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Peter Hegemann - One of the best experts on this subject based on the ideXlab platform.

  • crystal structure of the red light activated Channelrhodopsin chrimson
    Nature Communications, 2018
    Co-Authors: Kazumasa Oda, Johannes Vierock, Satomi Oishi, Silvia Rodriguezrozada, Reiya Taniguchi, Keitaro Yamashita, J S Wiegert, Tomohiro Nishizawa, Peter Hegemann
    Abstract:

    Channelrhodopsins are light-activated ion channels that mediate cation permeation across cell membranes upon light absorption. Red-light-activated Channelrhodopsins are of particular interest, because red light penetrates deeper into biological tissues and also enables dual-color experiments in combination with blue-light-activated optogenetic tools. Here we report the crystal structure of the most red-shifted Channelrhodopsin from the algae Chlamydomonas noctigama, Chrimson, at 2.6 A resolution. Chrimson resembles prokaryotic proton pumps in the retinal binding pocket, while sharing similarity with other Channelrhodopsins in the ion-conducting pore. Concomitant mutation analysis identified the structural features that are responsible for Chrimson’s red light sensitivity; namely, the protonation of the counterion for the retinal Schiff base, and the polar residue distribution and rigidity of the retinal binding pocket. Based on these mechanistic insights, we engineered ChrimsonSA, a mutant with a maximum activation wavelength red-shifted beyond 605 nm and accelerated closing kinetics. Channelrhodopsins are light-activated ion channels that mediate cation permeation across cell membranes upon light absorption. Here, the authors report the crystal structure of the most red-shifted Channelrhodopsin from the algae Chlamydomonas noctigama at 2.6 A resolution.

  • The form and function of Channelrhodopsin
    Science (New York N.Y.), 2017
    Co-Authors: Karl Deisseroth, Peter Hegemann
    Abstract:

    BACKGROUND Channelrhodopsins (ChRs) are naturally occurring light-gated ion channels that are important for allowing motile algal cells to find suitable light levels. In neuroscience, ChRs have become broadly significant for helping to enable the control of specific circuit elements with light (i.e., optogenetics). Research into how sensation, cognition, and behavior arise from neuronal activity dynamics has been enabled by the expression of ChRs, and other members of the microbial opsin family, in specific cells or in specific connections within nervous systems of behaving animals. Both the unique light-gated channels themselves and opportunities for their biological application have been under intense investigation. The resulting studies of atomic-scale structure-function relationships have led not only to sophisticated understanding of the underlying chemical processes governing these unique seven-transmembrane channels from the plant kingdom, but also (via optogenetics) to the discovery of fundamental neural circuit principles underlying adaptive and maladaptive behavior in animals. ADVANCES The atomic-scale understanding of light-gated ion channel function has spanned the key processes of activation/deactivation gating, light adaptation, color tuning, and ion selectivity. A ChR crystal structure–derived, molecular dynamics–calculated pore snapshot (top left panel of the figure) summarizes the wide scope of biophysical and biochemical discoveries. Molecular modeling and redesign have created multiple modes of coupling between delivered photons and spikes in an approach that has illuminated basic principles of protein function and also created new tools for optogenetics. In the top right panel of the figure, the top trace shows a photon-spike transduction mode arising from the ChETA mutation, which results in high-speed, high-fidelity single blue flash–single spike coupling. The second trace shows red photon-spike transduction arising from a redshifted ChR found in nature and then engineered for stronger, more redshifted performance (C1V1). The third trace shows bistable excitation photon-spike logic, in which step-function opsin (SFO) mutations were introduced to create stalled photocycles, allowing stable excitation without continuous light delivery. The bottom trace shows bistable inhibition photon-spike logic; ChRs that are normally cation-conducting, and are therefore excitatory in neural systems, were converted to anion-conducting (inhibitory) ChRs by replacing negatively charged pore residues, followed by SFO mutations for bistability. The C1V1 and SFO designs together allowed us to determine that the medial prefrontal neocortex modulates interactions between two distant subcortical structures to control reward-mediating physiology and behavior (clarityresourcecenter.org/ofMRI.html; www.optogenetics.org). OUTLOOK The ChR light-gated pore will continue to be studied for its own elegant properties, which are paradigmatic among ion channels because light-gated systems allow structure-function analysis on the femtosecond time scale. Meanwhile, psychiatry has already yielded some of its deepest mysteries to ChR pore structural insights, including in explorations of clinically relevant behavioral states such as anhedonia. Many more opportunities for ChRs in basic neuroscience remain untapped, with the potential for precision redesign to achieve new applications and new roles integrated with other advanced technologies.

