Monitoring light-induced structural changes of Channelrhodopsin-2 by UV-visible and Fourier transform infrared spectroscopy

Channelrhodopsin-2 (ChR2) is a microbial type rhodopsin and a light-gated cation channel that controls phototaxis in Chlamydomonas. We expressed ChR2 in COS-cells, purified it, and subsequently investigated this unusual photoreceptor by flash photolysis and UV-visible and Fourier transform infrared difference spectroscopy. Several transient photoproducts of the wild type ChR2 were identified, and their kinetics and molecular properties were compared with those of the ChR2 mutant E90Q. Based on the spectroscopic data we developed a model of the photocycle comprising six distinguishable intermediates. This photocycle shows similarities to the photocycle of the ChR2-related Channelrhodopsin of Volvox but also displays significant differences. We show that molecular changes include retinal isomerization, changes in hydrogen bonding of carboxylic acids, and large alterations of the protein backbone structure. These alterations are stronger than those observed in the photocycle of other microbial rhodopsins like bacteriorhodopsin and are related to those occurring in animal rhodopsins. UV-visible and Fourier transform infrared difference spectroscopy revealed two late intermediates with different time constants of tau = 6 and 40 s that exist during the recovery of the dark state. The carboxylic side chain of Glu(90) is involved in the slow transition. The molecular changes during the ChR2 photocycle are discussed with respect to other members of the rhodopsin family.     

Structural model of channelrhodopsin

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.     

Platymonas subcordiformis Channelrhodopsin-2 Function: I. THE PHOTOCHEMICAL REACTION CYCLE*

The photocycle kinetics of Platymonas subcordiformis channelrhodopsin-2 (PsChR2), among the most highly efficient light-gated cation channels and the most blue-shifted channelrhodopsin, was studied by time-resolved absorption spectroscopy in the 340–650-nm range and in the 100-ns to 3-s time window. Global exponential fitting of the time dependence of spectral changes revealed six lifetimes: 0.60 μs, 5.3 μs, 170 μs, 1.4 ms, 6.7 ms, and 1.4 s. The sequential intermediates derived for a single unidirectional cycle scheme based on these lifetimes were found to contain mixtures of K, L, M, O, and P molecular states, named in analogy to photointermediates in the bacteriorhodopsin photocycle. The photochemistry is described by the superposition of two independent parallel photocycles. The analysis revealed that 30% of the photoexcited receptor molecules followed Cycle 1 through the K, M, O, and P states, whereas 70% followed Cycle 2 through the K, L, M, and O states. The recovered state, R, is spectrally close, but not identical, to the dark state on the seconds time scale. The two-cycle model of this high efficiency channelrhodopsin-2 (ChR) opens new perspectives in understanding the mechanism of channelrhodopsin function.     

Platymonas subcordiformis Channelrhodopsin-2 (PsChR2) Function: II. RELATIONSHIP OF THE PHOTOCHEMICAL REACTION CYCLE TO CHANNEL CURRENTS*

Channelrhodopsins, such as the algal phototaxis receptor Platymonas subcordiformis channelrhodopsin-2 (PsChR2), are light-gated cation channels used as optogenetic tools for photocontrol of membrane potential in living cells. Channelrhodopsin (ChR)-mediated photocurrent responses are complex and poorly understood, exhibiting alterations in peak current amplitude, extents and kinetics of inactivation, and kinetics of the recovery of the prestimulus dark current that are sensitive to duration and frequency of photostimuli. From the analysis of time-resolved optical absorption data, presented in the accompanying article, we derived a two-cycle model that describes the photocycles of PsChR2. Here, we applied the model to evaluate the transient currents produced by PsChR2 expressed in HEK293 cells under both fast laser excitation and step-like continuous illumination. Interpretation of the photocurrents in terms of the photocycle kinetics indicates that the O states in both cycles are responsible for the channel current and fit the current transients under the different illumination regimes. The peak and plateau currents in response to a single light step, a train of light pulses, and a light step superimposed on a continuous light background observed for ChR2 proteins are explained in terms of contributions from the two parallel photocycles. The analysis shows that the peak current desensitization and recovery phenomena are inherent properties of the photocycles. The light dependence of desensitization is reproduced and explained by the time evolution of the concentration transients in response to step-like illumination. Our data show that photocycle kinetic parameters are sufficient to explain the complex dependence of photocurrent responses to photostimuli.     

