Traffic regulatory Proteins and ENaC

上皮钠通道(ENaC)是钠进入远端肾单位上皮细胞顶膜的速率决定步骤，在那里发生了钠平衡的精细调节。ENaC在这一过程中的重要性通过遗传病得到了说明，在遗传病中，通道突变会导致盐和水的动态平衡缺陷。ENaC的克隆及其亚基结构的描述使旨在确定通道功能和调节机制的实验成为可能。顶膜ENaC密度的控制是控制Na进入速率的重要机制，但我们并不了解调节ENaC在顶膜插入和循环的途径和蛋白质相互作用。我们的工作表明，ENaC与公认的交通调节(SNARE)蛋白(Synaxins，MUNC 18，VAMPS和半胱氨酸串蛋白，CSP)的物理相互作用(结合)为ENaC的调节插入和循环提供了条件。在这个项目中，我们将有选择地操纵极化皮质收集管细胞系(MCCD)和MDCK细胞中ENaC与SNARES(和其他交通蛋白)之间的物理相互作用，以研究这些相互作用控制顶膜ENaC密度的生理机制。肉毒杆菌或破伤风毒素将提供对几种蛋白质活性的急性操纵；其影响将通过毒素不敏感的陷阱来挽救。FRET将被用来确定在调节的根尖ENaC插入中相互作用的圈套对。结合位点将被定位在ENaC和SNARE蛋白上，结合不具功能的ENaC亚基和SNARs将被表达，以选择性地评估这些相互作用在ENaC交通中的作用。我们将检验这样的假设，即构成和调节的交通通路由不同的SNAR对介导，ENaC和SNARE蛋白聚集在不同的微域中以调节心尖通道的插入，以及MUNC 18的磷酸化对醛固酮或加压素的响应控制着心尖ENaC运输的合成素的可用性。我们将评估ENaC-CSP-αGDI复合体控制介导ENaC插入的顶端Rab3循环的可能性。借助于一种新的功能ENaC通道循环的方法，我们将表达蛋白质运输和降解特定步骤的显性负调控因子，以确定调节顶端ENaC插入和循环的控制点。通过电流波动分析、膜片钳和细胞表面生物素化等方法，研究在基础状态下，以及在ENaC调节剂、加压素和醛固酮的作用下，交通蛋白的表达对极化的MCCD和MDCK上皮细胞顶膜通道数(N)和开放概率(P0)的影响。这些研究将确定ENaC与交通调节蛋白的相互作用在正常条件下如何控制心尖膜通道密度，以及对钠转运的生理调节剂的反应。

The epithelial sodium channel (ENaC) is the rate-determining step in Na entry at the apical membranes of distal nephron epithelial cells where the fine regulation of Na balance occurs. The importance of ENaC in this process is illustrated by genetic diseases, in which channel mutations produce defects in salt and water homeostasis. The cloning of ENaC and delineation of its subunit structure has enabled experiments designed to define the mechanisms of channel function and regulation. The control of apical membrane ENaC density is an important, if not central, mechanism governing Na entry rate; yet, we do not understand the pathways and protein interactions that regulate ENaC insertion and recycling at the apical membrane. Our work has indicated that physical interactions (binding) of ENaC with acknowledged traffic regulatory (SNARE) proteins (syntaxins, munc 18, VAMPs and cysteine string protein, Csp) provide for regulated insertion and recycling of ENaC. In this project, we will selectively manipulate the physical interactions between ENaC and SNAREs (and other traffic proteins) in a polarized cortical collecting duct cell line (mCCD) and in MDCK cells to examine the physiological mechanisms by which these interactions control apical membrane ENaC density. Botulinum or Tetanus toxins will provide for acute manipulation of the activity of several proteins; effects will be rescued by toxin-insensitive SNAREs. FRET will be used to determine SNARE pairs that interact in regulated apical ENaC insertion. Binding sites will be mapped on both ENaC and SNARE proteins and binding incompetent ENaC subunits and SNAREs will be expressed to selectively assess the role of these interactions in ENaC traffic. We will examine the hypotheses that constitutive and regulated traffic pathways are mediated by different SNARE pairs, that ENaC and SNARE proteins are clustered in different microdomains to regulate apical channel insertion, and that phosphorylation of munc 18 in response to aldosterone or vasopressin controls syntaxin availability for apical ENaC trafficking. We will evaluate the possibility that an ENaC-Csp-alphaGDI complex controls an apical Rab3 cycle that mediates ENaC insertion. With the help of a new assay for the recycling of functional ENaC channels, we will express dominant negative regulators of specific steps in protein trafficking and degradation to determine the control points for regulated apical ENaC insertion and recycling. Current fluctuation analysis, patch clamp and cell surface biotinylation will be used to assess the effects of manipulating traffic protein expression on apical membrane channel number (N) and open probability (P0) in polarized mCCD and MDCK epithelia under basal conditions and in response to the ENaC modulators, vasopressin and aldosterone. These studies will determine how ENaC's interactions with traffic regulatory proteins controls apical membrane channel density under normal conditions and in response to physiological modulators of Na transport.