Molecular Investigation of Chicken Acid-Sensing Ion Channel 1 β11-12 Linker Isomerization and Channel Kinetics.

Structures of the trimeric acid-sensing ion channel have been solved in the resting, toxin-bound open and desensitized states. Within the extracellular domain, there is little difference between the toxin-bound open state and the desensitized state. The main exception is that a loop connecting the 11th and 12th β-strand, just two amino acid residues long, undergoes a significant and functionally critical re-orientation or flipping between the open and desensitized conformations. Here we investigate how specific interactions within the surrounding area influence linker stability in the “flipped” desensitized state using all-atom molecular dynamics simulations. An inherent challenge is bringing the relatively slow channel desensitization and recovery processes (in the milliseconds to seconds) within the time window of all-atom simulations (hundreds of nanoseconds). To accelerate channel behavior, we first identified the channel mutations at either the Leu414 or Asn415 position with the fastest recovery kinetics followed by molecular dynamics simulations of these mutants in a deprotonated state, accelerating recovery. By mutating one residue in the loop and examining the evolution of interactions in the neighbor, we identified a novel electrostatic interaction and validated prior important interactions. Subsequent functional analysis corroborates these findings, shedding light on the molecular factors controlling proton-mediated transitions between functional states of the channel. Together, these data suggest that the flipped loop in the desensitized state is stabilized by interactions from surrounding regions keeping both L414 and N415 in place. Interestingly, very few mutations in the loop allow for equivalent channel kinetics and desensitized state stability. The high degree of sequence conservation in this region therefore indicates that the stability of the ASIC desensitized state is under strong selective pressure and underlines the physiological importance of desensitization.

三聚体酸敏感离子通道的结构已在静息态、毒素结合的开放态和脱敏态下被解析。在胞外结构域内，毒素结合的开放态和脱敏态之间差异很小。主要的例外是连接第11和第12条β链的一个环（仅两个氨基酸残基长）在开放和脱敏构象之间发生显著且对功能至关重要的重新定向或翻转。在此我们利用全原子分子动力学模拟研究周围区域内的特定相互作用如何影响处于“翻转”脱敏态的连接子稳定性。一个固有的挑战是将相对缓慢的通道脱敏和恢复过程（在毫秒到秒的时间尺度）纳入全原子模拟的时间窗口（数百纳秒）内。为了加速通道行为，我们首先确定了在Leu414或Asn415位置具有最快恢复动力学的通道突变，然后对这些去质子化状态的突变体进行分子动力学模拟，以加速恢复。通过突变环中的一个残基并检查其邻近区域相互作用的演变，我们确定了一种新的静电相互作用，并验证了先前的重要相互作用。随后的功能分析证实了这些发现，阐明了控制通道功能状态之间质子介导转变的分子因素。总之，这些数据表明脱敏态下翻转的环通过周围区域的相互作用得以稳定，从而使L414和N415保持在原位。有趣的是，环中很少有突变能使通道具有相同的动力学和脱敏态稳定性。因此，该区域高度的序列保守性表明酸敏感离子通道脱敏态的稳定性受到强烈的选择压力，并强调了脱敏的生理重要性。