PDF(Pubmed)
Abstract：
KCR channelrhodopsins (K+-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K+ selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K+ selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K+ selectivity; rather than forming the symmetrical filter of canonical K+ channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K+ selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K+ selectivity also provides a framework for next-generation optogenetics.
摘要：
KCR通道视紫红质（K选择性光门控离子通道）已作为潜在的抑制性光遗传学工具受到关注，但更广泛地提出了有关如何实现其K选择性的基本谜团。这里,我们展示了HcKCR1和HcKCR2的2.5-2.7µ低温电子显微镜结构，以及具有增强的K选择性的结构指导突变体。结构,电生理学,计算，光谱学,和生化分析揭示了K+选择性的独特机制；而不是形成经典K+通道的对称过滤器，实现选择性和脱水，相反，每个单体内的三个细胞外前庭残基形成一个灵活的不对称选择性门，而不同的脱水途径在细胞内延伸。结构比较揭示了诱导视网膜旋转的视网膜结合口袋（考虑HcKCR1/HcKCR2光谱差异），和设计具有增加的K+选择性的相应KCR变体(KALI-1/KALI-2)为体外和体内光遗传学抑制提供了关键优势。因此，离子通道K+选择性机制的发现也为下一代光遗传学提供了框架。
