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.     

Regulation of neuron cholinoreceptor plasticity of Helix lucorum by second messengers and opioids

The rhythmical local ionophoretic applications of acetylcholine (ACh) to the somatic membrane of Helix lucorum identified neurons evokes the reversible depression of the ACh-induced response which shows cholinoreceptor (ChR) desensitization. ChR desensitization is regulated not by one but by several known second messengers and G-proteins. The endogenous opioids perform the excitation or inhibitory tonic control of the membrane potential in some neurons constantly activating the ionotropic opiate receptors. The direction of neuron ChR desensitization modulation by opioids depends on the type of the activated modulatory opiate receptors (mu or kappa) on the neuron membrane. Second messengers are involved in intracellular mechanism of modulation of the ChR desensitization by opiate kappa-agonist bremazocine.     

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.     

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.     

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.     

Characterization of Engineered Channelrhodopsin Variants with Improved Properties and Kinetics

Channelrhodopsin 2 (ChR2), a light-activated nonselective cationic channel from Chlamydomonas reinhardtii, has become a useful tool to excite neurons into which it is transfected. The other ChR from Chlamydomonas, ChR1, has attracted less attention because of its proton-selective permeability. By making chimeras of the transmembrane domains of ChR1 and ChR2, combined with site-directed mutagenesis, we developed a ChR variant, named ChEF, that exhibits significantly less inactivation during persistent light stimulation. ChEF undergoes only 33% inactivation, compared with 77% for ChR2. Point mutation of Ile¹⁷⁰ of ChEF to Val (yielding “ChIEF”) accelerates the rate of channel closure while retaining reduced inactivation, leading to more consistent responses when stimulated above 25 Hz in both HEK293 cells and cultured hippocampal neurons. In addition, these variants have altered spectral responses, light sensitivity, and channel selectivity. ChEF and ChIEF allow more precise temporal control of depolarization, and can induce action potential trains that more closely resemble natural spiking patterns.     

C2 domain protein MIN1 promotes eyespot organization in Chlamydomonas reinhardtii

Assembly and asymmetric localization of the photosensory eyespot in the biflagellate, unicellular green alga Chlamydomonas reinhardtii requires coordinated organization of photoreceptors in the plasma membrane and pigment granule/thylakoid membrane layers in the chloroplast. min1 (mini-eyed) mutant cells contain abnormally small, disorganized eyespots in which the chloroplast envelope and plasma membrane are no longer apposed. The MIN1 gene, identified here by phenotypic rescue, encodes a protein with an N-terminal C2 domain and a C-terminal LysM domain separated by a transmembrane sequence. This novel domain architecture led to the hypothesis that MIN1 is in the plasma membrane or the chloroplast envelope, where membrane association of the C2 domain promotes proper eyespot organization. Mutation of conserved C2 domain loop residues disrupted association of the MIN1 C2 domain with the chloroplast envelope in moss cells but did not abolish eyespot assembly in Chlamydomonas. In min1 null cells, channelrhodopsin-1 (ChR1) photoreceptor levels were reduced, indicating a role for MIN1 in ChR1 expression and/or stability. However, ChR1 localization was only minimally disturbed during photoautotrophic growth of min1 cells, conditions under which the pigment granule layers are disorganized. The data are consistent with the hypothesis that neither MIN1 nor proper organization of the plastidic components of the eyespot is essential for localization of ChR1.     

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.     

Kinetics of proton release and uptake by channelrhodopsin-2

Electrophysiological experiments showed that the light-activated cation channel channelrhodopsin-2 (ChR2) pumps protons in the absence of a membrane potential. We determined here the kinetics of transient pH change using a water-soluble pH-indicator. It is shown that ChR2 released protons prior to uptake with a stoichiometry of 0.3 protons per ChR2. Comparison to the photocycle kinetics revealed that proton release and uptake match rise and decay of the P(3)(520) intermediate. As the P(3)(520) state also represents the conductive state of cation channeling, the concurrence of proton pumping and channel gating implies an intimate mechanistic link of the two functional modes. Studies on the E123T and S245E mutants show that these residues are not critically involved in proton translocation.     

