Levitz Joshua, Popescu Andrei T, Reiner Andreas, Isacoff Ehud Y
Department of Molecular and Cell Biology, University of California, Berkeley Berkeley, CA, USA.
Department of Molecular and Cell Biology, University of California, BerkeleyBerkeley, CA, USA; Department of Biology and Biotechnology, Ruhr-University BochumBochum, Germany.
Front Mol Neurosci. 2016 Feb 2;9:2. doi: 10.3389/fnmol.2016.00002. eCollection 2016.
The ability to optically manipulate specific neuronal signaling proteins with genetic precision paves the way for the dissection of their roles in brain function, behavior, and disease. Chemical optogenetic control with photoswitchable tethered ligands (PTLs) enables rapid, reversible and reproducible activation or block of specific neurotransmitter-gated receptors and ion channels in specific cells. In this study, we further engineered and characterized the light-activated GluK2 kainate receptor, LiGluR, to develop a toolbox of LiGluR variants. Low-affinity LiGluRs allow for efficient optical control of GluK2 while removing activation by native glutamate, whereas variant RNA edited versions enable the synaptic role of receptors with high and low Ca(2+) permeability to be assessed and spectral variant photoswitches provide flexibility in illumination. Importantly, we establish that LiGluR works efficiently in the cortex of awake, adult mice using standard optogenetic techniques, thus opening the door to probing the role of specific synaptic receptors and cellular signals in the neural circuit operations of the mammalian brain in normal conditions and in disease. The principals developed in this study are widely relevant to the engineering and in vivo use of optically controllable proteins, including other neurotransmitter receptors.
以基因精准度对特定神经元信号蛋白进行光学操控的能力，为剖析它们在脑功能、行为及疾病中的作用铺平了道路。利用光开关连接配体（PTL）进行化学光遗传学控制，能够在特定细胞中快速、可逆且可重复地激活或阻断特定神经递质门控受体及离子通道。在本研究中，我们进一步设计并表征了光激活的谷氨酸钾离子受体2（GluK2），即LiGluR，以开发一套LiGluR变体工具箱。低亲和力的LiGluR能够在去除内源性谷氨酸激活的同时，实现对GluK2的高效光学控制，而经RNA编辑的变体版本则能够评估具有高钙（Ca2+）通透性和低钙通透性的受体的突触作用，光谱变体光开关则提供了光照的灵活性。重要的是，我们证实，使用标准光遗传学技术，LiGluR在成年清醒小鼠的皮层中能有效发挥作用，从而为探究特定突触受体和细胞信号在正常及疾病状态下哺乳动物脑的神经回路运作中的作用打开了大门。本研究中所建立的原理与包括其他神经递质受体在内的光学可控蛋白的工程设计及体内应用广泛相关。
