SODIUM ENTRY INTO AMILORIDE-SENSITIVE EPITHELIA

拟议的研究的主要目标是在分子水平上理解 离子流过导电钠的机理及调控 外膜(或顶端)质膜中存在的进入通道 大多数是重吸收钠的上皮细胞。这个钠进入通道是限速的 对整个跨上皮钠的重吸收，并受到调节 在激素方面，特别是通过多肽激素血管加压素和 类固醇激素--醛固酮。这个进入过程是被动的，而且是 对利尿剂阿米洛利的抑制敏感。实验是 旨在检验从A6细胞中分离出的蛋白质 而牛肾起着阿米洛利敏感离子通道的作用。 具体目标有五个：1)运动特性分析 利用重组技术对纯化的可能的钠通道蛋白进行重组 平面脂质中单通道特性的测量方法 双层的。这些实验将阐明阳离子选择性。 其通道、性质、侧向和电压依赖性 阿米洛利阻断，以及是否磷酸化或甲基化 蛋白质本身具有任何功能后果。2)确定 加压素和醛固酮是否调节钠通道的生物化学 通过检测功能性钠通道密度的增加是否 从细胞质池中招募通道的结果或 激活已驻留在膜中的静止通道。3)至 研究皮质肌动蛋白细丝网络的作用及其 与上皮细胞钠通道调节相关的蛋白质 活动。4)克隆并测定各亚基的全长cDNAs。 一旦完成，针对每个亚基的多克隆抗体将 准备用来研究 顶膜内的亚基。使用cdna探针和多克隆 抗体、信使RNA和蛋白表达的调节 肾A6细胞中加压素和醛固酮的不同亚基 使用Northern印迹分析、核糖核酸酶保护分析和 将对蛋白质印迹分析进行研究。5)确定功能 正义膜和正义膜上各亚基在阿米洛利敏感钠转运中的作用 反义寡核苷酸实验。这些研究将进一步推动我们的 生理、生化和分子生物学知识 这一无处不在的交通系统的特性，并增加我们的 对阿米洛利等利尿剂作用方式的认识 化合物。

The main goal of the proposed research is to understand at the molecular level the mechanisms and regulation of ion flow through conductive Na+ entry channels that exist in the outer (or apical) plasma membrane of most Na+ reabsorbing epithelia. This Na+ entry channel is rate-limiting for overall transepithelial Na+ reabsorption, and is regulated hormonally, specifically by the peptide hormone vasopressin and the steroid hormone aldosterone. This entry process is passive, and is sensitive to inhibition by the diuretic drug amiloride. Experiments are designed to test the hypothesis that the protein isolated from A6 cells and bovine kidney functions as an amiloride-sensitive ion channel. There are five specific aims: 1) To analyze the kinetic characteristics of this purified putative Na+ channel protein, utilizing reconstitution procedures to measure single channel characteristics in planar lipid bilayers. These experiments will elucidate the cation selectivity of the channel, the nature, the sidedness, and voltage dependence of the amiloride block, and whether phosphorylation or methylation of the protein per se has any functional consequence. 2) To determine biochemically whether vasopressin and aldosterone regulate Na+ channels by examining whether the increased density of functional Na+ channels results from a recruitment of channels from a cytoplasmic pool or by activation of quiescent channels already resident in the membrane. 3) To examine the contribution of the cortical actin filament network and its associated proteins to the regulation of epithelial Na+ channel activity. 4)To clone and sequence full-length cDNA's of each subunit. Once accomplished, polyclonal antibodies against each subunit will be prepared in order to investigate the topological arrangement of the subunits within the apical membrane. Using cDNA probes and polyclonal antibodies, the regulation of messenger RNA and protein expression of the different subunits by vasopressin and aldosterone in renal A6 cells using Northern blot analysis, ribonuclease protection assays, and Western blot analysis will be studied. 5) To determine the functional role of each subunit in amiloride-sensitive Na+ transport in sense and anti-sense oligonucleotide experiments. These studies will further our knowledge of the physiological, biochemical, and molecular biological properties of this ubiquitous transport system, and increase our understanding of the mode of action of amiloride and other diuretic compounds.