  • active site structure and absorption spectrum of Channelrhodopsin 2 wild type and c128t mutant
    Chemical Science, 2016
    Co-Authors: Yanan Guo, Peter Hegemann, Hiroshi Watanabe, Franziska E Beyle, Beatrix M Bold, Axel Koslowski, Walter Thiel, Marco Marazzi, Marcus Elstner
    Abstract:

    In spite of considerable interest, the active site of Channelrhodopsin still lacks a detailed atomistic description, the understanding of which could strongly enhance the development of novel optogenetics tools. We present a computational study combining different state-of-the-art techniques, including hybrid quantum mechanics/molecular mechanics schemes and high-level quantum chemical methods, to properly describe the hydrogen-bonding pattern between the retinal chromophore and its counterions in Channelrhodopsin-2 Wild-Type and C128T mutant. Especially, we show by extensive ground state dynamics that the active site, containing a glutamic acid (E123) and a water molecule, is highly dynamic, sampling three different hydrogen-bonding patterns. This results in a broad absorption spectrum that is representative of the different structural motifs found. A comparison with bacteriorhodopsin, characterized by a pentagonal hydrogen-bonded active site structure, elucidates their different absorption properties.

  • identification of a natural green light absorbing chloride conducting Channelrhodopsin from proteomonas sulcata
    Journal of Biological Chemistry, 2016
    Co-Authors: Jonas Wietek, Matthias Broser, Benjamin S Krause, Peter Hegemann
    Abstract:

    Chloride conducting Channelrhodopsins (ChloCs) are new members of the optogenetic toolbox that enable neuronal inhibition in target cells. Originally, ChloCs have been engineered from cation conducting Channelrhodopsins (ChRs), and later identified in a cryptophyte alga genome. We noticed that the sequence of a previously described Proteomonas sulcata ChR (PsChR1) was highly homologous to the naturally occurring and previously reported ChloCs GtACR1/2, but was not recognized as an anion conducting channel. Based on electrophysiological measurements obtained under various ionic conditions, we concluded that the PsChR1 photocurrent at physiological conditions is strongly inward rectifying and predominantly carried by chloride. The maximum activation was noted at excitation with light of 540 nm. An initial spectroscopic characterization of purified protein revealed that the photocycle and the transport mechanism of PsChR1 differ significantly from cation conducting ChRs. Hence, we concluded that PsChR1 is an anion conducting ChR, now renamed PsACR1, with a red-shifted absorption suited for multicolor optogenetic experiments in combination with blue light absorbing cation conducting ChRs.

  • Time-resolved infrared spectroscopic techniques as applied to Channelrhodopsin
    Frontiers in molecular biosciences, 2015
    Co-Authors: Eglof Ritter, Peter Hegemann, Ljiljana Puskar, Franz Bartl, Emad F. Aziz, Ulrich Schade
    Abstract:

    Among optogenetic tools, Channelrhodopsins, the light gated ion channels of the plasma membrane from green algae, play the most important role. Properties like channel selectivity, timing parameters or color can be influenced by the exchange of selected amino acids. Although widely used, in the field of neurosciences for example, there is still little known about their photocycles and the mechanism of ion channel gating and conductance. One of the preferred methods for these studies is infrared spectroscopy since it allows observation of proteins and their function at a molecular level and in near-native environment. The absorption of a photon in Channelrhodopsin leads to retinal isomerization within femtoseconds, the conductive states are reached in the microsecond time scale and the return into the fully dark-adapted state may take more than minutes. To be able to cover all these time regimes, a range of different spectroscopical approaches are necessary. This mini-review focuses on time-resolved applications of the infrared technique to study Channelrhodopsins and other light triggered proteins. We will discuss the approaches with respect to their suitability to the investigation of Channelrhodopsin and related proteins.

Joachim Heberle - One of the best experts on this subject based on the ideXlab platform.

  • photoexcitation of the p4480 state induces a secondary photocycle that potentially desensitizes Channelrhodopsin 2
    Journal of the American Chemical Society, 2018
    Co-Authors: Mattia Saita, Tom Resler, Ramona Schlesinger, Joachim Heberle, Franziska Prangasellnau, Victor A Lorenzfonfria
    Abstract:

    Channelrhodopsins (ChRs) are light-gated cation channels. In spite of their wide use to activate neurons with light, the photocurrents of ChRs rapidly decay in intensity under both continuous illum...

