SODIUM ENTRY INTO AMILORIDE-SENSITIVE EPITHELIA

拟议研究的主要目标是阐明 导电性钠离子进入的分子基础 存在于外(或顶端)质膜中的机制 所有的高电阻上皮细胞中。这个钠的入口 步骤是整个主动运输的重要组成部分 负责盐分和水的净移动的系统 这些纸巾。这个进入过程是被动的，是受监管的 对激素敏感，对利尿剂的抑制敏感 阿米洛利。具体目标分为三类。第一, 将进行研究，以确定生物化学和 生理上分离纯化的Na+通道蛋白。 这些研究将需要改进隔离和 纯化方案，亚基数目的测定，分子 阿米洛利的重量、碳水化合物组成和位置 结合部位(S)。研究通道蛋白是否可以 在体外和体内被磷酸化或甲基化，将 也将被承担。第二，将进行研究，分析 利用Na+进入通道的动力学特性 测量中单通道属性的重建过程 平面脂双层和脂泡内。这些实验将 阐明通道的阳离子选择性、性质、 侧性，以及阿米洛利块的电压依赖性，以及 无论是通道蛋白PER的磷酸化还是甲基化 SE具有任何功能后果。第三，单克隆化 针对纯化的Na+通道的多克隆抗体 蛋白质将会被制备出来。这些抗体将被用来构建 免疫亲和柱进一步完善通道分离 协议，并作为探针定位通道蛋白在不同的 细胞类型(培养的A6上皮细胞和大鼠皮质收集 小管)。这些研究将有助于我们了解 它的动力学、生化和生理特性 无处不在的交通系统，并增加我们对 阿米洛利和其他利尿剂化合物的作用方式。

The main goal of the proposed research is to elucidate the molecular basis of operation of the conductive sodium entry mechanism that exists in the outer (or apical) plasma membrane of all electrically high resistance epithelia. This sodium entry step is an essential component of the overall active transport system responsible for the net movement of salt and water across these tissues. This entry process is passive, is regulated hormonally, and is sensitive to inhibition by the diuretic drug amiloride. The specific aims fall into three categories. First, studies will be done to characterize both biochemically and physiologically the isolated and purified Na+ channel protein. These studies will entail improvements in the isolation and purification scheme, determination of subunit number, molecular weight, carbohydrate composition, and location of amiloride binding site(s). Investigations of whether the channel protein can be phosphorylated or methylated, both in vitro and in vivo, will also be undertaken. Second, studies will be done to analyze the kinetic characteristics of the Na+ entry channel, utilizing reconstitution procedures to measure single channel properties in planar lipid bilayers and in lipid vesicles. These experiments will elucidate the cation selectivity of the channel, the nature, the sidedness, and the voltage-dependence of the amiloride block, and whether phosphorylation or methylation of the channel protein per se has any functional consequence. Third, monoclonal and polyclonal antibodies directed against the purified Na+ channel protein will be prepared. These antibodies will be used to build an immunoaffinity column to improve further the channel isolation protocols, and as probes to localize channel protein in different cell types (cultured A6 epithelia and rat cortical collecting tubule). These studies will contribute to our knowledge of the kinetic, biochemical, and physiological properties of this ubiquitous transport system, and increase our understanding of the mode of action of amiloride and other diuretic compounds.