Sodium Entry into Amiloride-Sensitive Epithelia

描述（由申请人提供）：钠转运上皮细胞，例如肾远端和肾小管，具有控制全身稳态的功能。上皮钠通道 (ENaC) 已在其他钠转运上皮细胞中发现，例如唾液腺、结肠、支气管和气管上皮细胞，以及许多非上皮细胞，如淋巴细胞、神经元和星形胶质细胞。阿米洛利抑制是这些特定通道的一个标志，无论它们存在于哪个系统中。然而，当使用膜片钳技术检查宏观和单通道特性时，会出现无数的生物物理特征。本申请的中心假设是，观察到的阿米洛利敏感钠通道的功能多样性部分是由离子通道 Degenerin(DEG)/ENaC 超家族亚基的不同组合造成的。具体目标有三个。在第一个具体目标中，我们将确定在上皮细胞中发现的阿米洛利敏感阳离子通道的生化组成，该通道表现出与 ENaC 不同的生物物理特性。我们使用 RT-PCR 分析提供初步数据，显示这些细胞中存在各种 DEG/ENaC 成员的信息。因此，我们将使用这些细胞作为模型系统来确定该通道的生化成分。此外，我们将采用表面生物素化、表面化学发光、免疫共沉淀分析和大分子组装分析来验证亚基相互作用。我们还将使用细胞蛋白敲除方法（和 MTS 试剂敏感性研究）来建立亚基相互作用。第二个具体目标是：a.) 确定混合 ENaC/ASIC 的生物物理特征，b.) 使用 MTS 试剂敏感性识别 ENaC/ASIC 相互作用。第三个具体目标将确定 ?ENaC 的高分辨率晶体结构。这些结果将为了解阿米洛利敏感钠通道的性质和最终调节，以及通过插入或删除该 DEG/ENaC 超家族的特定亚基来调节这些混合通道的方式提供新的见解。因此，了解 ENaC 多样性的分子基础将为不断增加的 EWNaC 相关疾病的治疗干预提供独特的机会。 公共健康相关性：上皮钠通道 (ENaC) 蛋白和酸敏感离子通道 (ASIC) 蛋白分布在人体各处，负责维持体内的盐和水平衡，并在高血压、癌症、学习和许多其他正常和异常过程中发挥作用。通过分子生物学、生物化学、细胞生物学和电生理学的现代技术，我们的研究结果将深入了解天然组织（尤其是肾上皮细胞）中这些通道的细胞机制。这项特殊研究的相关性延伸到常染色体隐性遗传性多囊肾病，这种疾病影响着美国每 20,000 名婴儿中就有 1 名。

DESCRIPTION (provided by applicant): Sodium transporting epithelia, such as renal distal and collecyting tubules, function to control whole-body homeostasis. Epithelial sodium channels (ENaC) have been found in other sodium transportinmg epithelia, e.g., salivary glands, colon, bronchial and tracheal epithelia, as well as in many non-epithelial cells, like lymphocytes, neurons and astrocytes. Amiloride inhibition is a hallmark of these particular channels, regardless of the system in which they are found. Yet, when macroscopic and single channel properties are examined using the patch clamp technique, a myriad of biophysical characteristics emerge. The central hypothesis of this application is that the observed functional diversitiy of amiloride-sensitive sodium channels results, in part, from different combinations of subunits of the Degenerin(DEG)/ENaC superfamily of ion channels. There are three specific aims. In the first specific aim, we will detrermine the biochemical composition of an amiloride-sensitive cation channel found in epithelial cells that exhibit biophysical properties different from ENaC. We present preliminary data using RT-PCRprofiling showing that message for a variety of DEG/ENaC memebers are present in these cells. Thus, we will use these cells as ,model systems to determine the biochemical composition of this channel. In addition, we will employ surface biotinylation, surface chemiluminescence, co-immunoprecipitation analysis, and macromolecular assembly assays, to verify subunit interactions. We will also use ellular protein knockout approaches, (and MTS reagent susceptibility studies), to establish subunit interaction. The second specific aim is to: a.) determine the biophysical characteristics of hybrid ENaC/ASIC, b.) identify ENaC/ASIC interaction using MTS reagent susceptibility. The third specific aim will determine the high resolution crystal structure of ?ENaC. These results wil offer new insights into the nature and ultimately the regulation of amiloride-sensitive sodium channels, and the ways that these hybrid channels can be modulated by inserting or deleting specific subunits of this DEG/ENaC superfamily. Thus understanding the molecular basis for ENaC diversity will provide unique opportunities for therapeutic interventions in an ever-increasing plethora of EWNaC-related diseases. PUBLIC HEALTH RELEVANCE: Epithelial sodium channel (ENaC) proteins and acid-sensing ion channel (ASIC) proteins are distributed all over the human body, are responsible for maintaining the body's salt and water balance, and play a role in hypertension, cancer, learning, and many other normal and abnormal processes. Through modern techniques of molecular biology, biochemistry, cell biology, and electrophysiology, our study results will provide insight into the cellular mechanisms of these channels in native tissues, especially in renal epithelia. The relevance of this particular study extends to autosomal recessive polycystic kidney disease, which affects 1 in 20,000 babies in the United States.