Re-Introduction of Transmembrane Serine Residues Reduce the Minimum Pore Diameter of Channelrhodopsin-2

Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin found in the green algae Chlamydomonas reinhardtii. Under physiological conditions, ChR2 is an inwardly rectifying cation channel that permeates a wide range of mono- and divalent cations. Although this protein shares a high sequence homology with other microbial-type rhodopsins, which are ion pumps, ChR2 is an ion channel. A sequence alignment of ChR2 with bacteriorhodopsin, a proton pump, reveals that ChR2 lacks specific motifs and residues, such as serine and threonine, known to contribute to non-covalent interactions within transmembrane domains. We hypothesized that reintroduction of the eight transmembrane serine residues present in bacteriorhodopsin, but not in ChR2, will restrict the conformational flexibility and reduce the pore diameter of ChR2. In this work, eight single serine mutations were created at homologous positions in ChR2. Additionally, an endogenous transmembrane serine was replaced with alanine. We measured kinetics, changes in reversal potential, and permeability ratios in different alkali metal solutions using two-electrode voltage clamp. Applying excluded volume theory, we calculated the minimum pore diameter of ChR2 constructs. An analysis of the results from our experiments show that reintroducing serine residues into the transmembrane domain of ChR2 can restrict the minimum pore diameter through inter- and intrahelical hydrogen bonds while the removal of a transmembrane serine results in a larger pore diameter. Therefore, multiple positions along the intracellular side of the transmembrane domains contribute to the cation permeability of ChR2.     

Optogenetic long-term manipulation of behavior and animal development

Channelrhodopsin-2 (ChR2) is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e.g. for manipulations of behavior, or photoactivation of neurons during development. We used "slow" ChR2 variants with mutations in the C128 residue, that exhibit delayed off-kinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days) with low-intensity light. Photoactivation of ChR2(C128S) in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the "dauer" larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development.     

Regulation of the plastic properties of an electro-excitable neuronal membrane by serotonin

The action of serotonin on plastic properties of electroexcitable membrane was studied in Helix lucorum parietal ganglia on neurones of two types: habituating (HC) and nonhabituating (NHC) to rhythmic intracellular stimulation by impulses of depolarizing current. Serotonin produced an effect of facilitation on HC (increase of responses to stimulation against the background of depolarization and rise of input resistance of the cell). Besides, serotonin completely blocked the ability of these cells to habituate to rhythmic stimulation. The obtained data testify that such action of serotonin may be based on suppression by it of C-dependent K-conductivity. Serotonin suppresses responses of NHC to stimulation and contributes to their habituation to rhythmic stimulation. Such action is due to serotonin suppression of Ca-conductivity, and, consequently, to elimination of the mechanism of action potential generation.     

Chimeras of Channelrhodopsin-1 and -2 from Chlamydomonas reinhardtii Exhibit Distinctive Light-induced Structural Changes from Channelrhodopsin-2

Channelrhodopsin-2 (ChR2) from the green alga Chlamydomonas reinhardtii functions as a light-gated cation channel that has been developed as an optogenetic tool to stimulate specific nerve cells in animals and control their behavior by illumination. The molecular mechanism of ChR2 has been extensively studied by a variety of spectroscopic methods, including light-induced difference Fourier transform infrared (FTIR) spectroscopy, which is sensitive to structural changes in the protein upon light activation. An atomic structure of channelrhodopsin was recently determined by x-ray crystallography using a chimera of channelrhodopsin-1 (ChR1) and ChR2. Electrophysiological studies have shown that ChR1/ChR2 chimeras are less desensitized upon continuous illumination than native ChR2, implying that there are some structural differences between ChR2 and chimeras. In this study, we applied light-induced difference FTIR spectroscopy to ChR2 and ChR1/ChR2 chimeras to determine the molecular basis underlying these functional differences. Upon continuous illumination, ChR1/ChR2 chimeras exhibited structural changes distinct from those in ChR2. In particular, the protonation state of a glutamate residue, Glu-129 (Glu-90 in ChR2 numbering), in the ChR chimeras is not changed as dramatically as in ChR2. Moreover, using mutants stabilizing particular photointermediates as well as time-resolved measurements, we identified some differences between the major photointermediates of ChR2 and ChR1/ChR2 chimeras. Taken together, our data indicate that the gating and desensitizing processes in ChR1/ChR2 chimeras are different from those in ChR2 and that these differences should be considered in the rational design of new optogenetic tools based on channelrhodopsins.     

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.     

Effects on capacitance by overexpression of membrane proteins

Functional Channelrhodopsin-2 (ChR2) overexpression of about 10⁴ channels/μm² in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C_m). The C_m increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C_m of 1.15 ± 0.08 μF/cm² in ChR2-expressing cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (0.79 ± 0.02 μF/cm²). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C_m (0.85 ± 0.07 μF/cm²) as compared to non-expressing control cells (0.70 ± 0.03 μF/cm²). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins.     

