Molecular mechanisms of cation and anion conducting channelrhodopsins
Final Report Abstract
In optogenetics, activatable cells such as neurons are stimulated by light with high temporal and spatial resolution. For this purpose, Microbial rhodopsins are widley used, although their molecular mechanisms are still largely unexplained. As part of the funding programme, we have contributed to the elucidation of the molecular reaction mechanisms of cation- and anionconducting channelrhodopsins (CCRs and ACRs). For this matter we used time-resolved stepscan FTIR spectroscopy supplemented by UV/VIS and Raman spectroscopy together with biomolecular simulations. The spectroscopic methods provided us with mechanistic insights with high spatial-temporal resolution and the structural details of which were subsequently deciphered using biomolecular simulations. The knowledge gained was correlated with existing electrophysiological data. A key protein for optogenetics is the CCR CrChR2, which, however, only generates very low photocurrents when it undergoes the low-conducitng syn-cycle of its branched photocycle. During the funding period, time-resolved step-scan FTIR spectroscopy was used to demonstrate that the homologous ACR GtACR1 only engages in the highly conductive anticycle. The correlation between spectroscopy and electrophysiology provided information about molecular gating mechanisms and their role in the characteristic high photocurrents. The mechanistic importance of the amino acid E68 in the central gate was shown in this context. In addition, we discovered that a photocycle can already be induced from the O-intermediate of GtACR1. Further FTIR-spectroscopic studies revealed conformational- and environmental changes of the protonated Schiff base, on the basis of which we postulate a ‘pre-opened’ state of GtACR1 that enables rapid channel opening and explains the high efficiency of the channel. Furthermore, we have developed an optimized computational approach to calculate the isomerisation of the retinal and the associated conformational changes. We applied this strategy to study the isomerisation of the retinal in CrChR2 and observed the spontaneous formation of a mixture of two isomerisation states, as well as the re-formation of the nonisomerised state. These results are consistent with the observed cleaved photocycle of CrChR2. In addition, structural models for early intermediates of both cycles could be predicted. The insights gained from spectroscopic measurements and biomolecular simulations contribute to a better understanding and prediction of the molecular mechanisms within the retinal proteins with high spatial and temporal resolution. On this basis, better optogenetic tools can be developed.
Publications
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Time-resolved spectroscopic and electrophysiological data reveal insights in the gating mechanism of anion channelrhodopsin. Communications Biology, 4(1).
Dreier, Max-Aylmer; Althoff, Philipp; Norahan, Mohamad Javad; Tennigkeit, Stefan Alexander; El-Mashtoly, Samir F.; Lübben, Mathias; Kötting, Carsten; Rudack, Till & Gerwert, Klaus