Molecular Dynamics of Channelrhodopsin at the Early Stages of Channel Opening

Channelrhodopsin (ChR) is a light-gated cation channel that responds to blue light. Since ChR can be readily expressed in specific neurons to precisely control their activities by light, it has become a powerful tool in neuroscience. Although the recently solved crystal structure of a chimeric ChR, C1C2, provided the structural basis for ChR, our understanding of the molecular mechanism of ChR still remains limited. Here we performed electrophysiological analyses and all-atom molecular dynamics (MD) simulations, to investigate the importance of the intracellular and central constrictions of the ion conducting pore observed in the crystal structure of C1C2. Our electrophysiological analysis revealed that two glutamate residues, Glu122 and Glu129, in the intracellular and central constrictions, respectively, should be deprotonated in the photocycle. The simulation results suggested that the deprotonation of Glu129 in the central constriction leads to ion leakage in the ground state, and implied that the protonation of Glu129 is important for preventing ion leakage in the ground state. Moreover, we modeled the 13-cis retinal bound; i.e., activated C1C2, and performed MD simulations to investigate the conformational changes in the early stage of the photocycle. Our simulations suggested that retinal photoisomerization induces the conformational change toward channel opening, including the movements of TM6, TM7 and TM2. These insights into the dynamics of the ground states and the early photocycle stages enhance our understanding of the channel function of ChR.     

The Branched Photocycle of the Slow-Cycling Channelrhodopsin-2 Mutant C128T

Channelrhodopsins (ChRs) of green algae such as Chlamydomonas are used as neuroscience tools to specifically depolarize cells with light. A crude model of the ChR2 photocycle has been recently established, but details of the photoreactions are widely unknown. Here, we present the photoreactions of a slow-cycling ChR2 mutant (step function rhodopsin), with C128 replaced by threonine and 200-fold extended lifetime of the conducting-state P520. At a late state of the photocycle, a fraction of the proteins branches off into an inactive species, P380, which accumulates during prolonged illumination. At neutral pH, P380 is converted into P353, a species with a characteristic fine-structured spectrum that is interpreted as retroretinyl chromophore. The described branching reactions should be considered, when ChR is used as a neuroscience tool, especially in the case of fluorescence imaging at high light intensities.     

Characterization of a Highly Efficient Blue-shifted Channelrhodopsin from the Marine Alga Platymonas subcordiformis

Rhodopsin photosensors of phototactic algae act as light-gated cation channels when expressed in animal cells. These proteins (channelrhodopsins) are extensively used for millisecond scale photocontrol of cellular functions (optogenetics). We report characterization of PsChR, one of the phototaxis receptors in the alga Platymonas (Tetraselmis) subcordiformis. PsChR exhibited ∼3-fold higher unitary conductance and greater relative permeability for Na⁺ ions, as compared with the most frequently used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Photocurrents generated by PsChR in HEK293 cells showed lesser inactivation and faster peak recovery than those by CrChR2. Their maximal spectral sensitivity was at 445 nm, making PsChR the most blue-shifted channelrhodopsin so far identified. The λ_max of detergent-purified PsChR was 437 nm at neutral pH and exhibited red shifts (pK_a values at 6.6 and 3.8) upon acidification. The purified pigment undergoes a photocycle with a prominent red-shifted intermediate whose formation and decay kinetics match the kinetics of channel opening and closing. The rise and decay of an M-like intermediate prior to formation of this putative conductive state were faster than in CrChR2. PsChR mediated sufficient light-induced membrane depolarization in cultured hippocampal neurons to trigger reliable repetitive spiking at the upper threshold frequency of the neurons. At low frequencies spiking probability decreases less with PsChR than with CrChR2 because of the faster recovery of the former. Its blue-shifted absorption enables optogenetics at wavelengths even below 400 nm. A combination of characteristics makes PsChR important for further research on structure-function relationships in ChRs and potentially useful for optogenetics, especially for combinatorial applications when short wavelength excitation is required.     

