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端膜的速率决定步骤，在那里发生钠平衡的精细调节。ENaC在这一过程中的重要性可以通过遗传疾病来说明，在遗传疾病中，通道突变会导致盐和水稳态的缺陷。ENaC的克隆及其亚基结构的描述使得旨在确定通道功能和调节机制的实验成为可能。顶端膜ENaC密度的控制是一个重要的，如果不是中央，机制管理钠进入率，然而，我们不了解的途径和蛋白质相互作用，调节ENaC插入和回收在顶端膜。我们的工作表明，ENaC与公认的交通调节（SNARE）蛋白（syntaxins，munc 18，VAMP和半胱氨酸串蛋白，Csp）的物理相互作用（结合）提供了ENaC的插入和回收的调节。在这个项目中，我们将选择性地操纵极化皮质集合管细胞系（mCCD）和MDCK细胞中ENaC和SNARE（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;效果将被毒素不敏感的SNARE拯救。FRET将用于确定在调控的顶端ENaC插入中相互作用的SNARE对。将在ENaC和SNARE蛋白上定位结合位点，并表达无结合能力的ENaC亚基和SNARE，以选择性地评估这些相互作用在ENaC运输中的作用。我们将研究的假设，组成和调节交通途径介导的不同的SNARE对，ENaC和SNARE蛋白聚集在不同的微域，以调节顶端通道插入，和磷酸化的munc 18响应醛固酮或加压素控制突触融合蛋白的可用性顶端ENaC贩运。我们将评估ENaC-Csp-alphaGDI复合物控制介导ENaC插入的顶端Rab 3循环的可能性。借助功能性ENaC通道再循环的新检测方法，我们将表达蛋白质运输和降解中特定步骤的显性负调控因子，以确定调控顶端ENaC插入和再循环的控制点。将使用电流波动分析、膜片钳和细胞表面生物素化来评估在基础条件下以及响应于ENaC调节剂、加压素和醛固酮，操纵交通蛋白表达对极化mCCD和MDCK上皮中的顶端膜通道数目（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如何在正常条件下控制顶端膜通道密度，并响应Na转运的生理调节剂。

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.