Transmembrane Domain Three Contributes to the Ion Conductance Pathway of Channelrhodopsin-2

Channelrhodopsin-2 (ChR2) is a light-activated nonselective cation channel that is found in the eyespot of the unicellular green alga Chlamydomonas reinhardtii. Despite the wide employment of this protein to control the membrane potential of excitable membranes, the molecular determinants that define the unique ion conductance properties of this protein are not well understood. To elucidate the cation permeability pathway of ion conductance, we performed cysteine scanning mutagenesis of transmembrane domain three followed by labeling with methanethiosulfonate derivatives. An analysis of our experimental results as modeled onto the crystal structure of the C1C2 chimera demonstrate that the ion permeation pathway includes residues on one face of transmembrane domain three at the extracellular side of the channel that face the center of ChR2. Furthermore, we examined the role of a residue at the extracellular side of transmembrane domain three in ion conductance. We show that ion conductance is mediated, in part, by hydrogen bonding at the extracellular side of transmembrane domain three. These results provide a starting point for examining the cation permeability pathway for ChR2.     

Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration

The death of photoreceptor cells caused by retinal degenerative diseases often results in a complete loss of retinal responses to light. We explore the feasibility of converting inner retinal neurons to photosensitive cells as a possible strategy for imparting light sensitivity to retinas lacking rods and cones. Using delivery by an adeno-associated viral vector, here, we show that long-term expression of a microbial-type rhodopsin, channelrhodopsin-2 (ChR2), can be achieved in rodent inner retinal neurons in vivo. Furthermore, we demonstrate that expression of ChR2 in surviving inner retinal neurons of a mouse with photoreceptor degeneration can restore the ability of the retina to encode light signals and transmit the light signals to the visual cortex. Thus, expression of microbial-type channelrhodopsins, such as ChR2, in surviving inner retinal neurons is a potential strategy for the restoration of vision after rod and cone degeneration.     

Optically controlled contraction of photosensitive skeletal muscle cells

As the skeletal muscle cell is an efficient force transducer, it has been incorporated in bio-microdevices using electrical field stimulation for generating contractile patterns. To improve both the spatial and temporal resolutions, we made photosensitive skeletal muscle cells from murine C2C12 myoblasts, which express channelrhodopsin-2 (ChR2), one of archaea-type rhodopsins derived from green algae Chlamydomonas reinhardtii. The cloned ChR2-expressing C2C12 myoblasts were made and fused with untransfected C2C12 to form multinucleated myotubes. The maturation of myotubes was facilitated by electrical field stimulation. Blue LED light pulse depolarized the membrane potential of a ChR2-expressing myotube and eventually evoked an action potential. It also induced a twitch-like contraction in a concurrent manner. A contraction pattern was thus made with a given pattern of LED pulses. This technique would have many applications in the bioengineering field, such as wireless drive of muscle-powered actuators/microdevices.     

A chimeric receptor of the insulin-like growth factor receptor type 1 (IGFR1) and a single chain antibody specific to myelin oligodendrocyte glycoprotein activates the IGF1R signalling cascade in CG4 oligodendrocyte progenitors

In order to generate neural stem cells with increased ability to survive after transplantation in brain parenchyma we developed a chimeric receptor (ChR) that binds to myelin oligodendrocyte glycoprotein (MOG) via its ectodomain and activates the insulin-like growth factor receptor type 1 ‎‎(IGF1R) signalling cascade. Activation of this pro-survival pathway in response to ligand broadly available in the brain might increase neuroregenerative potential of transplanted precursors. The ChR was produced by fusing a MOG-specific single ‎chain antibody with the extracellular boundary of the IGF1R transmembrane segment. The ChR is expressed on the cellular surface, predominantly as a monomer, and is not N-glycosylated. To show MOG-dependent functionality of the ChR, neuroblastoma cells B104 expressing this ChR were stimulated with monolayers of cells expressing recombinant MOG. The ChR undergoes MOG-dependent tyrosine phosphorylation and homodimerisation. It promotes insulin and IGF-independent growth of the oligodendrocyte progenitor cell line CG4. The proposed mode of the ChR activation is by MOG-induced dimerisation which promotes kinase domain transphosphorylation, by-passing the requirement of conformation changes known to be important for IGF1R activation. Another ChR, which contains a segment of the β-chain ectodomain, was produced in an attempt to recapitulate some of these conformational changes, but proved non-functional.     

Chemical nature of functional cholinoreceptor groups of Lymnaea stagnalis neurons.

1. The effect of pH and the influence of some alkylating agents on the properties of the cholinoreceptive membrane of the mollusc Lymnaea stagnalis neurons have been studied using the microelectrode voltage clamp technique. 2. Lowering below 7.5 of the pH of the bathing solution had to decrease the neuronal responses to ACh. A twofold decrease in cholinoreceptive membrane conductivity was found at the pH 6.7 +/- 0.1 (n=10). Raising the pH to 10.6 did not influence the response to ACh. 3. The pH effect is not associated with the influence on the properties of ionic channels but appears to be due to reduction of a functional group at the ChR active site by proton. 4. No highly reactive SH-groups were found at the ChR active site, but some functionally important carboxyl groups have been discovered. 5. The effect of pH is probably connected with reduction of --COO-- or imidazol group with a pKa of about 6.7.