Kinetic and Vibrational Isotope Effects of Proton Transfer Reactions in Channelrhodopsin-2

Channelrhodopsins (ChRs) are light-gated cation channels. After blue-light excitation, the protein undergoes a photocycle with different intermediates. Here, we have recorded transient absorbance changes of ChR2 from Chlamydomonas reinhardtii in the visible and infrared regions with nanosecond time resolution, the latter being accomplished using tunable quantum cascade lasers. Because proton transfer reactions play a key role in channel gating, we determined vibrational as well as kinetic isotope effects (VIEs and KIEs) of carboxylic groups of various key aspartic and glutamic acid residues by monitoring their C=O stretching vibrations in H₂O and in D₂O. D156 exhibits a substantial KIE (>2) in its deprotonation and reprotonation, which substantiates its role as the internal proton donor to the retinal Schiff base. The unusual VIE of D156, upshifted from 1736 cm⁻¹ to 1738 cm⁻¹ in D₂O, was scrutinized by studying the D156E variant. The C=O stretch of E156 shifted down by 8 cm⁻¹ in D₂O, providing evidence for the accessibility of the carboxylic group. The C=O stretching band of E90 exhibits a VIE of 9 cm⁻¹ and a KIE of ∼2 for the de- and the reprotonation reactions during the lifetime of the late desensitized state. The KIE of 1 determined in the time range from 20 ns to 5 ms is incompatible with early deprotonation of E90.     

Projection structure of channelrhodopsin-2 at 6 Å resolution by electron crystallography

Channelrhodopsin-2 (ChR2) is the prototype of a new class of light-gated ion channels that is finding widespread applications in optogenetics and biomedical research. We present a  6-Å projection map of ChR2, obtained by cryo-electron microscopy of two-dimensional crystals grown from pure, heterologously expressed protein. The map shows that ChR2 is the same dimer with non-crystallographic 2-fold symmetry in three different membrane crystals. This is consistent with biochemical analysis, which shows a stable dimer in detergent solution. Comparison to the projection map to bacteriorhodopsin indicates a similar structure of seven transmembrane alpha helices. Based on the projection map and sequence alignments, we built a homology model of ChR2 that potentially accounts for light-induced channel gating. Although a monomeric channel is not ruled out, comparison to other membrane channels and transporters suggests that the ChR2 channel is located at the dimer interface on the 2-fold axis, lined by transmembrane helices 3 and 4.     

Multiple photocycles of channelrhodopsin

Two rhodopsins with intrinsic ion conductance have been identified recently in Chlamydomonas reinhardtii. They were named "channelrhodopsins" ChR1 and ChR2. Both were expressed in Xenopus laevis oocytes, and their properties were studied qualitatively by two electrode voltage clamp techniques. ChR1 is specific for H+, whereas ChR2 conducts Na+, K+, Ca2+, and guanidinium. ChR2 responds to the onset of light with a peak conductance, followed by a smaller steady-state conductance. Upon a second stimulation, the peak is smaller and recovers to full size faster at high external pH. ChR1 was reported to respond with a steady-state conductance only but is demonstrated here to have a peak conductance at high light intensities too. We analyzed quantitatively the light-induced conductance of ChR1 and developed a reaction scheme that describes the photocurrent kinetics at various light conditions. ChR1 exists in two dark states, D1 and D2, that photoisomerize to the conducting states M1 and M2, respectively. Dark-adapted ChR1 is completely arrested in D1. M1 converts into D1 within milliseconds but, in addition, equilibrates with the second conducting state M2 that decays to the second dark state D2. Thus, light-adapted ChR1 represents a mixture of D1 and D2. D2 thermally reconverts to D1 in minutes, i.e., much slower than any reaction of the two photocycles.     

