Krause Benjamin S, Grimm Christiane, Kaufmann Joel C D, Schneider Franziska, Sakmar Thomas P, Bartl Franz J, Hegemann Peter
Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Berlin, Germany.
Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Berlin, Germany.
Biophys J. 2017 Mar 28;112(6):1166-1175. doi: 10.1016/j.bpj.2017.02.001.
Channelrhodopsins (ChRs) are light-activated ion channels widely employed for photostimulation of excitable cells. This study focuses on ReaChR, a chimeric ChR variant with optimal properties for optogenetic applications. We combined electrophysiological recordings with infrared and UV-visible spectroscopic measurements to investigate photocurrents and photochemical properties of ReaChR. Our data imply that ReaChR is green-light activated (λ = 532 nm) with a non-rhodopsin-like action spectrum peaking at 610 nm for stationary photocurrents. This unusual spectral feature is associated with photoconversion of a previously unknown light-sensitive, blue-shifted photocycle intermediate L (λ = 495 nm), which is accumulated under continuous illumination. To explain the complex photochemical reactions, we propose a symmetrical two-cycle-model based on the two C=N isomers of the retinal cofactor with either syn- or anti-configuration, each comprising six consecutive states D, K, L, M, N, and O. Ion conduction involves two states per cycle, the late M- (M) with a deprotonated retinal Schiff base and the consecutive green-absorbing N-state that both equilibrate via reversible reprotonation. In our model, a fraction of the deprotonated M-intermediate of the anti-cycle may be photoconverted-as the L-state-back to its inherent dark state, or to its M-state pendant (M') of the syn-cycle. The latter reaction pathway requires a C=C, C=N double-isomerization of the retinal chromophore, whereas the intracircular photoconversion of M back to D involves only one C=C double-bond isomerization.
通道视紫红质（ChRs）是一类光激活离子通道，广泛应用于可兴奋细胞的光刺激。本研究聚焦于ReaChR，这是一种嵌合型ChR变体，具有用于光遗传学应用的最佳特性。我们将电生理记录与红外和紫外可见光谱测量相结合，以研究ReaChR的光电流和光化学性质。我们的数据表明，ReaChR由绿光激活（λ = 532 nm），其非视紫红质样作用光谱在610 nm处达到峰值，产生稳定的光电流。这种不寻常的光谱特征与一种先前未知的、对光敏感的、蓝移光循环中间体L（λ = 495 nm）的光转化有关，该中间体在持续光照下会积累。为了解释复杂的光化学反应，我们基于视黄醛辅因子的两个具有顺式或反式构型的C=N异构体，提出了一个对称的双循环模型，每个异构体包含六个连续状态D、K、L、M、N和O。离子传导每个循环涉及两个状态，即晚期M-（M）状态，其视网膜席夫碱去质子化，以及连续的绿色吸收N状态，两者通过可逆的质子化达到平衡。在我们的模型中，反循环中去质子化的M中间体的一部分可能会被光转化——作为L状态——回到其固有的暗状态，或者回到顺循环中其M状态的对应物（M'）。后一种反应途径需要视网膜发色团的C=C、C=N双键异构化，而M回到D的内环光转化仅涉及一个C=C双键异构化。
