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首页> 外文期刊>Biochemistry >Structural guidance of the photocycle of channelrhodopsin-2 by an interhelical hydrogen bond
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Structural guidance of the photocycle of channelrhodopsin-2 by an interhelical hydrogen bond

机译：螺旋间氢键对Channelrhodopsin-2光周期的结构指导

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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 C128 in the transmembrane (TM) helix 3 and D156 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 C128 and D156 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.
机译：Channelrhodopsin-2（ChR2）是一个光门控阳离子通道，是视黄叉烯类光感受器家族的成员。自从证明光诱导表达ChR2的动物细胞膜去极化以来，人们就越来越多地利用它来激发动作电位。 ChR2在光吸收时会传导阳离子，这体现了视网膜异构化为主要反应和结构未知的打开机制。从光谱数据可以明显看出，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，生色团位点处的过程与通道孔之间的联系仍然令人费解。在这里，我们使用了ChR2的慢突变体，该突变体是通过使假定的跨膜（TM）螺旋3中的C128和TM螺旋4中的D156发生突变而干扰假定的氢键而生成的。突变体打开状态的寿命增加了100倍以上。我们研究了慢突变体的光谱特性。 Schiff碱基的去质子化（产生P390）与野生型发生在相同的时间尺度，而慢突变体中P520的质子化受到阻碍，其光循环分裂，导致存在两种光中间体P390和P520 ，处于打开状态。 P390和P520的光反应在电生理测量中导致电流猝灭。我们得出结论，C128和D156之间的假定氢键是通道关闭反应的重要结构决定因素。此外，我们显示D156A突变体比C128A更适合于可激发细胞的光控制。

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