Variant ChRmine proteins with accelerated kinetics and / or red-shifted spectra

Abstract

Provided is a fast variant ChRmine protein having faster kinetics compared to parent ChRmine protein, the fast variant ChRmine protein having one or more amino acid substitutions compared to parent ChRmine protein. Also provided is a red-shifted variant ChRmine protein having a red-shifted spectrum compared to parent ChRmine protein, the fast variant ChRmine protein having one or more amino acid substitutions compared to parent ChRmine protein. Further provided is a nucleic acid encoding the variant ChRmine protein disclosed herein, as well as a genetically modified cell comprising such a nucleic acid. Additionally provided is an optogenetic method, comprising genetically modifying a subject to express a variant ChRmine protein disclosed herein in brain cells of the subject, applying stimulating light to the subject's brain, and imaging the subject's brain.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 302,419 (filed January 24, 2022), the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing The Sequence Listing is provided herewith as Sequence Listing XML, "STAN-1931WO_SEQ_LIST," created on January 20, 2023, and having a size of 39,000 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. [Background technology]

[0003] Introduction ChRmine, a pump-like cation-permeable channelrhodopsin, exhibits enigmatic properties such as large photocurrents, red-shifted spectra, and extreme light sensitivity. Although ChRmine and its homologs function as ion channels, in primary sequence they are more similar to ion pump rhodopsins, and the mechanism of passive channel permeation in this family is unclear. Summary of the Invention

[0004] overview This disclosure provides a 2.0-Å resolution cryo-EM structure of ChRmine, revealing structural features that are not typical of channelrhodopsins, namely, trimeric assembly, short transmembrane helices 3, a twisted extracellular loop 1, a large vestibule within the monomer, and an unprecedented opening at the trimer interface. Based on the structure of ChRmine, we designed three proteins with desirable characteristics in optogenetics, such as rsChRmine and hsChRmine, which have further red-shifted and fast properties, respectively, and frChRmine, which has faster and more red-shifted performance. These proteins can be used in neuroscience research, particularly using optogenetics.

[0005] Thus, certain embodiments of the present disclosure provide a fast variant ChRmine protein with faster kinetics compared to the parent ChRmine protein, the fast variant ChRmine protein having one or more amino acid substitutions compared to the parent ChRmine protein. Certain embodiments of the present disclosure also provide a red-shifted variant ChRmine protein with a red-shifted spectrum compared to the parent ChRmine protein, the red-shifted variant ChRmine protein having one or more amino acid substitutions compared to the parent ChRmine protein. Further embodiments of the present disclosure provide a nucleic acid encoding the variant ChRmine protein disclosed herein, as well as a genetically modified cell comprising such a nucleic acid. Further embodiments of the present disclosure provide an optogenetic method comprising genetically modifying a subject to express a variant ChRmine protein disclosed herein in brain cells of the subject, applying stimulating light to the subject's brain, and imaging the subject's brain.

[0006] Additional embodiments of the present disclosure provide a method comprising genetically modifying a subject to express a variant ChRmine protein disclosed herein in a cell and / or organ. The method can further comprise applying a stimulating light to the modified cell and / or organ and imaging the cell and / or organ of the subject. The cell and / or organ can belong to the cardiovascular system, the digestive system, the urinary system, the respiratory system, the reproductive system, the musculoskeletal system, or the pancreatic / endocrine system.

[0007] A brief description of the sequence SEQ ID NO: 1: ChRmine protein from Tiarina fusus (GenBank QDS02893.1).

[0008] SEQ ID NO: 2: ChRmine protein from Hyphochytrium catenoides (HcKCR1: GenBank MZ826862).

[0009] Sequence number 3: ChRmine protein from Hypochytrium catenoides (HcKCR2: GenBank MZ826861).

[0010] SEQ ID NO: 4: ChRmine protein from Rhodomonas abbreviata (RaCCR1: GenBank QIU80793.1).

[0011] SEQ ID NO: 5: ChRmine protein from Rhodomonas salina strain CCMP1319 (RsCCR1: GenBank QIU80800.1).

[0012] Sequence number 6: ChRmine protein from Rhodomonas abreviata (RaCCR2: GenBank QIU80796.1).

[0013] SEQ ID NO: 7: ChRmine protein from Rhodomonas salina strain CCMP1319 (RsCCR2: GenBank QIU80801.1).

