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响应几种环境线索，包括Na+，Cl-，质子，蛋白酶和剪切应力。ENaC细胞外结构域与细胞外环境相互作用以感知这些信号。因此，具有ENaC结构的准确模型对于理解细胞外因子对ENaC的调节至关重要。酸敏感离子通道1（ASIC 1）是唯一与ENaC同源的蛋白质，其结构已被报道。ENaC亚基胞外结构域与相应的ASIC 1结构域在很大程度上同源，除了所谓的指状结构域，其中ENaC亚基具有大的插入。ENaC指结构域与通道对Na+、蛋白酶和剪切应力的响应有关。这个建议的目标是改善后的ENaC胞外结构域的结构模型？亚单位拟议的研究将调查的结构相互作用，涉及一段残基，其中包括两个半胱氨酸的指状结构域插入。然后我们将使用这些信息来完善ENaC的结构模型？亚单位我们先前假设这两个指状结构域半胱氨酸形成二硫键桥接对，但我们的初步数据与这一概念相矛盾。实验提出了定义这些半胱氨酸和通道中的其他特定位点之间的特定相互作用，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和联合收割机的基础上同源ASIC 1和其他可用的数据来完善ENaC模型的约束距离的限制？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.