Max-Planck-Institut für Biophysik, Max-von-Laue Strasse 3, 60438 Frankfurt, Germany.
Biochemistry. 2010 Jan 19;49(2):267-78. doi: 10.1021/bi901634p.
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.
通道视紫红质 2（ChR2）是一种光门控阳离子通道，是视黄醛光受体家族的成员。自从证明了表达 ChR2 的动物细胞膜的光诱导去极化以来，它越来越多地被用于光触发动作电位。ChR2 在光吸收时传导阳离子，这体现了视黄醛异构化为主要反应和结构未知的开放机制。从光谱数据可以明显看出，质子化反应在席夫碱部位是光循环的一部分，类似于其他微生物型视紫红质。然而，色素部位的过程与通道孔之间的联系仍然是神秘的。在这里，我们使用通过突变跨膜（TM）螺旋 3 中的 C128 和 TM 螺旋 4 中的 D156 来干扰假定氢键而产生的 ChR2 慢突变体。突变体的开放状态的寿命增加了 100 多倍。我们研究了慢突变体的光谱特性。虽然席夫碱的去质子化（产生 P390）与野生型的时间尺度相同，但在慢突变体中质子化到 P520 被延迟，并且它们的光循环被分裂，导致在开放状态下存在两个光中间体，P390 和 P520。P390 和 P520 的光反应导致在电生理学测量中电流的猝灭。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，我们表明 D156A 突变体甚至比 C128A 更适合光控制可兴奋细胞。