[0014] Sequence number 8: ChRmine protein from Guillardia theta (GtCCR1: GenBank ANC73520.1).

[0015] Sequence number 9: ChRmine protein from Guillardia theta (GtCCR1: GenBank ANC73518.1).

[0016] Sequence number 10: ChRmine protein from Guillardia theta (GtCCR3: GenBank ANC73519.1).

[0017] Sequence number 11: ChRmine protein from Guillardia theta (GtCCR4: GenBank ARQ20888.1).

[0018] SEQ ID NO:12: ChRmine protein from Halobacterium salinarum NRC-1 (HsBR: PDB 5ZIM).

[0019] SEQ ID NO: 13: ChRmine protein from Chlamydomonas reinhardtii (C1C2: PDB:3UG9).

[0020] Sequence number 14: ChRmine protein from Chlamydomonas reinhardtii (CrChR2: PDB:6EID).

[0021] SEQ ID NO: 15: ChRmine protein from Chlamydomonas reinhardtii, Chlamydomonas noctigama (C1Chrimson: PDB:5ZIH).

[0022] Sequence number 16: ChRmine protein from Guillardia theta CCMP2712 (GtACR1: PDB:6CSM).

[0023] SEQ ID NO: 17: ChRmine protein from Chlamydomonas reinhardtii (CrChR1: GenBank AAL08946.1).

[0024] SEQ ID NO: 18: ChRmine protein from Volvox carteri f. nagariensis (VChR1: GenBank ABZ90900.1).

[0025] SEQ ID NO: 19: ChRmine protein from Volvox carteri f. nagariensis (VChR2: GenBank ABZ90902.1).

[0026] SEQ ID NO: 20: ChRmine protein from Stigeoclonium helveticum (Chronos: GenBank KF992040.1)

[0027] Sequence number 21: ChRmine protein from Guillardia theta (GtACR2: GenBank AKN63095.1).

[0028] SEQ ID NO: 22: ChRmine protein from Rhodomonas lens (RlACR: GenBank APZ76712.1).

[0029] Sequence number 23: Synthetic ChRmine protein (MerMAID1: GenBank QCW06519.1).

[0030] SEQ ID NO: 24: ChRmine protein from Pyramimonas melkonianii CCMP772 (PymeACR1: GenBank QNU12853.1).

[0031] SEQ ID NO: 25: ChRmine protein from the metagenome (vPyACR_21821: GenBank QNU12854.1).

[0032] Sequence number 26: ChRmine protein from Halobacterium salinarum (HsHR: PDB: 1E12).

[0033] SEQ ID NO: 27: ChRmine protein from uncultured bacteria (BPRMed12: PDB:4JQ6).

[0034] SEQ ID NO:28: ChRmine protein from Salinibacter ruber (XR:PDB:3DDL).

[0035] SEQ ID NO: 29: ChRmine protein from Dokdonia eikasta KR2 (PDB: 3X3B).

[0036] SEQ ID NO: 30: Example of a high-rate variant ChRmine protein.

[0037] Sequence number 31: Example of a redshifted variant ChRmine protein.

[0038] SEQ ID NO:32: Example of a fast and redshifted variant ChRmine protein. [Brief description of the drawings]

[0039] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0040] Color references in the Brief Description of the Drawings refer to color drawings that may be provided on a jurisdictional basis.

