Bühl Elena, Resler Tom, Lam Rebecca, Asido Marvin, Bamberg Ernst, Schlesinger Ramona, Bamann Christian, Heberle Joachim, Wachtveitl Josef
Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt am Main, Max-von-Laue Strasse 7, 60438 Frankfurt, Germany.
Department of Physics, Experimental Molecular Biophysics, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany.
J Am Chem Soc. 2023 Oct 11;145(40):21832-21840. doi: 10.1021/jacs.3c05399. Epub 2023 Sep 29.
The light-gated ion channel channelrhodopsin-2 from (ChR2) is the most frequently used optogenetic tool in neurosciences. However, the precise molecular mechanism of the channel opening and the correlation among retinal isomerization, the photocycle, and the channel activity of the protein are missing. Here, we present electrophysiological and spectroscopic investigations on the R120H variant of ChR2. R120 is a key residue in an extended network linking the retinal chromophore to several gates of the ion channel. We show that despite the deficient channel activity, the photocycle of the variant is intact. In a comparative study for R120H and the wild type, we resolve the vibrational changes in the spectral range of the retinal and amide I bands across the time range from femtoseconds to seconds. Analysis of the amide I mode reveals a significant impairment of the ultrafast protein response after retinal excitation. We conclude that channel opening in ChR2 is prepared immediately after retinal excitation. Additionally, chromophore isomerization is essential for both photocycle and channel activities, although both processes can occur independently.
来自莱茵衣藻的光门控离子通道通道视紫红质-2（ChR2）是神经科学中最常用的光遗传学工具。然而，通道开放的确切分子机制以及视网膜异构化、光循环和该蛋白通道活性之间的关联尚不清楚。在此，我们展示了对ChR2的R120H变体的电生理和光谱研究。R120是将视网膜发色团与离子通道的几个门连接起来的扩展网络中的一个关键残基。我们表明，尽管该变体的通道活性不足，但其光循环是完整的。在对R120H和野生型的比较研究中，我们解析了从飞秒到秒的时间范围内视网膜和酰胺I带光谱范围内的振动变化。对酰胺I模式的分析揭示了视网膜激发后超快蛋白质反应的显著受损。我们得出结论，ChR2中的通道开放在视网膜激发后立即开始准备。此外，发色团异构化对于光循环和通道活性都至关重要，尽管这两个过程可以独立发生。