Spectral characteristics of the photocycle of channelrhodopsin-2 and its implication for channel function

In 2003, channelrhodopsin-2 (ChR2) from Chlamydomonas reinhardtii was discovered to be a light-gated cation channel, and since that time the channel became an excellent tool to control by light neuronal cells in culture as well as in living animals with high temporal and spatial resolution in a noninvasive manner. However, little is known about the spectral properties and their relation to the channel function. We have expressed ChR2 in the yeast Pichia pastoris and purified the protein. Flash-photolysis data were combined with patch-clamp studies to elucidate the photocycle. The protein absorbs maximally at ∼ 480 nm before light excitation and shows flash-induced absorbance changes with at least two different photointermediates. Four relaxation processes can be extracted from the time course that we have analysed in a linear model for the photocycle leading to the kinetic intermediates P₁ to P₄. A short-lived photointermediate at 400 nm, suggesting a deprotonation of the retinal Schiff base, is followed by a red-shifted (520 nm) species with a millisecond lifetime. The first three kinetic intermediates in the photocycle, P₁ to P₃, are described mainly by the red-shifted 520-nm species. The 400-nm species contributes to a smaller extent to P₁ and P₂. The fourth one, P₄, is spectroscopically almost identical with the ground state and lasts into the seconds time region. We compared the spectroscopic data to current measurements under whole-cell patch-clamp conditions on HEK 293 cells. The lifetimes of the spectroscopically and electrophysiologically determined intermediates are in excellent agreement. The intermediates P₂ and P₃ (absorbing at 520 nm) are identified as the cation permeating states of the channel. Under stationary light, a modulation of the photocurrent by green light (540 nm) was observed. We conclude that the red-shifted spectral species represents the open channel state, and the thermal relaxation of this intermediate, the transition from P₃ to P₄, is coupled to channel closing.     

Fast,repetitive light-activation of CaV3.2 using Channelrhodopsin 2

Channelrhodopsin-2 (ChR2) is a light-gated ion channel that is successfully used in neurosciences to depolarize cells with blue light. In this regard control of membrane voltage with light opens new perspectives for the characterization of ion channels and the search for inhibitors or modulators. Here, we report a control of membrane potential with ChR2 and the potassium channel mTrek for the purpose of screening for ion channel specific drugs. To verify principle we have chosen the voltage gated calcium channel Ca_V3.2 as potential drug target. For this purpose we transfected the ChR2 gene into a HEK293T-cell line that permanently expresses Ca_V3.2 and the K-channel mTrek. The resting potential was adjusted with low concentration of extracellular potassium ions whereas transient depolarization was achieved by activation of ChR2 with short pulses of blue light. Calcium ion influx through Ca_V3.2 was monitored by observing fura-2 fluorescence. This approach allowed a repetitive activation of Ca_V3.2. The Ca²⁺ influx was specifically blocked by the inhibitor mibefradil. Since this assay is genetically-encoded, it may be employed for a variety of voltage-gated calcium channels and should be applicable to multi-well reader formats for high-throughput screening.     

Kinetic Evaluation of Photosensitivity in Bi-Stable Variants of Chimeric Channelrhodopsins

Channelrhodopsin-1 and 2 (ChR1 and ChR2) form cation channels that are gated by light through an unknown mechanism. We tested the DC-gate hypothesis that C167 and D195 are involved in the stabilization of the cation-permeable state of ChRWR/C1C2 which consists of TM1-5 of ChR1 and TM6-7 of ChR2 and ChRFR which consists of TM1-2 of ChR1 and TM3-7 of ChR2. The cation permeable state of each ChRWR and ChRFR was markedly prolonged in the order of several tens of seconds when either C167 or D195 position was mutated to alanine (A). Therefore, the DC-gate function was conserved among these chimeric ChRs. We next investigated the kinetic properties of the ON/OFF response of these bi-stable ChR mutants as they are important in designing the photostimulation protocols for the optogenetic manipulation of neuronal activities. The turning-on rate constant of each photocurrent followed a linear relationship to 0–0.12 mWmm⁻² of blue LED light or to 0–0.33 mWmm⁻² of cyan LED light. Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance. As the magnitude of the photocurrent was mostly determined by the turning-on rate constant and the irradiation time, the minimal irradiance that effectively evoked an action potential (threshold irradiance) was decreased with time only if the neuron, which expresses bi-stable ChRs, has a certain large membrane time constant (eg. τ_m > 20 ms). On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time. Based on these quantitative data, we would propose that these bi-stable ChRs would be most suitable for enhancing the intrinsic activity of excitatory pyramidal neurons at a minimal magnitude of irradiance.     

