Biospectroscopy, Center for Protein Diagnostics (PRODI), Ruhr University Bochum, Bochum, Germany.
Department of Biophysics, Ruhr University Bochum, Bochum, Germany.
Commun Biol. 2021 May 14;4(1):578. doi: 10.1038/s42003-021-02101-5.
Channelrhodopsins are widely used in optogenetic applications. High photocurrents and low current inactivation levels are desirable. Two parallel photocycles evoked by different retinal conformations cause cation-conducting channelrhodopsin-2 (CrChR2) inactivation: one with efficient conductivity; one with low conductivity. Given the longer half-life of the low conducting photocycle intermediates, which accumulate under continuous illumination, resulting in a largely reduced photocurrent. Here, we demonstrate that for channelrhodopsin-1 of the cryptophyte Guillardia theta (GtACR1), the highly conducting C = N-anti-photocycle was the sole operating cycle using time-resolved step-scan FTIR spectroscopy. The correlation between our spectroscopic measurements and previously reported electrophysiological data provides insights into molecular gating mechanisms and their role in the characteristic high photocurrents. The mechanistic importance of the central constriction site amino acid Glu-68 is also shown. We propose that canceling out the poorly conducting photocycle avoids the inactivation observed in CrChR2, and anticipate that this discovery will advance the development of optimized optogenetic tools.
通道蛋白视紫红质被广泛应用于光遗传学领域。高的光电流和低的电流失活水平是理想的。两种不同的视黄醛构象引起阳离子通道蛋白视紫红质-2（CrChR2）失活的两个平行光循环：一个具有高效导性；一个具有低导性。由于低导性光循环中间体的半衰期较长，它们在连续光照下积累，导致光电流大大降低。在这里，我们证明了对于隐甲藻的通道蛋白视紫红质-1（GtACR1），高导性的 C ⁇ = N - 反光循环是唯一使用时间分辨分步扫描 FTIR 光谱的作用循环。我们的光谱测量结果与之前报道的电生理数据之间的相关性，为分子门控机制及其在特征高光电流中的作用提供了深入的了解。还表明了中央收缩位点氨基酸 Glu-68 的机械重要性。我们提出消除不良的光循环可以避免在 CrChR2 中观察到的失活，预计这一发现将推动优化光遗传学工具的发展。