[0041] [Figure 1A]Figure 1A-1G: Cryo-EM structure of ChRmine and comparison with HsBR and C1C2. [A] Cryo-EM density map (top) and ribbon representation (bottom) of the ChRmine homotrimer, colored according to protomers (red (darker grey shades, mostly on the left side of the density map), yellow (lighter grey shades, mostly on the right side of the density map), and light green (intermediate grey shades, in the middle part of the density map)), ATR (green, as shown, indicated), and lipids (grey, as shown, indicated). [B] Ribbon representation of the ChRmine homotrimer as viewed from inside the cell (top, red monomer at bottom left, yellow monomer at bottom right, and light green monomer at top) and outside the cell (bottom, red monomer at top left, yellow monomer at top right, and light green monomer at bottom). [C] Overall structures of ChRmine (left), HsBR (middle), and C1C2 (right) viewed parallel to the membrane (top) and from the inside of the cell (bottom). [D] Representative HS-AFM images of ChRmine. [E] ChRmine monomer viewed parallel to the membrane (left) and from the outside of the cell (right). ATR (stick model) is in green (pointed out as shown) and ECL1 is in cyan (pointed out as shown). [F-G] ChRmine (red, lighter grey) superimposed from two angles on HsBR (blue, darker grey) (F) and C1C2 (yellow, light grey) (G). [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 1E] See legend to Figure 1A. [Figure 1F] See legend to Figure 1A. [Figure 1G] See legend to Figure 1A. [Figure 2A]Figures 2A-2D: Schiff base regions. [A] Schiff base regions of ChRmine (top), HsBR (bottom left), and C1C2 (bottom right). Spheres and black dashed lines represent water molecules and H-bonds, respectively. [B] Photocurrent amplitudes of wild-type (WT) ChRmine and the two mutants. Mean ± sem (n = 5-6); one-way ANOVA with Dunnett's test. ****p < 0.0001. [C] Absorption spectra of ChRmine WT (top), D115N (middle), and D253N (bottom) at pH 7.5 (black) and pH 4.0 (red, grey). [D] Time series traces of absorbance changes of ChRmine WT (top), D115N (middle), and D253N (bottom) at probe wavelengths of 363–366 (blue, line with highest peak), 406 (cyan, line with second highest peak), 505–520 (green, line with lowest peak), and 579–588 nm (red, essentially horizontal line). [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 3A]Figure 3A-3I: Ion permeation pathways in monomers. [A] Extracellular and intracellular vestibules (EV and IV, respectively) of ChRmine (left), C1C2 (middle), and HsBR (right) in monomers. Vestibule: solid grey. Intracellular and central constriction sites (ICS and CCS): dashed boxes (green and orange, respectively, and indicated). [B-C] Ion permeation pore and conserved negatively charged residues (E121, E122, E129, E136, and E140) along the ion permeation pathway of C1C2 (C), and the corresponding residues of ChRmine (B). The pore is colored according to electrostatic potential. [D] Ion permeation pore and pore-aligned negatively charged residues of ChRmine, C1C2, CrChR2, C1Chrimson, and positively charged residues of GtACR1. Grey mesh: ion permeable pore. [E] ICS (top) and CCS (bottom) of ChRmine (left) and C1C2 (right). [F] Variation of the constriction site. Data are mean ± sem (n (cells) = 5-9); one-way ANOVA with Dunnett's test. *p<0.05, ***p<0.001, ****p<0.0001. [G] Superimposed intracellular regions of ChRmine (red, lighter grey) and HsBR (blue, darker grey) (left), and the intracellular region of ChRmine (right). IV1 and IV2 of ChRmine: grey mesh. [H] Overall structure of the intracellular region of HsBR. Intracellular vestibule of HsBR: grey mesh. [I] Enlargement of the blue boxed region in HsBR shown in (A). Comparison of the overall structure (left) and key residues (right) of ECL1 between ChRmine (red and indicated), C1C2 (yellow and indicated), and HsBR (blue and indicated). Extracellular cavity of ChRmine: grey mesh. ECL1 in ChRmine adopts a different structure (left) as indicated, whereas in HsBR, Y79 and R82 efficiently occlude the cavity. