Photoexcitation of the P<sub>4</sub><sup>480</sup> State Induces a Secondary Photocycle That Potentially Desensitizes Channelrhodopsin-2

Saita, Mattia; Pranga-Sellnau, Franziska; Resler, Tom; Schlesinger, Ramona; Heberle, Joachim; Lórenz-Fonfrı́a, Vı́ctor A.

doi:10.1021/jacs.8b03931

Cited by 22 publications

(35 citation statements)

References 29 publications

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“…Indeed, the opening and closing kinetics of ChR2 are slower than those of voltage-gated Na + and K + channels. Photocycle models for ChR2 describe two open states (O1–O2) and at least two non-conductive “D” sub-states characterized by a slow recovery –from 10 s to more than 20 s– after a dark period (Ernst et al, 2008; Stehfest and Hegemann, 2010; Grossman et al, 2011; Saita et al, 2018). This requirement of a long-lasting dark period to recruit ChR2 activation upon a new light stimulus partially explains the limited neuronal response to high frequency photostimulation (Ernst et al, 2008; Grossman et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

In vivo Optogenetic Approach to Study Neuron-Oligodendroglia Interactions in Mouse Pups

Ortolani

Manot-Saillet

Orduz

et al. 2018

Front. Cell. Neurosci.

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Optogenetic and pharmacogenetic techniques have been effective to analyze the role of neuronal activity in controlling oligodendroglia lineage cells in behaving juvenile and adult mice. This kind of studies is also of high interest during early postnatal (PN) development since important changes in oligodendroglia dynamics occur during the first two PN weeks. Yet, neuronal manipulation is difficult to implement at an early age because high-level, specific protein expression is less reliable in neonatal mice. Here, we describe a protocol allowing for an optogenetic stimulation of neurons in awake mouse pups with the purpose of investigating the effect of neuronal activity on oligodendroglia dynamics during early PN stages. Since GABAergic interneurons contact oligodendrocyte precursor cells (OPCs) through bona fide synapses and maintain a close relationship with these progenitors during cortical development, we used this relevant example of neuron-oligodendroglia interaction to implement a proof-of-principle optogenetic approach. First, we tested Nkx2.1-Cre and Parvalbumin (PV)-Cre lines to drive the expression of the photosensitive ion channel channelrhodopsin-2 (ChR2) in subpopulations of interneurons at different developmental stages. By using patch-clamp recordings and photostimulation of ChR2-positive interneurons in acute somatosensory cortical slices, we analyzed the level of functional expression of ChR2 in these neurons. We found that ChR2 expression was insufficient in PV-Cre mouse at PN day 10 (PN10) and that this channel needs to be expressed from embryonic stages (as in the Nkx2.1-Cre line) to allow for a reliable photoactivation in mouse pups. Then, we implemented a stereotaxic surgery to place a mini-optic fiber at the cortical surface in order to photostimulate ChR2-positive interneurons at PN10. In vivo field potentials were recorded in Layer V to verify that photostimulation reaches deep cortical layers. Finally, we analyzed the effect of the photostimulation on the layer V oligodendroglia population by conventional immunostainings. Neither the total density nor a proliferative fraction of OPCs were affected by increasing interneuron activity in vivo, complementing previous findings showing the lack of effect of GABAergic synaptic activity on OPC proliferation. The methodology described here should provide a framework for future investigation of the role of early cellular interactions during PN brain maturation.

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Section: Discussionmentioning

confidence: 99%

In vivo Optogenetic Approach to Study Neuron-Oligodendroglia Interactions in Mouse Pups

Ortolani

Manot-Saillet

Orduz

et al. 2018

Front. Cell. Neurosci.

