Allosteric ENaC Regulation

描述(申请人提供)：上皮Na+通道(ENaC)在调节细胞外液容量和血压方面起着关键作用。ENAC是一个感知外部环境的离子通道家族，它对几种环境信号做出反应，包括Na+、Cl-、质子、蛋白酶和切应力。这些通道如何对这些细胞外信号做出反应，目前还知之甚少。这项建议的目的是确定外源Na+和蛋白水解酶调节ENaC的分子机制。它们结合在一起创造了特定的通道池，这些通道是结构性活跃的，而其他通道只有在细胞外Na+浓度较低时才会被招募。拟议中的基于结构模型的研究将确定这两个因素调节的分子机制。将定义Na+和抑制肽的结合位置，以及Na+或抑制肽结合时引起的动态变化。实验将测试酸性口袋是否有Na+抑制结合部位，以及与酸性口袋相邻的区域是否有抑制性多肽的结合部位。对于任何一个效应器都要引起通道活性的改变，它们必须在结合传播到通道孔时引起局部构象变化。提出了利用化学交联和静电效应捕获通道功能构象的实验。对细胞外因子如何调节ENaC的机械性理解将有助于我们理解ENaC的功能调节以及相关通道如何感知其环境。

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.