Sodium Entry into Amiloride-Sensitive Epithelia

描述（由申请人提供）：钠转运上皮，如肾远端和集合小管，具有控制全身稳态的功能。上皮钠通道（Epithelial sodium channels, ENaC）存在于其他钠转运上皮中，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。阿米洛利抑制作用是这些特殊通道的标志，不管它们在哪个系统中被发现。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性。本应用的中心假设是，观察到的酰胺敏感钠通道的功能多样性部分是由于离子通道的Degenerin(DEG)/ENaC超家族的亚基的不同组合。有三个具体目标。在第一个特定目标中，我们将确定在上皮细胞中发现的具有不同于ENaC的生物物理特性的阿米洛利敏感阳离子通道的生化组成。我们使用rt - pcr分析的初步数据显示，这些细胞中存在多种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ENaC/ASIC杂交的生物物理特性，b.)使用MTS试剂敏感性鉴定ENaC/ASIC相互作用。第三个具体目标是确定高分辨率晶体结构？钠。这些结果将为了解酰胺敏感钠通道的性质和最终调控提供新的见解，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越来越多的ewac相关疾病的治疗干预提供独特的机会。

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.