Refining a structural model of ENaC

描述（由申请人提供）：上皮Na+通道（ENaC）在细胞外液容量和血压的调节中起关键作用。ENaC属于感知外部环境的离子通道家族。ENaC响应多种环境因素，包括Na+， Cl-，质子，蛋白酶和剪切应力。ENaC胞外结构域与胞外环境相互作用以感知这些信号。因此，有一个准确的ENaC结构模型对于理解ENaC通过细胞外因子调控至关重要。酸感离子通道1 （ASIC1）是目前报道的唯一与ENaC同源的蛋白。ENaC亚基胞外结构域与相应的ASIC1结构域在很大程度上是同源的，除了所谓的手指结构域，ENaC亚基在手指结构域有一个大的插入。ENaC手指结构域与通道对Na+、蛋白酶和剪切应力的反应有关。本提案的目标是改进ENaC细胞外结构域的结构模型。亚基。提出的研究将调查涉及残基延伸的结构相互作用，包括两个半胱氨酸在手指结构域插入。然后，我们将使用这些信息来完善ENaC的结构模型。亚基。我们之前假设这两个手指结构域半胱氨酸形成了二硫桥接对，但我们的初步数据与这一概念相矛盾。实验被提出来定义这些半胱氨酸和通道中其他特定位点之间以及这些半胱氨酸和抑制肽之间的特定相互作用。我们将从我们的数据中得出距离约束，并将它们与基于ASIC1同源性和其他可用数据的约束结合起来，以改进ENaC ？亚基胞外结构域。然后，我们将通过开发新的功能假设来评估我们的模型。

DESCRIPTION (provided by applicant): The epithelial Na+ channel (ENaC) has a key role in the regulation of extracellular fluid volume and blood pressure. ENaC belongs to a family of ion channels that sense the external environment. ENaC responds to several environmental cues including Na+, Cl-, protons, proteases and shear stress. The ENaC extracellular domains interact with the extracellular environment to sense these cues. Having an accurate model of ENaC structure is therefore critical to understanding ENaC regulation by extracellular factors. Acid sensing ion channel 1 (ASIC1) is the only protein homologous to ENaC whose structure has been reported. The ENaC subunit extracellular domains are largely homologous the corresponding ASIC1 domains, except for the so called finger domain where ENaC subunits have a large insertion. The ENaC finger domain has been implicated in the channel's response to Na+, proteases, and shear stress. The goal of this proposal is to improve upon a structural model of the extracellular domains of the ENaC ? subunit. The proposed studies will investigate the structural interactions that involve a stretch of residues that includes two cysteines in the finger domain insertion. We will then use this information to refine a structural model of the ENaC ? subunit. We previously hypothesized that these two finger domain cysteines formed a disulfide bridged pair, but our preliminary data contradicts this notion. Experiments are proposed to define specific interactions between these cysteines and other specific sites in the channel, and between these cysteines and an inhibitory peptide. We will derive distance constraints from our data and combine them with constraints based on homology to ASIC1 and on other available data to refine a model of the ENaC ? subunit extracellular domains. We will then assess our model by developing novel hypotheses of function to test.