  • vibronic dynamics of the ultrafast all trans to 13 cis photoisomerization of retinal in Channelrhodopsin 1
    Journal of the American Chemical Society, 2016
    Co-Authors: Christoph Schnedermann, Ramona Schlesinger, Vera Muders, D Ehrenberg, Philipp Kukura, Joachim Heberle
    Abstract:

    Channelrhodopsins are light-gated ion channels with extensive applications in optogenetics. Channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) exhibits a red-shifted absorption spectrum as compared to Channelrhodopsin-2, which is highly beneficial for optogenetic application. The primary event in the photocycle of CaChR1 involves an isomerization of the protein-bound retinal chromophore. Here, we apply highly time-resolved vibronic spectroscopy to reveal the electronic and structural dynamics associated with the first step of the photocycle of CaChR1. We observe vibrationally coherent formation of the P1 intermediate exhibiting a twisted 13-cis retinal with a 110 ± 7 fs time constant. Comparison with low-temperature resonance Raman spectroscopy of the corresponding trapped photoproduct demonstrates that this rapidly formed P1 intermediate is stable for several hundreds of nanoseconds.

  • temporal evolution of helix hydration in a light gated ion channel correlates with ion conductance
    Proceedings of the National Academy of Sciences of the United States of America, 2015
    Co-Authors: Victor A Lorenzfonfria, Christian Bamann, Ernst Bamberg, Tom Resler, Ramona Schlesinger, Joachim Heberle
    Abstract:

    The discovery of Channelrhodopsins introduced a new class of light-gated ion channels, which when genetically encoded in host cells resulted in the development of optogenetics. Channelrhodopsin-2 from Chlamydomonas reinhardtii, CrChR2, is the most widely used optogenetic tool in neuroscience. To explore the connection between the gating mechanism and the influx and efflux of water molecules in CrChR2, we have integrated light-induced time-resolved infrared spectroscopy and electrophysiology. Cross-correlation analysis revealed that ion conductance tallies with peptide backbone amide I vibrational changes at 1,665(-) and 1,648(+) cm(-1). These two bands report on the hydration of transmembrane α-helices as concluded from vibrational coupling experiments. Lifetime distribution analysis shows that water influx proceeded in two temporally separated steps with time constants of 10 μs (30%) and 200 μs (70%), the latter phase concurrent with the start of ion conductance. Water efflux and the cessation of the ion conductance are synchronized as well, with a time constant of 10 ms. The temporal correlation between ion conductance and hydration of helices holds for fast (E123T) and slow (D156E) variants of CrChR2, strengthening its functional significance.

  • changes in the hydrogen bonding strength of internal water molecules and cysteine residues in the conductive state of Channelrhodopsin 1
    Journal of Chemical Physics, 2014
    Co-Authors: Victor A Lorenzfonfria, Ramona Schlesinger, Vera Muders, Joachim Heberle
    Abstract:

    Water plays an essential role in the structure and function of proteins, particularly in the less understood class of membrane proteins. As the first of its kind, Channelrhodopsin is a light-gated cation channel and paved the way for the new and vibrant field of optogenetics, where nerve cells are activated by light. Still, the molecular mechanism of Channelrhodopsin is not understood. Here, we applied time-resolved FT-IR difference spectroscopy to Channelrhodopsin-1 from Chlamydomonas augustae. It is shown that the (conductive) P2 380 intermediate decays with τ ≈ 40 ms and 200 ms after pulsed excitation. The vibrational changes between the closed and the conductive states were analyzed in the X-H stretching region (X = O, S, N), comprising vibrational changes of water molecules, sulfhydryl groups of cysteine side chains and changes of the amide A of the protein backbone. The O-H stretching vibrations of “dangling” water molecules were detected in two different states of the protein using H2 18O exchange. Uncoupling experiments with a 1:1 mixture of H2O:D2O provided the natural uncoupled frequencies of the four O-H (and O-D) stretches of these water molecules, each with a very weakly hydrogen-bonded O-H group (3639 and 3628 cm−1) and with the other O-H group medium (3440 cm−1) to moderately strongly (3300 cm−1) hydrogen-bonded. Changes in amide A and thiol vibrations report on global and local changes, respectively, associated with the formation of the conductive state. Future studies will aim at assigning the respective cysteine group(s) and at localizing the “dangling” water molecules within the protein, providing a better understanding of their functional relevance in CaChR1.

  • resonance raman and ftir spectroscopic characterization of the closed and open states of Channelrhodopsin 1
    FEBS Letters, 2014
    Co-Authors: Vera Muders, Christian Bamann, Victor A Lorenzfonfria, Joachim Heberle, Silke Kerruth, Ramona Schlesinger
    Abstract:

    Channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) is a light-activated cation channel, which is a promising optogenetic tool. We show by resonance Raman spectroscopy and retinal extraction followed by high pressure liquid chromatography (HPLC) that the isomeric ratio of all-trans to 13-cis of solubilized Channelrhodopsin-1 is with 70:30 identical to Channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Critical frequency shifts in the retinal vibrations are identified in the Raman spectrum upon transition to the open (conductive P2(380)) state. Fourier transform infrared spectroscopy (FTIR) spectra indicate different structures of the open states in the two Channelrhodopsins as reflected by the amide I bands and the protonation pattern of acidic amino acids.