Channelrhodopsin-2 localised to the axon initial segment

The light-gated cation channel Channelrhodopsin-2 (ChR2) is a powerful and versatile tool for controlling neuronal activity. Currently available versions of ChR2 either distribute uniformly throughout the plasma membrane or are localised specifically to somatodendritic or synaptic domains. Localising ChR2 instead to the axon initial segment (AIS) could prove an extremely useful addition to the optogenetic repertoire, targeting the channel directly to the site of action potential initiation, and limiting depolarisation and associated calcium entry elsewhere in the neuron. Here, we describe a ChR2 construct that we localised specifically to the AIS by adding the ankyrinG-binding loop of voltage-gated sodium channels (Na(v)II-III) to its intracellular terminus. Expression of ChR2-YFP-Na(v)II-III did not significantly affect the passive or active electrical properties of cultured rat hippocampal neurons. However, the tiny ChR2 currents and small membrane depolarisations resulting from AIS targeting meant that optogenetic control of action potential firing with ChR2-YFP-Na(v)II-III was unsuccessful in baseline conditions. We did succeed in stimulating action potentials with light in some ChR2-YFP-Na(v)II-III-expressing neurons, but only when blocking KCNQ voltage-gated potassium channels. We discuss possible alternative approaches to obtaining precise control of neuronal spiking with AIS-targeted optogenetic constructs and propose potential uses for our ChR2-YFP-Na(v)II-III probe where subthreshold modulation of action potential initiation is desirable.     

Bioinformatic and mutational analysis of channelrhodopsin-2 protein cation-conducting pathway

Channelrhodopsin-2 (ChR2) is a light-gated cation channel widely used as a biotechnological tool to control membrane depolarization in various cell types and tissues. Although several ChR2 variants with modified properties have been generated, the structural determinants of the protein function are largely unresolved. We used bioinformatic modeling of the ChR2 structure to identify the putative cationic pathway within the channel, which is formed by a system of inner cavities that are uniquely present in this microbial rhodopsin. Site-directed mutagenesis combined with patch clamp analysis in HeLa cells was used to determine key residues involved in ChR2 conductance and selectivity. Among them, Gln-56 is important for ion conductance, whereas Ser-63, Thr-250, and Asn-258 are previously unrecognized residues involved in ion selectivity and photocurrent kinetics. This study widens the current structural information on ChR2 and can assist in the design of new improved variants for specific biological applications.     

Role of a Helix B Lysine Residue in the Photoactive Site in Channelrhodopsins

In most studied microbial rhodopsins two conserved carboxylic acid residues (the homologs of Asp-85 and Asp-212 in bacteriorhodopsin) and an arginine residue (the homolog of Arg-82) form a complex counterion to the protonated retinylidene Schiff base, and neutralization of the negatively charged carboxylates causes red shifts of the absorption maximum. In contrast, the corresponding neutralizing mutations in some relatively low-efficiency channelrhodopsins (ChRs) result in blue shifts. These ChRs do not contain a lysine residue in the second helix, conserved in higher efficiency ChRs (Lys-132 in the crystallized ChR chimera). By action spectroscopy of photoinduced channel currents in HEK293 cells and absorption spectroscopy of detergent-purified pigments, we found that in tested ChRs the Lys-132 homolog controls the direction of spectral shifts in the mutants of the photoactive site carboxylic acid residues. Analysis of double mutants shows that red spectral shifts occur when this Lys is present, whether naturally or by mutagenesis, and blue shifts occur when it is replaced with a neutral residue. A neutralizing mutation of the Lys-132 homolog alone caused a red spectral shift in high-efficiency ChRs, whereas its introduction into low-efficiency ChR1 from Chlamydomonas augustae (CaChR1) caused a blue shift. Taking into account that the effective charge of the carboxylic acid residues is a key factor in microbial rhodopsin spectral tuning, these findings suggest that the Lys-132 homolog modulates their pK_a values. On the other hand, mutation of the Arg-82 homolog that fulfills this role in bacteriorhodopsin caused minimal spectral changes in the tested ChRs. Titration revealed that the pK_a of the Asp-85 homolog in CaChR1 lies in the alkaline region unlike in most studied microbial rhodopsins, but is substantially decreased by introduction of a Lys-132 homolog or neutralizing mutation of the Asp-212 homolog. In the three ChRs tested the Lys-132 homolog also alters channel current kinetics.     

