Molecular mechanisms of cation and anion conducting channelrhodopsins

阳离子和阴离子传导视紫红质通道的分子机制

• 批准号: 436212556
• 负责人: Professor Dr. Klaus Gerwert
• 金额: --
• 依托单位: Lehrstuhl für Biophysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/436212556?language=en
• 关键词: Molecular; mechanisms; cation; anion; conducting

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.

在光遗传学中，可激活的细胞如神经元被具有高时间和空间分辨率的光激发，主要使用微生物视紫红质。尽管它们在光遗传学中的应用越来越多，但详细的机制仍在争论中。在这里，我们打算揭示阳离子和阴离子传导通道视紫红质（CCR和ACR）的分子反应机制的时间分辨步进扫描FTIR光谱，辅以UV/维斯和拉曼光谱与分子生物学，和生物分子模拟。我们期望，ACR和CCR的分子机制的比较将揭示关键的结构基序，这决定了，尽管它们非常相似的结构，其不同的功能。这些见解是理解视网膜蛋白质的基本兴趣。我们最近发表的数据为研究最深入的光遗传学工具CrChR2提出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：光活化的CrChR2分为一个强质子和离子传导短寿命的暗适应（="反循环"）和一个质子传导差，但长寿命的光适应光循环（="同步循环"）。连续光照导致积累的缓慢衰减的同步循环解释的失活光电流由于低离子cancelity.In我们的建议，我们解决了以下关键问题：（1）是什么样的分子机制的阳离子通道CrChR2的开放的反循环？(2)阴离子通道GtACR 1开放的分子机制是什么？(3)为什么ACR的离子电导率比CCR高得多？(4)诱导ACC和CCR功能差异（例如光适应）的关键残基是什么，特别是与CrChR 2和GtACR 1相比？为了获得具有高时空分辨率的机械见解，我们采用时间分辨光谱测量。然后，使用生物分子模拟来解析光谱数据中编码的结构细节。通过我们的合作伙伴进行的电生理测量来探测通道的功能。实验和模拟的集成将澄清ACR和CCR的结构/功能关系的相似性和差异，负责其不同的性能，特别是关于离子电导率。利用这些知识，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的光适应顺式循环的形成。因此，失活效应应降低，并且应产生具有增加的离子传导性的CrChR2，从而产生显著改善的光遗传学潜力。