Institute for Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Invalidenstraße 42, 10115, Berlin, Germany.
Research Group Synaptic Wiring and Information Processing, Center for Molecular Neurobiology Hamburg, Falkenried 94, 20251, Hamburg, Germany.
Sci Rep. 2017 Nov 2;7(1):14957. doi: 10.1038/s41598-017-14330-y.
Genetic engineering of natural light-gated ion channels has proven a powerful way to generate optogenetic tools for a wide variety of applications. In recent years, blue-light activated engineered anion-conducting channelrhodopsins (eACRs) have been developed, improved, and were successfully applied in vivo. We asked whether the approaches used to create eACRs can be transferred to other well-characterized cation-conducting channelrhodopsins (CCRs) to obtain eACRs with a broad spectrum of biophysical properties. We generated 22 variants using two conversion strategies applied to 11 CCRs and screened them for membrane expression, photocurrents and anion selectivity. We obtained two novel eACRs, Phobos and Aurora, with blue- and red-shifted action spectra and photocurrents similar to existing eACRs. Furthermore, step-function mutations greatly enhanced the cellular operational light sensitivity due to a slowed-down photocycle. These bi-stable eACRs can be reversibly toggled between open and closed states with brief light pulses of different wavelengths. All new eACRs reliably inhibited action potential firing in pyramidal CA1 neurons. In Drosophila larvae, eACRs conveyed robust and specific light-dependent inhibition of locomotion and nociception.
基因工程的天然光门控离子通道已被证明是一种强大的方法，可用于产生各种应用的光遗传学工具。近年来，已经开发、改进了蓝光照应的工程化阴离子通道型视蛋白（eACRs），并成功地在体内应用。我们想知道，创建 eACRs 所使用的方法是否可以转移到其他经过充分研究的阳离子通道型视蛋白（CCRs）上，以获得具有广泛生物物理特性的 eACRs。我们使用两种转换策略生成了 22 种变体，应用于 11 种 CCRs，并对它们的膜表达、光电流和阴离子选择性进行了筛选。我们获得了两种新型的 eACRs，即 Phobos 和 Aurora，它们具有蓝移和红移的作用光谱以及类似于现有 eACRs 的光电流。此外，分步突变由于光循环减慢而大大提高了细胞操作的光敏感性。这些双稳态 eACRs 可以通过不同波长的短光脉冲在打开和关闭状态之间可逆地切换。所有新的 eACRs 都能可靠地抑制 CA1 锥体神经元的动作电位放电。在果蝇幼虫中，eACRs 可传递稳健且特定的光依赖性运动和痛觉抑制。