Karl Deisseroth - One of the best experts on this subject based on the ideXlab platform.

  • investigating the feasibility of Channelrhodopsin variants for nanoscale optogenetics
    New Phytologist, 2019
    Co-Authors: Markus A Stahlberg, Edward S Boyden, Karl Deisseroth, Charu Ramakrishnan, Katrin I Willig, Camin Dean
    Abstract:

    Optogenetics has revolutionized the study of circuit function in the brain, by allowing activation of specific ensembles of neurons by light. However, this technique has not yet been exploited extensively at the subcellular level. Here, we test the feasibility of a focal stimulation approach using stimulated emission depletion/reversible saturable optical fluorescence transitions-like illumination, whereby switchable light-gated channels are focally activated by a laser beam of one wavelength and deactivated by an overlapping donut-shaped beam of a different wavelength, confining activation to a center focal region. This method requires that activated Channelrhodopsins are inactivated by overlapping illumination of a distinct wavelength and that photocurrents are large enough to be detected at the nanoscale. In tests of current optogenetic tools, we found that ChR2 C128A/H134R/T159C and CoChR C108S and C108S/D136A-activated with 405-nm light and inactivated by coillumination with 594-nm light-and C1V1 E122T/C167S-activated by 561-nm light and inactivated by 405-nm light-were most promising in terms of highest photocurrents and efficient inactivation with coillumination. Although further engineering of step-function Channelrhodopsin variants with higher photoconductances will be required to employ this approach at the nanoscale, our findings provide a framework to guide future development of this technique.

  • The form and function of Channelrhodopsin
    Science (New York N.Y.), 2017
    Co-Authors: Karl Deisseroth, Peter Hegemann
    Abstract:

    BACKGROUND Channelrhodopsins (ChRs) are naturally occurring light-gated ion channels that are important for allowing motile algal cells to find suitable light levels. In neuroscience, ChRs have become broadly significant for helping to enable the control of specific circuit elements with light (i.e., optogenetics). Research into how sensation, cognition, and behavior arise from neuronal activity dynamics has been enabled by the expression of ChRs, and other members of the microbial opsin family, in specific cells or in specific connections within nervous systems of behaving animals. Both the unique light-gated channels themselves and opportunities for their biological application have been under intense investigation. The resulting studies of atomic-scale structure-function relationships have led not only to sophisticated understanding of the underlying chemical processes governing these unique seven-transmembrane channels from the plant kingdom, but also (via optogenetics) to the discovery of fundamental neural circuit principles underlying adaptive and maladaptive behavior in animals. ADVANCES The atomic-scale understanding of light-gated ion channel function has spanned the key processes of activation/deactivation gating, light adaptation, color tuning, and ion selectivity. A ChR crystal structure–derived, molecular dynamics–calculated pore snapshot (top left panel of the figure) summarizes the wide scope of biophysical and biochemical discoveries. Molecular modeling and redesign have created multiple modes of coupling between delivered photons and spikes in an approach that has illuminated basic principles of protein function and also created new tools for optogenetics. In the top right panel of the figure, the top trace shows a photon-spike transduction mode arising from the ChETA mutation, which results in high-speed, high-fidelity single blue flash–single spike coupling. The second trace shows red photon-spike transduction arising from a redshifted ChR found in nature and then engineered for stronger, more redshifted performance (C1V1). The third trace shows bistable excitation photon-spike logic, in which step-function opsin (SFO) mutations were introduced to create stalled photocycles, allowing stable excitation without continuous light delivery. The bottom trace shows bistable inhibition photon-spike logic; ChRs that are normally cation-conducting, and are therefore excitatory in neural systems, were converted to anion-conducting (inhibitory) ChRs by replacing negatively charged pore residues, followed by SFO mutations for bistability. The C1V1 and SFO designs together allowed us to determine that the medial prefrontal neocortex modulates interactions between two distant subcortical structures to control reward-mediating physiology and behavior (clarityresourcecenter.org/ofMRI.html; www.optogenetics.org). OUTLOOK The ChR light-gated pore will continue to be studied for its own elegant properties, which are paradigmatic among ion channels because light-gated systems allow structure-function analysis on the femtosecond time scale. Meanwhile, psychiatry has already yielded some of its deepest mysteries to ChR pore structural insights, including in explorations of clinically relevant behavioral states such as anhedonia. Many more opportunities for ChRs in basic neuroscience remain untapped, with the potential for precision redesign to achieve new applications and new roles integrated with other advanced technologies.