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“…Without considering parallel photocycles we initially attributed early pore hydration due to E90 deprotonation to a pregating step in a linear photocycle. At the same time early E90 deprotonation has been challenged by measurements of partly light-adapted ChR2 arguing that E90 is only deprotonated during the lifetime of P480 that was assumed to be a late intermediate of a linear photocycle or a late branching reaction (8,14,41). Comparing FTIR measurements of dark-and light-adapted ChR2 we were able to resolve the controversy regarding E90 deprotonation.…”

Section: Deprotonation Of E90 Is Essential For Proton Conductance Of mentioning

confidence: 93%

A unifying photocycle model for light adaptation and temporal evolution of cation conductance in Channelrhodopsin-2

Kühne

Vierock

Tennigkeit

et al. 2018

Preprint

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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.

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“…This band was hardly seen in T127A due to spectral overlap by a positive contribution that is absent in the WT. Instead, a broad negative band at 1533 cm −1 was registered, which was a marker for the contribution of the P4 480 photoreaction [35]. Figure 8.…”

Section: Ftir Difference Spectroscopy On the T127 Variantsmentioning

confidence: 98%

Atomistic Insight into the Role of Threonine 127 in the Functional Mechanism of Channelrhodopsin-2

et al. 2019

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Channelrhodopsins (ChRs) belong to the unique class of light-gated ion channels. The structure of channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) has been resolved, but the mechanistic link between light-induced isomerization of the chromophore retinal and channel gating remains elusive. Replacements of residues C128 and D156 (DC gate) resulted in drastic effects in channel closure. T127 is localized close to the retinal Schiff base and links the DC gate to the Schiff base. The homologous residue in bacteriorhodopsin (T89) has been shown to be crucial for the visible absorption maximum and dark–light adaptation, suggesting an interaction with the retinylidene chromophore, but the replacement had little effect on photocycle kinetics and proton pumping activity. Here, we show that the T127A and T127S variants of CrChR2 leave the visible absorption maximum unaffected. We inferred from hybrid quantum mechanics/molecular mechanics (QM/MM) calculations and resonance Raman spectroscopy that the hydroxylic side chain of T127 is hydrogen-bonded to E123 and the latter is hydrogen-bonded to the retinal Schiff base. The C=N–H vibration of the Schiff base in the T127A variant was 1674 cm−1, the highest among all rhodopsins reported to date. We also found heterogeneity in the Schiff base ground state vibrational properties due to different rotamer conformations of E123. The photoreaction of T127A is characterized by a long-lived P2380 state during which the Schiff base is deprotonated. The conservative replacement of T127S hardly affected the photocycle kinetics. Thus, we inferred that the hydroxyl group at position 127 is part of the proton transfer pathway from D156 to the Schiff base during rise of the P3530 intermediate. This finding provides molecular reasons for the evolutionary conservation of the chemically homologous residues threonine, serine, and cysteine at this position in all channelrhodopsins known so far.

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Photoexcitation of the P₄⁴⁸⁰ State Induces a Secondary Photocycle That Potentially Desensitizes Channelrhodopsin-2

Cited by 22 publications

References 29 publications

In vivo Optogenetic Approach to Study Neuron-Oligodendroglia Interactions in Mouse Pups

In vivo Optogenetic Approach to Study Neuron-Oligodendroglia Interactions in Mouse Pups

A unifying photocycle model for light adaptation and temporal evolution of cation conductance in Channelrhodopsin-2

Atomistic Insight into the Role of Threonine 127 in the Functional Mechanism of Channelrhodopsin-2

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Photoexcitation of the P4480 State Induces a Secondary Photocycle That Potentially Desensitizes Channelrhodopsin-2

Cited by 22 publications

References 29 publications

In vivo Optogenetic Approach to Study Neuron-Oligodendroglia Interactions in Mouse Pups

In vivo Optogenetic Approach to Study Neuron-Oligodendroglia Interactions in Mouse Pups

A unifying photocycle model for light adaptation and temporal evolution of cation conductance in Channelrhodopsin-2

Atomistic Insight into the Role of Threonine 127 in the Functional Mechanism of Channelrhodopsin-2

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Photoexcitation of the P₄⁴⁸⁰ State Induces a Secondary Photocycle That Potentially Desensitizes Channelrhodopsin-2