Allosteric ENaC Regulation

上皮Na+通道（ENaC）在细胞外液容量和血液的调节中具有关键作用 压力ENaC属于离子通道家族，其感测外部环境，并且其响应于 几种环境线索，包括Na+，Cl-，质子，蛋白酶和剪切应力。这些渠道如何 对这些细胞外信号的反应知之甚少。本提案的目的是确定 通过外部Na+和蛋白酶调节ENaC的分子机制。它们联合收割机 组成性激活的通道池，以及仅在细胞外Na+ 浓度很低。基于结构模型的拟议研究将确定分子 这两种因素的调节机制。Na+和抑制肽的结合位点将定义为： 以及在结合Na+或抑制肽时诱导的动态变化。实验将检验 酸性口袋容纳Na+抑制结合位点，并且与酸性口袋相邻的区域容纳结合位点。 抑制肽的位点。对于任一效应物诱导通道活性的变化，它们必须诱导局部 结合后的构象变化传播到通道孔。实验旨在捕获 使用化学交联和静电效应的通道的功能构象。一种机械的 了解细胞外因子如何调节ENaC将有助于我们了解ENaC的功能 监管以及相关通道如何感知其环境。

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.