBMV和来自人胎盘膜囊泡的基底外侧形式。的 从纯化的蛋白质获得的特定氨基酸序列， 将肽与cDNA文库获得的序列进行比较 由人肾皮质和人胎盘组成。转录 Na/H逆向转运蛋白活性的调节将在培养的 表达顶侧和/或基底侧交换器的细胞。位点特异性 诱变将进行检查的分子细节， 各种形式的Na/H反向转运蛋白的活性位点。体外 表达系统青蛙卵母细胞，横切测定）将用于评估 Na/H逆向转运蛋白的结构-功能及其极化 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.