Structural Guidance of the Photocycle of Channelrhodopsin-2 by an Interhelical Hydrogen Bond
Structural Guidance of the Photocycle of Channelrhodopsin-2 by an Interhelical Hydrogen Bond
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DOI：
10.1021/bi901634p
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发表时间：
2010-01-19
期刊：
影响因子：
2.9
通讯作者：
Bamberg, Ernst
中科院分区：
文献类型：
--
作者：
Bamann, Christian;Gueta, Ronnie;Bamberg, Ernst
视紫红质-2（ChR 2）是一种光门控阳离子通道，是亚视黄基光感受器家族的成员。由于ChR 2表达的动物细胞膜的光诱导去极化的证明，它越来越多地被用于光触发动作电位。ChR 2在光吸收时传导阳离子，其体现了作为主要反应的视网膜异构化和结构上未知的开放机制。从光谱数据中可以明显看出，席夫碱的质子化反应是光循环的一部分，与其他微生物型视紫红质相当。然而，发色团部位的突起和通道孔之间的联系仍然是个谜。在这里，我们使用ChR 2的缓慢突变体，这些突变体是通过在跨膜（TM）螺旋3中的C1 28和TM螺旋4中的D 156突变时扰乱假定的氢键而产生的。突变体开放状态的寿命增加了100倍以上。我们研究了慢突变体的光谱特性。而去质子化的席夫碱（产生P390）发生在相同的时间尺度上的野生型，reprotonation到P520被推迟在缓慢的突变体和他们的光循环分裂，导致存在两个光中间体，P390和P520，在开放状态。P390和P520的光反应导致电生理测量中电流的淬灭。我们的结论是，C128和D156之间的推定氢键是一个重要的结构决定因素的通道的关闭反应。此外，我们发现D156 A突变体比C128 A更适合于可兴奋细胞的光控制。
Channelrhodopsin-2 (ChR2) is a light-gated cation channel and a member of the family of retinylidene photoreceptors. Since the demonstration of light-induced depolarization of ChR2-expressing animal cell membranes, it was increasingly exploited for light triggering of action potentials. ChR2 conducts cations upon light absorption that embodies retinal isomerization as the primary reaction and a structurally unknown opening mechanism. It is evident from spectroscopic data that protonation reactions at the Schiff base are part of the photocycle, comparable to other microbial-type rhodopsins. However, the connection between the processes at the chromophore site and the channel's pore remained enigmatic. Here, we use slow mutants of ChR2 that we generated by disturbing a postulated hydrogen bond when mutating Cl 28 in the transmembrane (TM) helix 3 and D 156 in TM helix 4. The lifetime of the mutants' open state is increased more than 100 times. We investigated the spectral properties of the slow mutants. Whereas the deprotonation of the Schiff base (yielding P390) occurs on the same time scale as that of the wild type, reprotonation to P520 is retarded in the slow mutants and their photocycle is split, leading to the presence of two photointermediates, P390 and P520, in the open state. The photoreactions of P390 and P520 lead to a quenching of the current in electrophysiological measurements. We conclude that the putative hydrogen bond between C 128 and D 156 is an important structural determinant of the channel's closing reaction. Furthermore, we show that the D156A mutant is even more suitable for light control of excitable cells than C128A.