Atomic mutagenesis in ion channels with engineered stoichiometry
Atomic mutagenesis in ion channels with engineered stoichiometry
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DOI：
10.7554/elife.18976
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发表时间：
2016-10-06
期刊：
影响因子：
7.7
通讯作者：
Ahern, Christopher A.
中科院分区：
文献类型：
--
作者：
Lueck, John D.;Mackey, Adam L.;Ahern, Christopher A.
钾通道的C型失活通过由通道选择性过滤器中的氢键网络介导的内部计时机制来微调可兴奋细胞中的电信号。先前，我们使用无义抑制来突出保守的Trp 434-Asp 447吲哚氢键在Shaker钾通道中与色氨酸的非氢键同源物Ind的作用（Pless等人，2013年）。在这里，分子动力学模拟表明，Trp 434 Ind氢键合作伙伴，Asp 447，意外地“翻转”向细胞外环境，允许水渗透选择性过滤器后面的空间，同时减少局部负静电荷。此外，提出了一种蛋白质工程方法，其中分裂内含肽序列侧接内质网保留/回收基序（ERret），其被掺入Shaker单体的N-或C-末端或钠通道双结构域片段内。该系统使得能够化学计量控制振荡单体和编码通道四聚体内的多个氨基酸。
C-type inactivation of potassium channels fine-tunes the electrical signaling in excitable cells through an internal timing mechanism that is mediated by a hydrogen bond network in the channels' selectively filter. Previously, we used nonsense suppression to highlight the role of the conserved Trp434-Asp447 indole hydrogen bond in Shaker potassium channels with a non-hydrogen bonding homologue of tryptophan, Ind (Pless et al., 2013). Here, molecular dynamics simulations indicate that the Trp434Ind hydrogen bonding partner, Asp447, unexpectedly 'flips out' towards the extracellular environment, allowing water to penetrate the space behind the selectivity filter while simultaneously reducing the local negative electrostatic charge. Additionally, a protein engineering approach is presented whereby split intein sequences are flanked by endoplasmic reticulum retention/retrieval motifs (ERret) are incorporated into the N- or C- termini of Shaker monomers or within sodium channels two-domain fragments. This system enabled stoichiometric control of Shaker monomers and the encoding of multiple amino acids within a channel tetramer.