Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons
Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons
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DOI：
10.3791/53818
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发表时间：
2016-03-01
影响因子：
1.2
通讯作者：
Wang, Lei
中科院分区：
文献类型：
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作者：
Kang, Ji-Yong;Kawaguchi, Daichi;Wang, Lei
光刺激是控制具有出色空间和时间分辨率的生物事件的无创方法。需要新的方法来调节其本机环境中表达的内源性蛋白。在这里，我们提出了一种使用遗传编码的非天然氨基酸（UAA）直接在神经元中进行神经元蛋白功能的方法。通过使用正交tRNA/氨基酰基-TRNA合成酶对抑制琥珀色密码子，一种光反应的UAA 4,5-二甲氧基-2-硝基苯二苯甲酰苯基 - 结合蛋白（CMN）是位点特异性地融合在神经元蛋白kir2的孔中。 1，一个内部整流的钾通道。庞大的CMN物理阻断了通道孔，使KIR2.1无导体。光照明即时将CMN转换为较小的天然氨基酸Cys，激活Kir2.1通道功能。我们表达了大鼠海马原发性神经元中的这些光诱导的内部整流钾（PIRK）通道，并证明PIRK的光激活因通过激活的KIR2.1通道而导致K+电流的外流引起的神经元释放。在子宫电穿孔中，我们还在体内表达幼体新皮层中的小鼠，显示了新皮质神经元中珍珠的光激活。遗传编码UAA对靶蛋白类型或细胞位置没有任何限制，并且光电反应性UAA家族可用于调节不同的天然氨基酸残基。因此，该技术具有通常应用于许多神经元蛋白以实现大脑不同过程的光学调节的潜力。当前的方案为在体外和体内介绍了在神经元中复杂的UAA掺入的可访问程序，以实现分子水平上神经元蛋白活性的照片控制。
Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.