一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制
Summary
Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.
Abstract
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.
Introduction
相对于传统的电刺激，光刺激，提供带到标本生理系统最小干扰较大的时间和空间分辨率。由于使用激光来刺激神经 元于1971年1示范，许多创造性的方式被发明来控制外源性神经活动的光。 photocaged激动剂的光释放长期以来被用于研究神经元网络到配位体2,3,4的生理反应。这种技术的特异性有限，原因是笼激动剂的扩散。遗传特异性由异位表达光敏视蛋白渠道取得和泵5,6,7，并且已成功地应用到调制在不同的模式生物中选择的神经元网络。然而，这将是困难的应用此方法到光学控制各种其他神经元蛋白，由于从视蛋白的蛋白质与其它蛋白质接枝光响应竟被ð要求激烈工程可能改变研究中的蛋白质的自然特性。化学圈养的外源感光配体与蛋白质已经证明另一种方式来控制通道蛋白8,9,10的功能。该配体呈现或通过偶氮苯部分的光致异构化从蛋白质的结合位点抽出。束缚化学限制了应用主要是膜蛋白的胞外侧，不含细胞内侧面和细胞内蛋白质。
光敏Uaas，被纳入的蛋白质后，提供操纵蛋白质与光的一般策略。在早期的努力，tRNA的化学酰化photocaged Uaas显微注射入爪蟾卵母 ，以纳入Uaas到膜受体和离子通道11，它有先进的它们的结构-功能关系12,13,14的理解。这种注射方法主要限于大的卵母细胞。一个UAA的遗传掺入通过正交tRNA /合成酶对，通过内源性蛋白的翻译融合了UAA活细胞15,16,17,18绕过了技术上的挑战酰化tRNA的显微注射和。 UAA掺入神经 元蛋白已被证明在初级神经元和神经干细胞19,20。最近，光响应UAA已经遗传掺入神经 蛋白在哺乳动物脑中体内首次21。这些进步使得有可能研究与Uaas神经元蛋白在其天然细胞环境。
内向整流钾离子通道的Kir2.1是更容易通过K +电流成比出细胞的一个强有力的整流器，并且在调节生理过程，包括细胞的兴奋，血管张力，心脏速率，重是必不可少最终盐流和胰岛素释放22。的Kir2.1的过表达超极化的目标神经元，它变得不太可激发23,24的膜电位。以光学控制的Kir2.1在其天然细胞，Kang等人的基因掺入一个光响应UAA成的Kir2.1在哺乳动物细胞，神经元和胚胎鼠大脑21表示。闪光短暂的脉冲能够将UAA转化成天然氨基酸半胱氨酸，从而激活靶蛋白的Kir2.1。当这种光诱导内向整流钾（PIRK）通道蛋白在大鼠海马原代神经元中表达，它响应于光激活抑制神经元放电。此外，PIRK通道中的胚胎鼠新皮质中表达，并测定在皮层神经元的光激活PIRK电流。成功实现了UAA技术在哺乳动物大脑体内敞开了大门光学控制神经细胞的蛋白质在其天然环境中，这将使在分子水平上的神经元过程和机制的光学夹层。
在这个协议中，我们描述了Uaas遗传纳入文化和体内胚胎小鼠大脑初级神经元的程序。光响应UAA CMN和的Kir2.1用来说明该过程。方法评估成功的UAA整合并提供神经蛋白活性的光学控制。这个协议提供了一个明确的指导中的神经元和在体内基因编码Uaas，并以光学调节经由光敏UAA神经蛋白的功能。我们希望这个协议推动通过体内 UAA技术，神经科学和光遗传学生物学研究。
Protocol
Representative Results
Discussion
实现有效的光调制中，重要的初步步骤是决定，其中掺入光响应UAA中靶蛋白。目标蛋白的结构和功能的信息是非常有帮助，指导候选场址的选择。与此同时，光调节的目的将确定哪些位点是最合适的。选择候补的站点之后，建议在进行初级神经元和在体内之前测试在哺乳动物细胞系，如人胚肾（HEK）细胞更容易培养和操作，该位点。要确定的Kir2.1光激活站点，沿线的Kir2.1的孔隙多个站点已?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Dr. S. Szobota for helpful discussion on neuron culture and Ca-P transfection. L.W. acknowledges support from The Salk Innovation Grant, the California Institute for Regenerative Medicine (RN1-00577-1), and the National Institutes of Health (1DP2OD004744).
Materials
| Cover Glasses, Circles, 12 mm, Thickness 0.13-0.17 mm | Carolina Biologicals | 633029 |
| Corning BioCoat Poly-D-Lysine | Corning Discovery Labware | 354210 |
| D-(+)-Glucose solution | Sigma-Aldrich | G8769 |
| Iris Spatula-curved | Fine Science Tool | 10092-12 |
| Dissecting Knife – Fine Angled Tip | Fine Science Tool | 10056-12 |
| GlutaMAX-I Supplement | Life Technologies | 35050-061 |
| MITO+ Serum Extender | BD Biosciences | 355006 |
| Falcon 40 µm Cell Strainer | Corning Life Sciences | 352340 |
| BES (N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, N,N-Bis(2-hydroxyethyl)taurine) | Sigma-Aldrich | B6420 |
| LED LIGHT SOURCE – Black LED 385 | Prizmatix Ltd. | |
| Agarose, Low Melting Point, Analytical Grade | Promega | V2111 |
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