PDF(Pubmed)
Abstract：
Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is the first discovered natural light-gated ion channel that shows higher selectivity to K+ than to Na+ and therefore is used to silence neurons with light (optogenetics). Replacement of the conserved cysteine residue in the transmembrane helix 3 (Cys110) with alanine or threonine results in a >1,000-fold decrease in the channel closing rate. The phenotype of the corresponding mutants in channelrhodopsin 2 is attributed to breaking of a specific interhelical hydrogen bond (the \"DC gate\"). Unlike CrChR2 and other ChRs with long distance \"DC gates\", the HcKCR1 structure does not reveal any hydrogen bonding partners to Cys110, indicating that the mutant phenotype is likely caused by disruption of direct interaction between this residue and the chromophore. In HcKCR1_C110A, fast photochemical conversions corresponding to channel gating were followed by dramatically slower absorption changes. Full recovery of the unphotolyzed state in HcKCR1_C110A was extremely slow with two time constants 5.2 and 70 min. Analysis of the light-minus-dark difference spectra during these slow processes revealed accumulation of at least four spectrally distinct blue light-absorbing photocycle intermediates, L, M1 and M2, and a UV light-absorbing form, typical of bacteriorhodopsin-like channelrhodopsins from cryptophytes. Our results contribute to better understanding of the mechanistic links between the chromophore photochemistry and channel conductance, and provide the basis for using HcKCR1_C110A as an optogenetic tool.
摘要：
来自hyphochytriumcatenoides（HcKCR1）的钾通道视紫红质1是第一个发现的自然光门控离子通道，对K的选择性高于对Na的选择性，因此用于用光（光遗传学）沉默神经元。用丙氨酸或苏氨酸替换跨膜螺旋3(Cys110)中的保守半胱氨酸残基导致通道闭合速率降低>1,000倍。通道视紫红质2中相应突变体的表型归因于特定螺旋间氢键的断裂（“DC门”）。与CrChR2和其他具有长距离“DC门”的ChR不同，HcKCR1结构未显示任何与Cys110的氢键合配偶体，表明突变表型可能是由该残基与发色团之间的直接相互作用的破坏引起的。在HcKCR1_C110A中，对应于通道门控的快速光化学转换之后，吸收变化显着减慢。HcKCR1_C110A中未光解状态的完全恢复极其缓慢，两个时间常数为5.2和70min。在这些缓慢的过程中，对明暗差异光谱的分析显示，至少积累了四个光谱上不同的蓝光吸收光循环中间体，L,M1和M2，以及UV光吸收形式，典型的来自隐藻的细菌视紫红质样通道视紫红质。我们的结果有助于更好地理解生色团光化学和通道电导之间的机械联系，为HcKCR1_C110A作为光遗传学工具的应用提供了依据。