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 3F] See legend to Figure 3A. [Figure 3G] See legend to Figure 3A. [Figure 3H] See legend to Figure 3A. [Figure 3I] See legend to Figure 3A. [Figure 4A] Figure 4A-4K: Hydrophilic pores within the trimer interface. [A] Location of the pore within the trimer interface in ChRmine (left) and HsBR (right). The trimer pore pathway is shown as a grey mesh, with only two protomers shown for clarity. The pore in HsBR is hydrophobic and filled with several lipid molecules, whereas for ChRmine, the pore is hydrophilic and negatively charged. [B] Electrostatic potential surfaces and cross sections for ChRmine (left) and HsBR (right). [C] Trimer pore radius of ChRmine as a function of distance along the pore axis, calculated with HOLE. [D] Zoom-in from the blue box region in (A) and the constriction formed by ECL1, from two angles. [E-F] Comparison of the ECL of ChRmine with the ion-selective filters of NavM (E) and ASIC (F). [GH] Enlarged view of the boxed region in (A) showing hydrogen-bonding interactions between protomers on the intracellular (G) and extracellular (H) sides. [I] Fluorescence size-exclusion chromatography (FSEC) traces of ChRmine WT, S138W, Y156F, and S138W / Y156F mutants, suggesting destabilization of the trimer by the mutation. [J] Photocurrent amplitudes of the mutants shown in (GH) and R136H (as a negative control) (data are means ± s.e.m., n = 4–7; one-way analysis of variance (ANOVA) followed by Dunnett's test, **p < 0.01). [K] Reversal potentials of ChRmine WT, R136H, and S138W mutants, showing that the S138W mutation modulates cation selectivity. Data are means ± s.e.m. (n = 3–9); unpaired t test, **p < 0.01 and ****p < 0.0001. NS, not significant. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Figure 4F] See legend to Figure 4A. [Figure 4G] See legend to Figure 4A. [Figure 4H] See legend to Figure 4A. [Figure 4I] See legend to Figure 4A. [Figure 4J] See legend to Figure 4A. [Figure 4K] See legend to Figure 4A. [Figure 5A] Figure 5A-5F: Computational analysis of pore dynamics. [A] Visualization of the three F104 residues that define the trimer pore constriction site from representative frames of the dark- and light-state channels in the simulations. [B] Trimer pore radii were calculated for each of 10 independent 2 μs simulations. The pore radius is larger in the light-state simulations than in the dark-state stimulation (p<0.001, Welch test). [C-D] Water penetrating the trimer interface during MD simulations. Representative snapshot with water molecules around the constriction (C), and consecutive snapshots focusing on one water molecule (D). [E] The ChRmine monomer pore opens wider in the light-state simulations (13-cis-retinal and protonated D115) compared to the dark-state simulations (all-trans-retinal and deprotonated D115) (p<0.001, Welch test). The average minimum monomer pore radius for the three monomers was calculated for each of 10 independent 2 μs simulations. [F] In the light-state simulation, the pore radius is larger than in the dark-state simulation, as shown through representative frames from the two simulation conditions. Rotation of D115 and isomerization of retinal open up the inner monomer space. [Figure 5B] See legend to Figure 5A. [Figure 5C]See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 5E] See legend to Figure 5A. [Figure 5F] See legend to Figure 5A. [Figure 6A] Figure 6A-6K: Structure-informed design of ChRmine variants. [A] Residues including ChRmine EV (dark grey surface). Dashed lines indicate H-bonds. [B] RBPs of ChRmine (top left), C1C2 (top right) and HsBR (bottom). Key amino acids and all-trans retinal molecule shown by stick models. [C] On (left) and off (right) kinetics (n=4-23). ​​[D] Summary of photocurrent ratios (n=4-16). [E] Voltage clamp traces of WT- (top) and rsChRmine- (bottom) expressing neurons stimulated by the indicated light wavelengths. [F] Exemplary traces of opsins. [G] Normalized action spectra of WT and rsChRmine (n=16 for WT and 15 for rs). [H] 2P action spectra of WT (n=6) and rs (n=5). [I] Schematic of brain slice physiology. [J] Spike probability versus pulse width in slices (5Hz / 4 s, 1 mW / mm2, n=5 for WT, n=6 for rs). [K] Spike probability versus light power in slices (5Hz for 5 s, pulse width = 5 ms, n=5 for WT, n=6 for rs). All data are mean ± sem; sample size n indicates number of cells. Wilcoxon rank sum test for G, and Kruskal-Wallis test with Dunn's test for the rest. