Physiologic role of the gamma epithelial sodium channel subunit in the kidney

描述（由申请人提供）：上皮性钠通道（ENaC）在远端肾元中表达，在那里它们是钠重吸收的最终位点，因此在细胞外液容量和血压（BP）的调节中起关键作用。ENaC功能获得突变与高血压有关，而功能丧失突变与低血压有关。enac由三个结构相关的亚基组成，称为？,吗?和?。这些亚基在内质网（ER）内组装成三聚体，在内质网退出后，亚基进行翻译后加工。特别是，在细胞外结构域的特定位点上的切割。蛋白酶furin和prostasin的亚基在异种系统中激活ENaC中起关键作用，可能是通过在亚基的外域释放抑制结构域。我们建议进行实验，首先确定？ENaC通过制造小鼠远端肾细胞特异性？即破坏。接下来的研究将评估？ENaC亚基蛋白在体内水解产生表达？ENaC亚单位在远端肾元缺乏关键的确定的蛋白水解裂解位点的furin或前列腺素。具体目标是：1。缺乏？肾中ENaC表达细胞选择性表达ENaC。拟议的研究旨在产生一种小鼠模型，这种模型已经破坏了？ENaC在肾元中的表达。为了实现这一目标，将使用两只小鼠：1)一只小鼠在收集管的主要细胞或整个肾元中表达Cre重组酶；2)含有loxp -侧翼外显子的小鼠？博的功能。饲养这些动物会导致远端肾素敲除？钠。这些吗?ENaC敲除动物将允许我们定义的作用？ENaC在调节血压、肾脏电解质排泄和集管离子运输中起作用。2. 肾脏特异性重构野生型或突变型小鼠的产生和表征？钠。这些研究的目的是确定？ENaC蛋白水解在体内调节ENaC活性。前列腺素依赖性切割？亚基，配合糠蛋白裂解，充分激活通道。拟议的研究旨在产生一种表达a ？在肾元远端选择性的肾素或前列腺素切割位点发生突变的亚基。要做到这一点，原生肾？ENaC将会被破坏？重构ENaC表达；1) a ？在furin切割位点发生突变的亚基；2) a ？在前列腺蛋白酶裂解位点发生突变的亚基；还是野生型？子单元（作为控制）。介绍的？亚基将带有表位标签，以方便检测和蛋白水解处理。这些动物将允许我们(i)定义蛋白质水解加工的程度？在基础条件下和在体积耗尽的情况下，前列腺素或furin切割位点的亚基；（二）确定？功能通道表达中前列腺素或糠蛋白裂解位点的亚基蛋白水解过程。公共卫生相关性：拟议的研究旨在确定肾脏上皮钠通道切割的生理作用。这些研究将确定钠通道内两个不同的卵裂位点在调节肾脏钠和钾排泄和维持正常血压中的重要性。这项工作有可能揭示参与发展和/或维持高血压和钾排泄障碍的机制。

DESCRIPTION (provided by applicant): Epithelial Na channels (ENaC) are expressed in the distal nephron where they serve as the final sites of Na reabsorption, thereby playing a key role in the regulation of extracellular fluid volume and blood pressure (BP). ENaC gain-of-function mutations are associated with hypertension, whereas loss-of-function mutations are associated with hypotension. ENaCs are comprised of three structurally related subunits termed ?, ? and ?. These subunits are assembled into a trimer within the endoplasmic reticulum (ER) and, following ER exit, the subunits undergo post-translational processing. In particular, cleavage at defined sites of the extracellular domain of the ? subunit by the proteases furin and prostasin has a key role in activating ENaC in heterologous systems, presumably by releasing an inhibitory domain within the subunit's ectodomain. Experiments are proposed to first define the functional role of ? ENaC in vivo by generating mice with distal nephron-specific ? ENaC disruption. These will be followed by studies assessing the functional role of ? ENaC subunit proteolysis in vivo by generating mice that express ? ENaC subunits in the distal nephron that lack key defined proteolytic cleavage sites for furin or prostasin. The specific aims are: 1. Generation and characterization of mice lacking ? ENaC selectively in ENaC-expressing cells in the kidney. The proposed studies are directed at generating a mouse model that has disrupted ? ENaC expression in the nephron. To accomplish this, two mice will be used: 1) a mouse expressing Cre recombinase either in principal cells of the collecting duct or in the entire nephron; and 2) a mouse containing loxP-flanked exons critical to ? ENaC function. Breeding of these animals will result in distal nephron-knockout of ? ENaC. These ? ENaC knockout animals will allow us to define the role of ? ENaC in the regulation of blood pressure, renal electrolyte excretion, and collecting duct ion transport. 2. Generation and characterization of mice with renal-specific reconstitution of wild-type or mutant ? ENaC. These studies are designed to define the role of ? ENaC proteolysis in the in vivo regulation of ENaC activity. Prostasin-dependent cleavage of the ? subunit, in concert with furin cleavage, fully activates the channel. The proposed studies are directed at generating a mouse model that expresses a ? subunit with mutations in the furin or prostasin cleavage sites selectively within the distal nephron. To accomplish this, native renal ? ENaC will be disrupted and ? ENaC expression reconstituted using; 1) a ? subunit with a mutation in the furin cleavage site; 2) a ? subunit with a mutation within the prostasin cleavage site; or 3) a wild type ? subunit (as a control). The introduced ? subunits will bear epitope tags to facilitate detection and proteolytic processing. These animals will allow us to (i) define the extent of proteolytic processing of the ? subunit at the prostasin or furin cleavage sites under basal conditions and in the setting of volume depletion; and (ii) determine the role of ? subunit proteolytic processing at the prostasin or furin cleavage sites in the expression of functional channels. PUBLIC HEALTH RELEVANCE: The proposed studies are designed to determine the physiologic role of cleavage of the epithelial sodium channel in the kidney. The studies will define the importance of two different cleavage sites within the sodium channel in regulation of kidney sodium and potassium excretion and maintenance of normal blood pressure. This work has the potential to uncover mechanisms involved in the development and/or maintenance of hypertension and disorders of potassium excretion.