Molecular mechanisms of cation and anion conducting channelrhodopsins

在光遗传学中，像神经元这样的可激活细胞被高时间和空间分辨率的光激发，主要是利用微生物视紫红质。尽管它们在光遗传学中的应用越来越广泛，但其具体机制仍在争论中。在此，我们将通过时间分辨步进扫描FTIR光谱，结合UV/VIS和拉曼光谱，结合分子生物学和生物分子模拟，揭示阳离子和阴离子导电通道紫红质（CCRs和ACRs）的分子反应机制。我们期望通过对acr和ccr分子机制的比较，揭示它们的关键结构基序，尽管它们的结构非常相似，但它们的功能却截然不同。这些见解对于理解视网膜蛋白具有基本的意义。我们最近发表的数据为最深入研究的光遗传学工具CrChR2提供了一个平行光循环模型，这使得激烈讨论的相互矛盾的光谱和电生理数据达成了一致。我们的模型是理解通道视紫红质的一个里程碑：光激活的CrChR2分裂为一个强质子和离子传导的短时间黑暗适应（=“反循环”）和一个质子传导差但长时间光适应的光循环（=“同步循环”）。连续照明导致缓慢衰减的同步周期的积累，解释了由于低离子电导率而导致的失活光电流。在我们的提案中，我们解决了以下关键问题：(1)在反循环中打开阳离子通道CrChR2的分子机制是什么？(2)阴离子通道GtACR1打开的分子机制是什么？(3)为什么acr比ccr表现出更高的离子电导率？(4) ACRs和CCRs，尤其是CrChR2和GtACR1相比，哪些关键残基诱导了它们的功能差异（如光适应）？为了获得具有高时空分辨率的机制见解，我们采用了时间分辨光谱测量。然后，利用生物分子模拟来解析编码在光谱数据中的结构细节。通道的功能由我们的合作伙伴进行的电生理测量来探测。实验和模拟的结合将阐明acr和ccr的结构/功能关系的异同，这些异同导致了它们的不同性能，特别是在离子电导率方面。利用这些获得的知识，我们的目标是通过位点特异性诱变将功能从acr转移到ccr，反之亦然。我们期望抑制CrChR2的光适应同步循环的形成。因此，应该减少失活效应，并且应该创建具有更高离子电导率的CrChR2，从而显著提高光遗传潜力。

In optogenetics activatable cells like neurons are excited by light with high temporal and spatial resolution using mostly microbial rhodopsins. Despite their tremendously increasing application in optogenetics the detailed mechanism is still under debate. Here, we intend to unveil the molecular reaction mechanisms of cation and anion conducting channelrhodopsins (CCRs and ACRs) by time-resolved step-scan FTIR spectroscopy, complemented by UV/VIS and Raman spectroscopy together with molecular biology, and biomolecular simulations. We expect that the comparison of the molecular mechanisms of ACRs and CCRs will reveal the key structural motifs, which determine, despite their very similar structures, their distinct functions. Such insights are of basic interest to understand retinal proteins. Our recently published data gave rise to a parallel photocycle model for the most deeply studied optogenetic tool CrChR2, which brought intensely discussed conflicting spectroscopic and electrophysiological data to an agreement. Our model is a milestone in understanding channelrhodopsins: Photoactivated CrChR2 divides to a strongly proton and ion conducting short-lived dark-adapted (=“anti-cycle”) and a poorly proton conducting but long-living light-adapted photocycle (=“syn-cycle”). Continuous illumination leads to accumulation of the slower decaying syn-cycle explaining the inactivated photocurrents due to low ion conductivity.In our proposal we address the following key questions:(1) What are the molecular mechanisms underlying the opening of the cation channel CrChR2 in the anti-cycle?(2) What are the molecular mechanisms underlying the opening of the anion channel GtACR1?(3) Why do ACRs exhibit much higher ion conductivities than CCRs?(4) What are the key residues inducing the functional differences (e.g. light adaptation) of ACRs and CCRs, especially in comparison of CrChR2 and GtACR1?To obtain mechanistic insights with high spatio-temporal resolution we employ time-resolved spectroscopic measurements. Then, biomolecular simulations are used to resolve the structural details, which are encoded in the spectroscopic data. The functionality of the channels is probed by electrophysiological measurements performed by our collaboration partners. The integration of experiment and simulation will clarify the similarities and differences in the structure/function relationships of ACRs and CCRs being responsible for their distinct performances, in particular regarding ion conductivity. Using this acquired knowledge we aim to transfer the functionalities from ACRs to CCRs and vice versa by site-specific mutagenesis. We expect to suppress the formation of the light-adapted syn-cycle of CrChR2. Thus, the inactivation effect should be reduced, and a CrChR2 with increased ion conductivity should be created, giving rise to significantly improved optogenetic potential.