Sodium Entry into Amiloride-Sensitive Epithelia

描述（由申请方提供）：钠转运上皮细胞，如肾远端小管和集合小管，具有控制全身稳态的功能。上皮钠通道（ENaC）已经在其他钠转运上皮中发现，例如，唾液腺、结肠、支气管和气管上皮以及许多非上皮细胞如淋巴细胞、神经元和星形胶质细胞中。阿米洛利抑制是这些特殊通道的标志，无论它们是在什么系统中发现的。然而，当使用膜片钳技术检查宏观和单通道特性时，出现了无数的生物物理特性。本申请的中心假设是观察到的阿米洛利敏感性钠通道的功能多样性部分地由离子通道的变性蛋白（DEG）/ENaC超家族的亚基的不同组合引起。有三个具体目标。在第一个具体的目标，我们将detremine的生物化学成分的阿米洛利敏感的阳离子通道中发现的上皮细胞，表现出不同于ENaC的生物物理特性。我们目前的初步数据，使用RT-PCR分析表明，各种DEG/ENaC memebers的信息存在于这些细胞。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组成。此外，我们将采用表面生物素化，表面化学发光，共免疫沉淀分析，和大分子组装测定，以验证亚基相互作用。我们还将使用细胞蛋白质敲除方法（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试剂敏感性鉴定ENaC/ASIC相互作用。第三个具体的目标将确定高分辨率的晶体结构？ENaC。这些结果将为阿米洛利敏感性钠通道的性质和最终调节提供新的见解，以及通过插入或删除DEG/ENaC超家族的特定亚基来调节这些混合通道的方式。因此，了解ENaC多样性的分子基础将为日益增多的EWNaC相关疾病的治疗干预提供独特的机会。 公共卫生相关性：上皮钠通道（ENaC）蛋白和酸敏感离子通道（ASIC）蛋白分布于人体各处，负责维持人体的盐和水平衡，在高血压、癌症、学习等许多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，我们的研究结果将提供对这些通道在天然组织中，特别是在肾上皮中的细胞机制的深入了解。这项特别研究的相关性延伸到常染色体隐性遗传性多囊肾病，在美国每20，000名婴儿中就有1名患有这种疾病。

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.