  • Molecular and Cellular Mechanisms for Trapping and Activating Emotional Memories
    2016
    Co-Authors: Thomas Rogerson, Karl Deisseroth, Balaji Jayaprakash, Denise J. Cai, Yoshitake Sano, Yong-seok Lee, Yu Zhou, Pallavi Bekal, Alcino J. Silva
    Abstract:

    Recent findings suggest that memory allocation to specific neurons (i.e., neuronal allocation) in the amygdala is not random, but rather the transcription factor cAMP-response element binding protein (CREB) modulates this process, perhaps by regulating the transcription of channels that control neuronal excitability. Here, optogenetic studies in the mouse lateral amygdala (LA) were used to demonstrate that CREB and neuronal excitability regulate which neurons encode an emotional memory. To test the role of CREB in memory allocation, we overexpressed CREB in the lateral amygdala to recruit the encoding of an auditory-fear conditioning (AFC) memory to a subset of neurons. Then, post-training activation of these neurons with Channelrhodopsin-2 was sufficient to trigger recall of the memory for AFC, suggesting that CREB regulates memory allocation. To test the role of neuronal excitability in memory allocation, we used a step function opsin (SFO) to transiently increase neuronal excitability in a subset of LA neurons during AFC. Post-training activation of these neurons with Volvox Channelrhodopsin-1 was able to trigger recall of that memory. Importantly, our studies show that activation of the SFO did not affect AFC by either increasing anxiety or by strengthening the unconditioned stimulus. Our findings strongly support the hypothesis that CREB regulates memory allocation by modulating neuronal excitability.

  • color tuned Channelrhodopsins for multiwavelength optogenetics
    Journal of Biological Chemistry, 2012
    Co-Authors: Matthias Prigge, Jonas Wietek, Satoshi P Tsunoda, Karl Deisseroth, Franziska Schneider, Carrie Shilyansky, Peter Hegemann
    Abstract:

    Channelrhodopsin-2 is a light-gated ion channel and a major tool of optogenetics. It is used to control neuronal activity via blue light. Here we describe the construction of color-tuned high efficiency Channelrhodopsins (ChRs), based on chimeras of Chlamydomonas Channelrhodopsin-1 and Volvox Channelrhodopsin-1. These variants show superb expression and plasma membrane integration, resulting in 3-fold larger photocurrents in HEK cells compared with Channelrhodopsin-2. Further molecular engineering gave rise to chimeric variants with absorption maxima ranging from 526 to 545 nm, dovetailing well with maxima of Channelrhodopsin-2 derivatives ranging from 461 to 492 nm. Additional kinetic fine-tuning led to derivatives in which the lifetimes of the open state range from 19 ms to 5 s. Finally, combining green- with blue-absorbing variants allowed independent activation of two distinct neural cell populations at 560 and 405 nm. This novel panel of Channelrhodopsin variants may serve as an important toolkit element for dual-color cell stimulation in neural circuits.

  • structural model of Channelrhodopsin
    Journal of Biological Chemistry, 2012
    Co-Authors: Hiroshi Watanabe, Peter Hegemann, Satoshi P Tsunoda, Feng Zhang, Karl Deisseroth, Franziska Schneider, Kai Welke, Marcus Elstner
    Abstract:

    Channelrhodopsins (ChRs) are light-gated cation channels that mediate ion transport across membranes in microalgae (vectorial catalysis). ChRs are now widely used for the analysis of neural networks in tissues and living animals with light (optogenetics). For elucidation of functional mechanisms at the atomic level, as well as for further engineering and application, a detailed structure is urgently needed. In the absence of an experimental structure, here we develop a structural ChR model based on several molecular computational approaches, capitalizing on characteristic patterns in amino acid sequences of ChR1, ChR2, Volvox ChRs, Mesostigma ChR, and the recently identified ChR of the halophilic alga Dunaliella salina. In the present model, we identify remarkable structural motifs that may explain fundamental electrophysiological properties of ChR2, ChR1, and their mutants, and in a crucial validation of the model, we successfully reproduce the excitation energy predicted by absorption spectra.

Hiroshi Tomita - One of the best experts on this subject based on the ideXlab platform.

