Regulatory Interactions of CFTR and ENaC

描述（由申请人提供）：囊性纤维化跨膜传导调节剂（CFTR）是一种多功能蛋白，可在上皮细胞的顶质膜上运输氯化物。CFTR还调节其他蛋白质的离子运输，如上皮钠通道（Epithelial Sodium Channel, ENaC）。CFTR抑制ENaC在气道上皮内的钠转运，而囊性纤维化（CF）的主要特征之一是气道上皮内ENaC的过度活跃。然而，CFTR和ENaC调控相互作用发生的机制尚不清楚。对CFTR突变体功能的药理学修复研究主要集中在评估CFTR突变体氯离子转运特性的恢复，而在很大程度上忽略了CFTR的其他功能，如与ENaC的相互调节。因此，药物修复突变CFTR功能是否也会恢复CFTR和ENaC的关键调控相互作用仍然是一个悬而未决的问题。因此，了解这些关键调控相互作用的分子基础是实施改善CFTR功能的药理学策略的关键。也有人认为CFTR通过降低ENaC开放概率（Po）抑制ENaC活性。相比之下，我们的初步数据强烈表明，CFTR和ENaC也可能具有与细胞内运输相关的调节相互作用。因此，我们将验证以下假设：CFTR和ENaC的调节相互作用导致上皮细胞中这些通道的细胞内运输和表面表达的改变。修复突变CFTR的药物制剂可能调节这些贩运相互作用。一些此类药物可能通过改变细胞质分子伴侣的表达来影响贩运。由于CFTR和ENaC的某些胞质结构域对调节相互作用至关重要，伴侣蛋白与这些胞质结构域的结合可能调节这种相互作用。本提案将建立在这些调查和初步数据的基础上，并针对以下具体目标进行研究，以解决这一假设：1)确定CFTR和ENaC在上皮细胞中相互调节细胞内运输的动力学机制。2)确定胞质70千道尔顿分子伴侣影响和调节上皮细胞CFTR和ENaC转运相互作用的机制。相关性：肺和气道保护自身免受环境影响的主要机制取决于呼吸上皮中适当的离子运输。这种离子转运在囊性纤维化中是异常的，并导致显著的发病率和死亡率。这些数据将促进更好地了解囊性纤维化肺病的发病机制，以及许多其他气道疾病，以及其他分泌性上皮疾病。

DESCRIPTION (provided by applicant): The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a multifunctional protein that transports chloride across the apical plasma membrane of epithelial cells. CFTR also regulates ion transport by other proteins, such as the Epithelial Sodium Channel, ENaC. CFTR inhibits sodium transport by ENaC in airway epithelia, and one of the cardinal features of Cystic Fibrosis (CF) is hyperactivity of ENaC in the airway epithelia. However, the mechanism by which regulatory interactions of CFTR and ENaC occurs is not clear. Efforts at pharmacologic repair of mutant CFTR function have concentrated on assessing restoration of a mutant CFTR's chloride transport properties and have largely ignored other functions of CFTR, such as its interregulation with ENaC. It thus remains an open question whether pharmacologic repair of mutant CFTR function will also restore the critical regulatory interactions of CFTR and ENaC. Understanding the molecular basis of these critical regulatory interactions is therefore key in the implementation of pharmacologic strategies to improve CFTR function. Others have suggested that CFTR inhibits ENaC activity by decreasing ENaC open probability (Po). In contrast, our preliminary data strongly suggests that CFTR and ENaC may also have regulatory interactions related to intracellular trafficking. We will therefore test the hypothesis that: Regulatory interactions of CFTR and ENaC result in altered intracellular trafficking and surface expression of these channels in epithelial cells. Pharmacologic agents that repair mutant CFTR may modulate these trafficking interactions. Some such agents may influence trafficking by altering the expression of cytosolic molecular chaperones. As certain cytosolic domains of CFTR and ENaC appear critical for regulatory interactions, binding of chaperones to these cytosolic domains may regulate such interactions. The present proposal will build on these investigations and preliminary data and address this hypothesis with studies directed at the following Specific Aims: 1) To determine the kinetic mechanism by which CFTR and ENaC regulate each other's intracellular trafficking in epithelial cells. 2) To determine the mechanism by which cytosolic 70 kilodalton molecular chaperones influence and regulate these trafficking interactions of CFTR and ENaC in epithelial cells. Relevance: The major mechanism by which the lung and airway defends itself from the environment depends on proper ion transport in the respiratory epithelia. Such ion transport is aberrant in Cystic Fibrosis and leads to significant morbidity and mortality. These data will promote better understanding of the pathogenesis of not only Cystic Fibrosis lung disease, but also a number of other diseases of the airway, as well as diseases of other secretory epithelia.