SODIUM ENTRY INTO AMILORIDE-SENSITIVE EPITHELIA

拟议研究的主要目标是了解在分子 水平的机制和调节离子流通过导电Na + 进入通道存在于外（或顶端）质膜， 大多数Na+重吸收上皮细胞。 这个Na+进入通道是限速的 对于整个跨上皮Na+重吸收， 特别是通过肽类激素血管加压素和 类固醇激素醛固酮。 这种进入过程是被动的， 对利尿药阿米洛利的抑制作用敏感。 实验 旨在检验从A6细胞中分离的蛋白质 牛肾作为阿米洛利敏感的离子通道。 具体目的有五个：1）分析了运动学特性 这种纯化的推定的Na+通道蛋白，利用重建 测量平面脂质中的单通道特性的方法 双层。 这些实验将阐明的阳离子选择性 的通道、性质、侧性和电压依赖性。 阿米洛利阻断，以及是否磷酸化或甲基化的 蛋白质本身具有任何功能性后果。2)以确定 加压素和醛固酮对Na+通道的调节 通过检测功能性Na+通道密度的增加是否 从细胞质池募集通道或通过 激活已经存在于膜中的静止通道。3)到 检查皮质肌动蛋白丝网络的贡献及其 相关蛋白对上皮细胞Na+通道的调节 活动4）克隆和测序每个亚基的全长cDNA。 一旦完成，针对每个亚基的多克隆抗体将被制备。 为了研究的拓扑结构， 亚基在顶膜内。 使用cDNA探针和多克隆 抗体，信使RNA和蛋白质表达的调节， 加压素和醛固酮在肾A6细胞中的不同亚基 使用北方印迹分析，核糖核酸酶保护测定，和 将进行Western blot分析。5)为了确定函数 各亚基在阿米洛利敏感性Na+转运中的作用 反义寡核苷酸实验。 这些研究将进一步促进我们的 生理学、生物化学和分子生物学知识 这个无处不在的运输系统的属性，并增加我们的 了解阿米洛利和其他利尿剂的作用方式 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.