  • local and systemic responses following intravitreous injection of aav2 encoded modified volvox Channelrhodopsin 1 in a genetically blind rat model
    Gene Therapy, 2016
    Co-Authors: Eriko Sugano, Hiroshi Tomita, Fumiaki Nishiyama, Kitako Tabata, Maki Takahashi, Makoto Tamai, H Shimizu, M Sato
    Abstract:

    We previously designed a modified Channelrhodopsin-1 (mVChR1) protein chimera with a broader action than that of Chlamydomonas Channelrhodopsin-2 and reported that its transduction into retinal ganglion cells can restore visual function in genetically blind, dystrophic Royal College of Surgeons (RCS) rats, with photostimuli ranging from 486 to 640 nm. In the current study, we sought to investigate the safety and influence of mVChR1 transgene expression. Adeno-associated virus type 2 encoding mVChR1 was administered by intravitreous injection into dystrophic RCS rats. Reverse-transcription PCR was used to monitor virus and transgene dissemination and the results demonstrated that their expression was restricted specifically within the eye tissues, and not in non-target organs. Moreover, examination of the blood, plasma and serum revealed that no excess immunoreactivity was present, as determined using standard clinical hematological parameters. Serum antibodies targeting the recombinant adeno-associated virus (rAAV) capsid increased after the injection; however, no increase in mVChR1 antibody was detected during the observation period. In addition, retinal histological examination showed no signs of inflammation in rAAV-injected rats. In conclusion, our results demonstrate that mVChR1 can be exogenously expressed without harmful immunological reactions in vivo. These findings will aid in studies of AAV gene transfer to restore vision in late-stage retinitis pigmentosa.

  • restoration of the majority of the visual spectrum by using modified volvox Channelrhodopsin 1
    Molecular Therapy, 2014
    Co-Authors: Hiroshi Tomita, Eriko Sugano, Namie Murayama, Taku Ozaki, Fumiaki Nishiyama, Kitako Tabata, Maki Takahashi, Takehiko Saito, Makoto Tamai
    Abstract:

    We previously showed that blind rats whose vision was restored by gene transfer of Chlamydomonas Channelrhodopsin-2 (ChR2) could only detect wavelengths less than 540 nm because of the action spectrum of the transgene product. Volvox-derived Channelrhodopsin-1, VChR1, has a broader spectrum than ChR2. However, the VChR1 protein was mainly localized in the cytoplasm and showed weak ion channel properties when the VChR1 gene was transfected into HEK293 cells. We generated modified Volvox Channelrhodopsin-1 (mVChR1), which is a chimera of Volvox Channelrhodopsin-1 and Chlamydomonas Channelrhodopsin-1 and demonstrated increased plasma membrane integration and dramatic improvement in its channel properties. Under whole-cell patch clamp, mVChR1-expressing cells showed a photo-induced current upon stimulation at 468–640 nm. The evoked currents in mVChR1-expressing cells were ~30 times larger than those in VChR1-expressing cells. Genetically, blind rats expressing mVChR1 via an adeno-associated virus vector regained their visual responses to light with wavelengths between 468 and 640 nm and their recovered visual responses were maintained for a year. Thus, mVChR1 is a candidate gene for gene therapy for restoring vision, and gene delivery of mVChR1 may provide blind patients access to the majority of the visible light spectrum.

  • molecular determinants differentiating photocurrent properties of two Channelrhodopsins from chlamydomonas
    Journal of Biological Chemistry, 2009
    Co-Authors: Hongxia Wang, Hiroshi Tomita, Eriko Sugano, Yuka Sugiyama, Takuya Hikima, Tetsuo Takahashi, Toru Ishizuka, Hiromu Yawo
    Abstract:

    A light signal is converted into an electrical one in a single molecule named Channelrhodopsin, one of the archaea-type rhodopsins in unicellular green algae. Although highly homologous, two molecules of this family, Channelrhodopsin-1 (ChR1) and -2 (ChR2), are distinct in photocurrent properties such as the wavelength sensitivity, desensitization, and turning-on and -off kinetics. However, the structures regulating these properties have not been completely identified. Photocurrents were analyzed for several chimera molecules made by replacing N-terminal segments of ChR2 with the homologous counterparts of ChR1. We found that the wavelength sensitivity of the photocurrent was red-shifted with negligible desensitization and slowed turning-on and -off kinetics when replacement was made with the segment containing the fifth transmembrane helix of ChR1. Therefore, this segment is involved in the determination of photocurrent properties, the wavelength sensitivity, and the kinetics characterizing ChR1 and ChR2. Eight amino acid residues differentiating this segment were exchanged one-by-one, and the photocurrent properties of each targeted mutant ChR2 were further analyzed. Among them, position Tyr(226)(ChR1)/Asn(187)(ChR2) is one of the molecular determinants involved in the wavelength sensitivity, desensitization, and turning-on and -off kinetics. It is suggested that these amino acid residues directly or indirectly interact with the chromophore as well as with the protein structure determining the photocurrent kinetics. Some of the chimera Channelrhodopsins are suggested to have several advantages over the wild-type ChR2 in the introduction of light-induced membrane depolarization for the purpose of artificial stimulation of neurons in vivo and visual prosthesis for photoreceptor degeneration.

