Kinetic evaluation of photosensitivity in bi-stable variants of chimeric channelrhodopsins.
Kinetic evaluation of photosensitivity in bi-stable variants of chimeric channelrhodopsins.
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DOI：
10.1371/journal.pone.0119558
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Yawo H
中科院分区：
文献类型：
--
作者：
Hososhima S;Sakai S;Ishizuka T;Yawo H
视紫红质-1和2（ChR 1和ChR 2）形成阳离子通道，通过未知的机制被光门控。我们测试了DC门假设，即C167和D195参与了由ChR 1的TM 1 -5和ChR 2的TM 6 -7组成的ChRWR/C1 C2和由ChR 1的TM 1 -2和ChR 2的TM 3 -7组成的ChRFR的阳离子渗透状态的稳定。当C167或D195位置突变为丙氨酸（A）时，每个ChRWR和ChRFR的阳离子渗透状态显著延长几十秒。因此，DC门控功能在这些嵌合ChR中是保守的。我们接下来研究了这些双稳态ChR突变体的ON/OFF响应的动力学性质，因为它们在设计用于神经元活动的光遗传学操纵的光刺激方案中是重要的。每个光电流的开启速率常数与蓝色LED光的0-0.12 mWmm−2或青色LED光的0-0.33 mWmm−2呈线性关系。通过黄色或橙子LED光以依赖于辐照度的方式将双稳态ChR的每个光电流关闭到非导电状态。由于光电流的大小主要由开启速率常数和照射时间决定，因此只有当表达双稳态ChRs的神经元具有一定的大膜时间常数（例如，阈值辐射）时，有效诱发动作电位的最小辐射（阈值辐射）才随时间降低。τm > 20 ms）。另一方面，在另一组神经元中，阈值辐照度不依赖于照射时间。基于这些定量数据，我们建议，这些双稳态的ChRs将是最适合于增强兴奋性锥体神经元的内在活动，在最小幅度的辐照。
Channelrhodopsin-1 and 2 (ChR1 and ChR2) form cation channels that are gated by light through an unknown mechanism. We tested the DC-gate hypothesis that C167 and D195 are involved in the stabilization of the cation-permeable state of ChRWR/C1C2 which consists of TM1-5 of ChR1 and TM6-7 of ChR2 and ChRFR which consists of TM1-2 of ChR1 and TM3-7 of ChR2. The cation permeable state of each ChRWR and ChRFR was markedly prolonged in the order of several tens of seconds when either C167 or D195 position was mutated to alanine (A). Therefore, the DC-gate function was conserved among these chimeric ChRs. We next investigated the kinetic properties of the ON/OFF response of these bi-stable ChR mutants as they are important in designing the photostimulation protocols for the optogenetic manipulation of neuronal activities. The turning-on rate constant of each photocurrent followed a linear relationship to 0–0.12 mWmm−2 of blue LED light or to 0–0.33 mWmm−2 of cyan LED light. Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance. As the magnitude of the photocurrent was mostly determined by the turning-on rate constant and the irradiation time, the minimal irradiance that effectively evoked an action potential (threshold irradiance) was decreased with time only if the neuron, which expresses bi-stable ChRs, has a certain large membrane time constant (eg. τm > 20 ms). On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time. Based on these quantitative data, we would propose that these bi-stable ChRs would be most suitable for enhancing the intrinsic activity of excitatory pyramidal neurons at a minimal magnitude of irradiance.
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