Structural insights into ion conduction by channelrhodopsin 2
Structural insights into ion conduction by channelrhodopsin 2
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DOI：
10.1126/science.aan8862
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发表时间：
2017-11-24
期刊：
影响因子：
56.9
通讯作者：
Gordeliy, Valentin
中科院分区：
文献类型：
--
作者：
Volkov, Oleksandr;Kovalev, Kirill;Gordeliy, Valentin
离子通道是细胞膜上的一种蛋白质，在受到刺激时，它调节离子穿过细胞或细胞器膜的流动。产生的电信号参与生物功能，如神经元中的电化学传输和信息处理。视紫红质（ChRs）似乎是不寻常的通道。它们属于微生物视紫红质的大家族，视紫红质是含有作为发色团的视网膜的七螺旋跨膜蛋白。光子吸收引发视网膜异构化，导致光循环，具有不同的光谱可区分的中间体，从而控制通道的打开和关闭。在2003年，它被证明，光诱导电流的异源表达的ChR 2可以用来改变宿主的膜电位。该概念进一步应用于通过使用光诱导的去极化来触发表达ChR 2的神经元中的动作电位来精确控制肌肉和神经活动。这种具有ChR 2和其他ChR的光遗传学方法已广泛用于以高时空分辨率远程控制培养物和活体动物中的神经细胞。它也被用于生物医学研究，旨在治愈严重的疾病。理由尽管有丰富的生物化学和生物物理数据，高分辨率的结构和结构机制的天然ChR 2（和其他ChR）尚未被称为。一个进步是嵌合体（C1 C2）的结构。然而，最近的电生理学和傅立叶变换红外数据表明，C1 C2表现出光诱导的反应，在功能和机制上不同于ChR 2。鉴于ChR 2是光遗传学中最常用的工具，ChR 2的高分辨率结构非常重要。破译天然通道的结构将揭示视网膜席夫碱（RSB）处的光诱导变化如何与通道操作相关联，并且可以使增强的光遗传学工具的工程化更有效。我们在LEXSY中表达ChR 2，并用于介晶结晶方法中以确定野生型ChR 2和C128 T慢突变体在2.4和2.7 nm处的晶体结构，（C，半胱氨酸; T，苏氨酸）。两种不同的暗态构象的ChR 2在两个原异构体中的不对称单位被解决。原聚体的整体结构排列未显示骨架构象的可见差异。然而，某些氨基酸的构象和水分子的位置并不相同。二聚化是强的，主要通过螺旋3和4与N末端的相互作用提供。此外，原聚体通过二硫键C34/C36′连接。在这两个原型中，我们确定了离子传导途径，包括四个腔[细胞外腔1（EC 1）、EC 2、细胞内腔1（IC 1）和IC 2]，这些腔被三个门[细胞外门（ECG）、中央门（CG）和细胞内门（ICG）]隔开（图，A组）。精氨酸R120和R268分别是所有ChR中ECG和ICG的核心。席夫碱通过氢键与E123和D253氨基酸（E，谷氨酸; D，天冬氨酸）连接，并且是CG的关键部分，CG通过由许多水分子介导的延伸的氢键网络与另外两个门进一步连接（图，小图B）。DC门与通道路径中的门分离，并且通过水分子W5通过氢键桥接。DC对（C128和D156）的氢键有两个重要的结果。它稳定了螺旋3和4，并提供了从D156的连接，这是一个可能的...
INTRODUCTIONIon channels are integral membrane proteins that upon stimulation modulate the flow of ions across the cell or organelle membrane. The resulting electrical signals are involved in biological functions such as electrochemical transmission and information processing in neurons. Channelrhodopsins (ChRs) appear to be unusual channels. They belong to the large family of microbial rhodopsins, seven-helical transmembrane proteins containing retinal as chromophore. Photon absorption initiates retinal isomerization resulting in a photocycle, with different spectroscopically distinguishable intermediates, thereby controlling the opening and closing of the channel. In 2003, it was demonstrated that light-induced currents by heterologously expressed ChR2 can be used to change a host’s membrane potential. The concept was further applied to precisely control muscle and neural activity by using light-induced depolarization to trigger an action potential in neurons expressing ChR2. This optogenetic approach with ChR2 and other ChRs has been widely used for remote control of neural cells in culture and in living animals with high spatiotemporal resolution. It is also used in biomedical studies aimed to cure severe diseases.RATIONALEDespite the wealth of biochemical and biophysical data, a high-resolution structure and structural mechanisms of a native ChR2 (and other ChRs) have not yet been known. A step forward was the structure of a chimera (C1C2). However, recent electrophysiological and Fourier transform infrared data showed that C1C2 exhibits light-induced responses that are functionally and mechanistically different from ChR2. Given that ChR2 is the most frequently used tool in optogenetics, a high-resolution structure of ChR2 is of high importance. Deciphering the structure of the native channel would shed light on how the light-induced changes at the retinal Schiff base (RSB) are linked to the channel operation and may make engineering of enhanced optogenetic tools more efficient.RESULTSWe expressed ChR2 in LEXSY and used in the meso crystallization approach to determine the crystal structure of the wild-type ChR2 and C128T slow mutant at 2.4 and 2.7 Å, respectively (C, cysteine; T, threonine). Two different dark-state conformations of ChR2 in the two protomers in the asymmetric unit were resolved. The overall structure alignment of the protomers does not show a visible difference in backbone conformation. However, the conformation of some amino acids and the position of water molecules are not the same. The dimerization is strong and provided mainly through the interaction of helices 3 and 4 and the N termini. In addition, the protomers are connected with a disulfide bond, C34/C36′. In both protomers, we identified ion conduction pathway comprising four cavities [extracellular cavity 1 (EC1), EC2, intracellular cavity 1 (IC1), and IC2] that are separated by three gates [extracellular gate (ECG), central gate (CG), and intracellular gate (ICG)] (figure, panel A). Arginines R120 and R268 are the cores of ECG and ICG, respectively, in all ChRs. The Schiff base is hydrogen-bond–connected to E123 and D253 amino acids (E, glutamic acid; D, aspartic acid) and is a key part of the CG that is further connected with two other gates through an extended H-bond network mediated by numerous water molecules (figure, panel B). The DC gate is separate from the gates in the channel pathway and is bridged by hydrogen bonds through the water molecule w5. Hydrogen bonding of the DC pair (C128 and D156) has two important consequences. It stabilizes helices 3 and 4 and provides connection from D156, a possible …