Christian Bamann - One of the best experts on this subject based on the ideXlab platform.

  • structural insights into ion conduction by Channelrhodopsin 2
    Science, 2017
    Co-Authors: Oleksandr Volkov, Christian Bamann, K Kovalev, Vitaly Polovinkin, Valentin Borshchevskiy, R Astashkin, Egor Marin, A N Popov, Taras Balandin
    Abstract:

    INTRODUCTION Ion channels are integral membrane proteins that upon stimulation modulate the flow of ions across the cell or organelle membrane. The resulting electrical signals are involved in biological functions such as electrochemical transmission and information processing in neurons. Channelrhodopsins (ChRs) appear to be unusual channels. They belong to the large family of microbial rhodopsins, seven-helical transmembrane proteins containing retinal as chromophore. Photon absorption initiates retinal isomerization resulting in a photocycle, with different spectroscopically distinguishable intermediates, thereby controlling the opening and closing of the channel. In 2003, it was demonstrated that light-induced currents by heterologously expressed ChR2 can be used to change a host’s membrane potential. The concept was further applied to precisely control muscle and neural activity by using light-induced depolarization to trigger an action potential in neurons expressing ChR2. This optogenetic approach with ChR2 and other ChRs has been widely used for remote control of neural cells in culture and in living animals with high spatiotemporal resolution. It is also used in biomedical studies aimed to cure severe diseases. RATIONALE Despite the wealth of biochemical and biophysical data, a high-resolution structure and structural mechanisms of a native ChR2 (and other ChRs) have not yet been known. A step forward was the structure of a chimera (C1C2). However, recent electrophysiological and Fourier transform infrared data showed that C1C2 exhibits light-induced responses that are functionally and mechanistically different from ChR2. Given that ChR2 is the most frequently used tool in optogenetics, a high-resolution structure of ChR2 is of high importance. Deciphering the structure of the native channel would shed light on how the light-induced changes at the retinal Schiff base (RSB) are linked to the channel operation and may make engineering of enhanced optogenetic tools more efficient. RESULTS We expressed ChR2 in LEXSY and used in the meso crystallization approach to determine the crystal structure of the wild-type ChR2 and C128T slow mutant at 2.4 and 2.7 A, respectively (C, cysteine; T, threonine). Two different dark-state conformations of ChR2 in the two protomers in the asymmetric unit were resolved. The overall structure alignment of the protomers does not show a visible difference in backbone conformation. However, the conformation of some amino acids and the position of water molecules are not the same. The dimerization is strong and provided mainly through the interaction of helices 3 and 4 and the N termini. In addition, the protomers are connected with a disulfide bond, C34/C36′. In both protomers, we identified ion conduction pathway comprising four cavities [extracellular cavity 1 (EC1), EC2, intracellular cavity 1 (IC1), and IC2] that are separated by three gates [extracellular gate (ECG), central gate (CG), and intracellular gate (ICG)] (figure, panel A). Arginines R120 and R268 are the cores of ECG and ICG, respectively, in all ChRs. The Schiff base is hydrogen-bond–connected to E123 and D253 amino acids (E, glutamic acid; D, aspartic acid) and is a key part of the CG that is further connected with two other gates through an extended H-bond network mediated by numerous water molecules (figure, panel B). The DC gate is separate from the gates in the channel pathway and is bridged by hydrogen bonds through the water molecule w5. Hydrogen bonding of the DC pair (C128 and D156) has two important consequences. It stabilizes helices 3 and 4 and provides connection from D156, a possible proton donor, to the RSB. The presence of the hydrogen bonds provides structural insights into how the DC gate controls ChR2 gating lifetime. CONCLUSION The determined structures of ChR2 and its C128T mutant present the molecular basis for the understanding of ChR functioning. They provide insights into mechanisms of channel opening and closing. A plausible scenario is that the disruption of the H-bonds between E123 and D253 and the Schiff base and the protonation of D253 upon retinal isomerization trigger rearrangements in the extended hydrogen-bonded networks, stabilizing the ECG and CG and also rearranging the H-bonding network in the cavities. Upon retinal isomerization, these two gates are opened and the network is broken. This leads to the reorientation of helix 2. Additional changes in helices 6 and 7 induced by the isomerization could help with opening the ICG and channel pore formation.

