The Structure and Gating of the Epithelial Na+ Channel

描述（由申请人提供）：上皮Na+通道（ENaC）活性可以通过两种不同的机制调节：通过单通道门控特性的变化（即，开放概率），或通过顶膜中表达的通道数目的变化。ENaC门控的机制尚未确定。基于与其他阳离子通道的类比，已经提出了几种机制。这些机制通常分为两类，通道阻断或变构转换。在通道阻断假说中，ENaC本身的一部分或另一种分子插入通道孔中，阻止Na+转移。在该假设中，当通道从开放状态转变为闭合状态时，残留物接触的变化将局限于与阻断剂相互作用的区域。在变构转变假说中，形成孔的螺旋将经历构象变化，使得Na+不再能够穿过孔。外部刺激可以通过引起小的局部变化来诱导这种转变，这些变化可以传播到全局变化，从而驱动变构转变。在这种假设中，随着通道从开放状态转变为闭合状态，残留物接触的变化将是广泛的。我们假设ENaC的孔区域内的残基接触在通道的开放与闭合构象中发生改变。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域残基的图谱。这样的地图将有助于描绘出目前提出的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