Faculty of Advanced Life Science, Hokkaido University, Sapporo, 060-0810, Japan.
Global Station for Soft Matter, Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo, 001-0021, Japan.
Sci Rep. 2018 Sep 7;8(1):13445. doi: 10.1038/s41598-018-31742-6.
Natural anion channelrhodopsins (ACRs) have recently received increased attention because of their effectiveness in optogenetic manipulation for neuronal silencing. In this study, we focused on Proteomonas sulcata ACR1 (PsuACR1), which has rapid channel closing kinetics and a rapid recovery to the initial state of its anion channel function that is useful for rapid optogenetic control. To reveal the anion concentration dependency of the channel function, we investigated the photochemical properties of PsuACR1 using spectroscopic techniques. Recombinant PsuACR1 exhibited a Cl dependent spectral red-shift from 531 nm at 0.1 mM to 535 nm at 1000 mM, suggesting that it binds Cl in the initial state with a K of 5.5 mM. Flash-photolysis experiments revealed that the photocycle was significantly changed at high Cl concentrations, which led not only to suppression of the accumulation of the M-intermediate involved in the Cl non-conducting state but also to a drastic change in the equilibrium state of the other photo-intermediates. Because of this, the Cl conducting state is protracted by one order of magnitude, which implies an impairment of the rapid channel closing of PsuACR1 in the presence of high concentrations of Cl.
天然阴离子通道视紫红质（ACRs）最近因其在神经元沉默的光遗传学操作中的有效性而受到越来越多的关注。在这项研究中，我们专注于 Proteomonas sulcata ACR1（PsuACR1），它具有快速的通道关闭动力学和快速恢复其阴离子通道功能的初始状态的能力，这对于快速光遗传学控制非常有用。为了揭示通道功能对阴离子浓度的依赖性，我们使用光谱技术研究了 PsuACR1 的光化学性质。重组 PsuACR1 表现出 Cl 依赖性光谱红移，从 0.1 mM 时的 531nm 红移到 1000mM 时的 535nm，表明它在初始状态下与 Cl 结合，Kd 为 5.5mM。闪光光解实验表明，在高 Cl 浓度下，光循环发生了显著变化，这不仅导致参与 Cl 非传导状态的 M 中间体的积累受到抑制，而且还导致其他光中间体的平衡状态发生剧烈变化。因此，Cl 传导状态延长了一个数量级，这意味着在高浓度 Cl 存在下，PsuACR1 的快速通道关闭能力受损。
