Molecular mechanisms of cation and anion conducting channelrhodopsins

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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.