  • photochemical properties of the red shifted Channelrhodopsin chrimson
    Photochemistry and Photobiology, 2017
    Co-Authors: David Urmann, Charlotte Lorenz, Stephanie M Linker, Markus Braun, Josef Wachtveitl, Christian Bamann
    Abstract:

    Color-tuned variants of Channelrhodopsins allow for selective optogenetic manipulation of different host cell populations. Chrimson is the Channelrhodopsin with the longest wavelength absorbance maximum. We characterize its photochemical properties at different pH values corresponding to two protonation states of the counterion for the protonated Schiff base. Both states will lead to a functional channel opening, but the route is different as reflected in the photochemical states observed spectroscopically. The light-induced isomerization kinetics change with the local electrostatic environment, becoming faster with the presence of an anionic counterion. The spectral effect is stronger on the ground-state energy surface. From the excited state, a bifurcated pathway leads to the electronic ground state resulting in a pronounced excitation wavelength dependence. The subsequent steps in the photocycles at pH 6 and pH 9.5 differ in the accumulation of states with a protonated and deprotonated Schiff base, respectively, that can be correlated with the open channel. Therefore, different protonation states are preserved in the open and the initial states. Chrimson's photocycle at alkaline pH shows features observed in other rhodopsins without an internal proton donor to the Schiff base, but it accumulates an intermediate with an even longer lifetime reflecting slow recovery of the initial state.

  • temporal evolution of helix hydration in a light gated ion channel correlates with ion conductance
    Proceedings of the National Academy of Sciences of the United States of America, 2015
    Co-Authors: Victor A Lorenzfonfria, Christian Bamann, Ernst Bamberg, Tom Resler, Ramona Schlesinger, Joachim Heberle
    Abstract:

    The discovery of Channelrhodopsins introduced a new class of light-gated ion channels, which when genetically encoded in host cells resulted in the development of optogenetics. Channelrhodopsin-2 from Chlamydomonas reinhardtii, CrChR2, is the most widely used optogenetic tool in neuroscience. To explore the connection between the gating mechanism and the influx and efflux of water molecules in CrChR2, we have integrated light-induced time-resolved infrared spectroscopy and electrophysiology. Cross-correlation analysis revealed that ion conductance tallies with peptide backbone amide I vibrational changes at 1,665(-) and 1,648(+) cm(-1). These two bands report on the hydration of transmembrane α-helices as concluded from vibrational coupling experiments. Lifetime distribution analysis shows that water influx proceeded in two temporally separated steps with time constants of 10 μs (30%) and 200 μs (70%), the latter phase concurrent with the start of ion conductance. Water efflux and the cessation of the ion conductance are synchronized as well, with a time constant of 10 ms. The temporal correlation between ion conductance and hydration of helices holds for fast (E123T) and slow (D156E) variants of CrChR2, strengthening its functional significance.

  • enlightening the photoactive site of Channelrhodopsin 2 by dnp enhanced solid state nmr spectroscopy
    Proceedings of the National Academy of Sciences of the United States of America, 2015
    Co-Authors: Johanna Beckerbaldus, Christian Bamann, Ernst Bamberg, Josef Wachtveitl, Krishna Saxena, Henrik Gustmann, Lynda J Brown, Richard C D Brown, Christian Reiter, Harald Schwalbe
    Abstract:

    Channelrhodopsin-2 from Chlamydomonas reinhardtii is a light-gated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to (15)N-labeled Channelrhodopsin-2 carrying 14,15-(13)C2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early [Formula: see text] K-like state, the slowly decaying late intermediate [Formula: see text], and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of Channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray-based structure analysis, which is required for resolving molecular mechanisms.

  • Light-Induced Helix Movements in Channelrhodopsin-2
    Journal of molecular biology, 2014
    Co-Authors: Maria Müller, Christian Bamann, Ernst Bamberg, Werner Kühlbrandt
    Abstract:

    Channelrhodopsin-2 (ChR2) is a cation-selective light-gated channel from Chlamydomonas reinhardtii (Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, Berthold P, et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci USA 2003;100:13940-5), which has become a powerful tool in optogenetics. Two-dimensional crystals of the slow photocycling C128T ChR2 mutant were exposed to 473 nm light and rapidly frozen to trap the open state. Projection difference maps at 6A resolution show the location, extent and direction of light-induced conformational changes in ChR2 during the transition from the closed state to the ion-conducting open state. Difference peaks indicate that transmembrane helices (TMHs) TMH2, TMH6 and TMH7 reorient or rearrange during the photocycle. No major differences were found near TMH3 and TMH4 at the dimer interface. While conformational changes in TMH6 and TMH7 are known from other microbial-type rhodopsins, our results indicate that TMH2 has a key role in light-induced channel opening and closing in ChR2.