The Structure and Gating of the Epithelial Na+ Channel

描述(申请人提供)：上皮钠通道(ENaC)的活性可以由两种不同的机制调节：要么是通过单通道门控属性的变化(即开放概率)，要么是通过在根尖膜上表达的通道数量的变化。ENaC门控的机制尚未确定。在与其他阳离子通道类比的基础上，提出了几种作用机理。这些机制通常分为两类，要么是通道阻断，要么是变构转换。在通道阻断假说中，要么是ENaC本身的一部分，要么是另一个分子插入到通道孔中，阻止Na+转移。在这一假设中，当通道从开放状态转变为关闭状态时，残基接触的变化将局限于与阻滞剂相互作用的区域。在变构转换假说中，形成孔的螺旋将经历构象变化，使得Na+不能再穿过孔。外部刺激可以通过引起微小的局部变化来诱导这种转变，这可能会传播到全球变化，从而推动变构转变。在这个假设中，当通道从开放状态转变到关闭状态时，残基接触的变化将是广泛的。我们假设，在ENaC的孔区内进行的残基接触在通道的开放构象与闭合构象之间发生了变化。为了评估这些变化，我们将利用Ni2+选择性结合组氨酸的能力来生成孔区残基的地图。这样的地图将有助于描绘当前提出的ENaC门控模型中最有可能的模型

DESCRIPTION (provided by applicant): Epithelial Na+ Channel (ENaC) activity can be modulated by two distinct mechanisms: either by changes in single channel gating properties (i.e., open probability), or by changes in the number of channels expressed in the apical membrane. The mechanism for ENaC gating has yet to be determined. Based on analogy with other cation channels, several mechanisms have been proposed. These mechanisms generally fall into two categories, either channel blocking, or an allosteric transition. In the channel-blocking hypothesis, either a portion of ENaC itself, or another molecule inserts into the channel pore, preventing Na+ transfer. In this hypothesis, changes in residue contacts as the channel transitions from an open to a closed state would be localized to the area, which interacts with the blocker. In the allosteric transition hypothesis, the helices, which form the pore, would undergo a conformational change such that Na+ could no longer traverse the pore. External stimuli could induce such a transition by causing small local changes, which could propogate into global changes, driving the allosteric transition. In this hypothesis, changes in residue contacts as the channel transitions from an open to a closed state would be widespread. We hypothesize that residue contacts made within the pore region of ENaC are altered in the open vs. the closed conformation of the channel. In order to assess these changes, we will exploit the ability of Ni2+ to selectively bind histidine to generate a map of pore region residues. Such a map will help to delineate which amongst the currently proposed models for ENaC gating is most likely