Looking within for vision

Channelrhodopsin-2 (ChR2), a directly light-gated cation channel from the green alga Chlamydomonas reinhardtii has been shown to be a directly light-switched cation-selective ion channel, which employs 11-cis retinal as its chromophore. This is the same chromophore as the mammalian photoreceptor's visual pigment-rhodopsin. Previously, investigators demonstrated that ChR2 can be used to optically control neuronal firing by depolarizing the cell. In this issue of Neuron, Bi et al. apply viral-mediated gene transfer to deliver ChR2 to retinal ganglion cells (RGC) in a rodent model of inherited blindness. In this way, the authors have genetically engineered surviving retinal neurons to take on the lost photoreceptive function. The conversion of light-insensitive retinal interneurons into photosensitive cells introduces an entirely new direction for treatments of blinding retinal degeneration.     

Role of a Helix B Lysine Residue in the Photoactive Site in Channelrhodopsins

In most studied microbial rhodopsins two conserved carboxylic acid residues (the homologs of Asp-85 and Asp-212 in bacteriorhodopsin) and an arginine residue (the homolog of Arg-82) form a complex counterion to the protonated retinylidene Schiff base, and neutralization of the negatively charged carboxylates causes red shifts of the absorption maximum. In contrast, the corresponding neutralizing mutations in some relatively low-efficiency channelrhodopsins (ChRs) result in blue shifts. These ChRs do not contain a lysine residue in the second helix, conserved in higher efficiency ChRs (Lys-132 in the crystallized ChR chimera). By action spectroscopy of photoinduced channel currents in HEK293 cells and absorption spectroscopy of detergent-purified pigments, we found that in tested ChRs the Lys-132 homolog controls the direction of spectral shifts in the mutants of the photoactive site carboxylic acid residues. Analysis of double mutants shows that red spectral shifts occur when this Lys is present, whether naturally or by mutagenesis, and blue shifts occur when it is replaced with a neutral residue. A neutralizing mutation of the Lys-132 homolog alone caused a red spectral shift in high-efficiency ChRs, whereas its introduction into low-efficiency ChR1 from Chlamydomonas augustae (CaChR1) caused a blue shift. Taking into account that the effective charge of the carboxylic acid residues is a key factor in microbial rhodopsin spectral tuning, these findings suggest that the Lys-132 homolog modulates their pK_a values. On the other hand, mutation of the Arg-82 homolog that fulfills this role in bacteriorhodopsin caused minimal spectral changes in the tested ChRs. Titration revealed that the pK_a of the Asp-85 homolog in CaChR1 lies in the alkaline region unlike in most studied microbial rhodopsins, but is substantially decreased by introduction of a Lys-132 homolog or neutralizing mutation of the Asp-212 homolog. In the three ChRs tested the Lys-132 homolog also alters channel current kinetics.     

Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses

For studying the function of specific neurons in their native circuitry, it is desired to precisely control their activity. This often requires dissection to allow accurate electrical stimulation or neurotransmitter application , and it is thus inherently difficult in live animals, especially in small model organisms. Here, we employed channelrhodopsin-2 (ChR2), a directly light-gated cation channel from the green alga Chlamydomonas reinhardtii, in excitable cells of the nematode Caenorhabditis elegans, to trigger specific behaviors, simply by illumination. Channelrhodopsins are 7-transmembrane-helix proteins that resemble the light-driven proton pump bacteriorhodopsin , and they also utilize the chromophore all-trans retinal, but to open an intrinsic cation pore. In muscle cells, light-activated ChR2 evoked strong, simultaneous contractions, which were reduced in the background of mutated L-type, voltage-gated Ca2+-channels (VGCCs) and ryanodine receptors (RyRs). Electrophysiological analysis demonstrated rapid inward currents that persisted as long as the illumination. When ChR2 was expressed in mechanosensory neurons, light evoked withdrawal behaviors that are normally elicited by mechanical stimulation. Furthermore, ChR2 enabled activity of these neurons in mutants lacking the MEC-4/MEC-10 mechanosensory ion channel . Thus, specific neurons or muscles expressing ChR2 can be quickly and reversibly activated by light in live and behaving, as well as dissected, animals.