Allosteric ENaC Regulation

描述（申请人提供）：上皮钠离子通道（ENaC）在调节细胞外液容量和血压方面发挥关键作用。ENaC属于感测外部环境的离子通道家族，并且其响应于包括Na+、Cl-、质子、蛋白酶和剪切应力的若干环境线索。这些通道如何对这些细胞外信号作出反应还知之甚少。该提案的目标是确定外部Na+和蛋白酶调节ENaC的分子机制。它们联合收割机产生组成型活性的特定通道池，以及仅在细胞外Na+浓度低时招募的其他通道。基于结构模型的拟议研究将确定这两种因素的分子调控机制。将定义Na+和抑制肽的结合位点，以及Na+或抑制肽结合后诱导的动态变化。实验将测试酸性口袋是否具有Na+抑制性结合位点，以及与酸性口袋相邻的区域是否具有抑制性肽的结合位点。对于诱导通道活性变化的任一效应物，它们必须在结合后诱导局部构象变化，这些变化传播到通道孔。实验提出了使用化学交联和静电效应来捕获通道的功能构象。了解细胞外因子如何调节ENaC的机制将有助于我们了解ENaC的功能调节以及相关通道如何感知环境。

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 and it responds to several environmental cues including Na+, Cl-, protons, proteases and shear stress. How these channels respond to these extracellular cues is poorly understood. The goal of this proposal is to determine the molecular mechanisms of ENaC regulation by external Na+ and proteases. They combine to create specific pools of channels that are constitutively active, and others that are recruited only when extracellular Na+ concentrations are low. The proposed studies based on structural models will determine molecular mechanisms of regulation by both factors. Binding sites for Na+ and an inhibitory peptide will be defined, as well as dynamic changes induced upon binding of Na+ or an inhibitory peptide. Experiments will test whether an acidic pocket hosts a Na+ inhibitory binding site, and a region adjacent to the acidic pocket hosts the binding site for an inhibitory peptide. For either effector to induce changes in channel activity, they must induce local conformational changes upon binding that propagate to the channel pore. Experiments are proposed to trap functional conformations of the channel using chemical crosslinks and electrostatic effects. A mechanistic understanding of how extracellular factors regulate ENaC will inform our understanding of ENaC functional regulation and how related channels may sense their environments.