Although channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light adaptation, photocurrent inactivation, and alteration of the ion selectivity during continuous illumination are not well understood from a molecular perspective. Herein, we address these open questions using single-turnover electrophysiology, time-resolved step-scan FTIR, and Raman spectroscopy of fully dark-adapted ChR2. This yields a unifying parallel photocycle model integrating now all so far controversial discussed data. In dark-adapted ChR2, the protonated retinal Schiff base chromophore (RSBH+) adopts an all-trans,C=N-anti conformation only. Upon light activation, a branching reaction into either a 13-cis,C=N-anti or a 13-cis,C=N-syn retinal conformation occurs. The anti-cycle features sequential H+ and Na+ conductance in a late M-like state and an N-like open-channel state. In contrast, the 13-cis,C=N-syn isomer represents a second closed-channel state identical to the long-lived P480 state, which has been previously assigned to a late intermediate in a single-photocycle model. Light excitation of P480 induces a parallel syn-photocycle with an open-channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P480 and stays deprotonated in the C=N-syn cycle. Deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light adaptation. Parallel anti- and syn-photocycles now explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.

Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gα i1 , which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine "finger." We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gα i1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gα i1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gα i1 ·RGS4 mechanism. This movement induces a charge shift toward β-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis.GTPase | FTIR spectroscopy | QM/MM calculations | arginine finger | reaction mechanism

Optogenetics uses light‐sensitive proteins, so‐called optogenetic tools, for highly precise spatiotemporal control of cellular states and signals. The major limitations of such tools include the overlap of excitation spectra, phototoxicity, and lack of sensitivity. The protein characterized in this study, the Japanese lamprey parapinopsin, which we named UVLamP, is a promising optogenetic tool to overcome these limitations. Using a hybrid strategy combining molecular, cellular, electrophysiological, and computational methods we elucidated a structural model of the dark state and probed the optogenetic potential of UVLamP. Interestingly, it is the first described bistable vertebrate opsin that has a charged amino acid interacting with the Schiff base in the dark state, that has no relevance for its photoreaction. UVLamP is a bistable UV‐sensitive opsin that allows for precise and sustained optogenetic control of G protein‐coupled receptor (GPCR) pathways and can be switched on, but more importantly also off within milliseconds via lowintensity short light pulses. UVLamP exhibits an extremely narrow excitation spectrum in the UV range allowing for sustained activation of the Gi/o pathway with a millisecond UV light pulse. Its sustained pathway activation can be switched off, surprisingly also with a millisecond blue light pulse, minimizing phototoxicity. Thus, UVLamP serves as a minimally invasive, narrow‐bandwidth probe for controlling the Gi/o pathway, allowing for combinatorial use with multiple optogenetic tools or sensors. Because UVLamP activated Gi/o signals are generally inhibitory and decrease cellular activity, it has tremendous potential for health‐related applications such as relieving pain, blocking seizures, and delaying neurodegeneration.

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.

The primary goal of optogeneticsi st he light-controlled noninvasive and specific manipulation of various cellular processes. Herein, we present ah ybrid strategy for targeted protein engineering combining computational techniques with electrophysiologicala nd UV/visible spectroscopic experiments.W ev alidated our concept for channelrhodopsin-2 and applied it to modify the less-well-studied vertebrate opsin melanopsin. Melanopsin is ap romising optogenetic tool that functions as a selectivem olecular light switch forGprotein-coupled receptor pathways. Thus, we constructed am odel of the melanopsin G q protein complex and predicted an absorption maximum shift of the Y211F variant. This variant displays an arrow blue-shifted action spectrum and twofold faster deactivation kinetics compared to wild-type melanopsin on Gprotein-coupledi nward rectifying K + (GIRK) channels in HEK293c ells. Furthermore, we verifiedt he in vivo activity and optogenetic potential for the variant in mice. Thus, we proposet hat our developed concept will be generallyapplicable to designing optogenetic tools.

The primary goal of optogenetics is the light‐controlled noninvasive and specific manipulation of various cellular processes. Herein, we present a hybrid strategy for targeted protein engineering combining computational techniques with electrophysiological and UV/visible spectroscopic experiments. We validated our concept for channelrhodopsin‐2 and applied it to modify the less‐well‐studied vertebrate opsin melanopsin. Melanopsin is a promising optogenetic tool that functions as a selective molecular light switch for G protein‐coupled receptor pathways. Thus, we constructed a model of the melanopsin Gq protein complex and predicted an absorption maximum shift of the Y211F variant. This variant displays a narrow blue‐shifted action spectrum and twofold faster deactivation kinetics compared to wild‐type melanopsin on G protein‐coupled inward rectifying K+ (GIRK) channels in HEK293 cells. Furthermore, we verified the in vivo activity and optogenetic potential for the variant in mice. Thus, we propose that our developed concept will be generally applicable to designing optogenetic tools.

Although Channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light-adaptation, photocurrent inactivation, and alteration of the ion selectivity during continuous illumination are not well-understood from a molecular perspective. Herein, we address these open questions using single turn-over electrophysiology, time-resolved step-scan FTIR and Raman spectroscopy of fully dark adapted ChR2. This yields a unifying parallel photocycle model explaining all data: in dark-adapted ChR2, the protonated Schiff base retinal chromophore (RSBH + ) adopts an all-trans,C=N-anti conformation only. Upon light activation, a branching reaction into either a 13-cis,C=N-anti or a 13-cis,C=N-syn retinal conformation occurs. The anti-cycle features sequential H + and Na + conductance in a late M-like state and an N-like open-channel state. In contrast, the 13cis,C=N-syn isomer represents a second closed-channel state identical to the long lived P480-state, which has been previously assigned to a late intermediate in a single photocycle model. Light excitation of P480 induces a parallel syn-photocycle with an open channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P480 and stays deprotonated in the C=N-syn-cycle and we show that deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light-adaptation. Parallel anti-and syn-photocycles explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools. Significance statementUnderstanding the mechanisms of photoactivated biological processes facilitates the development of new molecular tools, engineered for specific optogenetic applications, allowing the control of neuronal activity with light. Here, we use a variety of experimental and theoretical techniques to examine the precise nature of the light-activated ion channel in one of the most important molecular species used in optogenetics, channelrhodopsin-2. Existing models for the photochemical and photophysical pathway after light absorption by the molecule fail to explain many aspects of its observed behavior including the inactivation of the photocurrent under continuous illumination. We resolve this by proposing a new branched photocycle explaining electrical and photochemical channel properties and establishing the structure of intermediates during channel turnover.

Optogenetic control of G protein‐coupled receptor pathways: The Japanese lamprey (Lethenteron camtschaticum) parapinopsin (“UVLamP”) serves as a minimally invasive, narrow‐bandwidth, bistable, next‐generation optogenetic probe for controlling the Gi/o pathway. A millisecond UV light pulse allows for sustained pathway activation that can be switched off with a millisecond blue light pulse on demand. The first structural model of parapinopsin in the dark state reveals novel interaction partners shedding light on the mechanisms responsible for opsin bistability. More information can be found in the communication by D. Eickelbeck, K. Gerwert, S. Herlitze et al. on page 612 in Issue 5, 2020 (DOI: 10.1002/cbic.201900485).