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 6F] See legend to Figure 6A. [Figure 6G]See legend to Figure 6A. [Figure 6H] See legend to Figure 6A. [Figure 6I] See legend to Figure 6A. [Figure 6J] See legend to Figure 6A. [Figure 6K] See legend to Figure 6A. [Figure 7A]Figure 7A-7R: In vitro demonstration of rsChRmine application. [A] Schematic: all optical physiology. [B] Representative traces of XCaMP-G responses to orange (585 nm) or red (635 nm) light stimulation in WT (left) or rsChRmine (right) neurons. [C] Peak XCaMP-G responses to orange and red light (n = 14 for rs; n = 15 for WT, two-tailed Mann-Whitney U test). [D] Peak XCaMP-B responses to blue (435 nm, left), cyan (488 nm, middle), and green (570 nm, right) light stimulation (n = 31 for rs; n = 32 for WT, two-tailed Mann-Whitney U test). [E] Summary of rise and decay kinetics of XCaMP-G transients (n = 14 for hs; n = 17 for rs; n = 24 for WT, one-way ANOVA with Tukey test). [F] Peak XCaMP-G response to 585 nm light stimulation (2.0 mW / mm2) (n=14 for hs and 10 for WT). All data are mean ± sem; sample size n indicates number of cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [G] Schematic: FIP rig for simultaneous Ca2+ recording and optogenetic stimulation. [H] Schematic: viral delivery and fiber placement in mPFC. [I] Expression of GCaMP6m and rsChRmine in neurons. [J] GCaMP6m trace in response to 594 nm stimulation in freely moving mice expressing opsin. (n=4 mice, 4 trials per mouse). [K] Left: average response to various light powers. Right: EPD50. [L] Quantification of baseline GCaMP6m fluorescence in rsChRmine, WT ChRmine, and ChrimsonR expressing mice. [M] Mean amplitude evoked at 470 nm normalized to the peak amplitude evoked with 594 nm stimulation for mice expressing each opsin. [N] GCaMP6m fluorescence at the start of imaging at different 470 nm optical powers. [O] GCaMP6m traces in response to 720 nm (top) and 750 nm (bottom) stimulation in freely moving mice expressing opsin. (n=4 mice, 4 trials per mouse).[P] Mean responses to 594, 720, and 750 nm stimulation for the indicated opsins. [Q and R] Ca2+ recordings from Pyr to PV (Q) and from PV to Pyr (R). Left, schematic of transgene expression in mPFC PV and CaMKIIα-positive Pyr neurons. Right, representative average simultaneous two-color photometric traces (green: GCaMP6, blue: XCaMP-B) aligned to the onset of 594 nm stimulation. All data are means (curves) ± s.e.m. (shading around curves); sample size n indicates number of cells unless otherwise stated. One-way ANOVA with Tukey's test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 7E] See legend to Figure 7A. [Figure 7F] See legend to Figure 7A. [Figure 7G] See legend to Figure 7A. [Figure 7H] See legend to Figure 7A. [Figure 7I] See legend to Figure 7A. [Figure 7J] See legend to Figure 7A. [Figure 7K] See legend to Figure 7A. [Figure 7L] See legend to Figure 7A. [Figure 7M] See legend to Figure 7A. [Figure 7N] See legend to Figure 7A. [Figure 7O] See legend to Figure 7A. [Figure 7P] See legend to Figure 7A. [Figure 7Q] See legend to Figure 7A. [Figure 7R] See legend to Figure 7A. [Figure 8A]Figures 8A-8F: Structure-based sequence alignment, phylogenetic tree, and predicted structure of ChRmine. [A] The sequences are ChRmine (GenBank QDS02893.1, SEQ ID NO: 1), HcKCR1 (GenBank MZ826862, SEQ ID NO: 2) (Govorunova et al., 2021), HcKCR2 (GenBank MZ826861, SEQ ID NO: 3) (Govorunova et al., 2021), RsCCR1 (GenBank QIU80800.1, SEQ ID NO: 5), RaCCR1 (GenBank QIU80793.1, SEQ ID NO: 4), RsCCR2 (GenBank QIU80801.1, SEQ ID NO: 7), RaCCR2 (GenBank QIU80796.1, SEQ ID NO: 6), GtCCR1 (GenBank ANC73520.1, SEQ ID NO: 8), GtCCR2 (GenBank ANC73518.1, SEQ ID NO: 9), and GtCCR3 (GenBank ANC73519.1, SEQ ID NO: 10), GtCCR4 (GenBank ARQ20888.1, SEQ ID NO: 11), HsBR (PDB:5ZIM, SEQ ID NO: 12) (Hasegawa et al., 2018), C1C2 (PDB:3UG9, SEQ ID NO: 13) (Kato et al., 2012), CrChR2 (PDB:6EID, SEQ ID NO: 14) (Volkov et al., 2017), C1Chrimson (PDB:5ZIH, SEQ ID NO: 15) (Oda et al., 2018), GtACR1 (PDB:6CSM, SEQ ID NO: 16) (Kim et al., 2018). Sequence alignments were made using the PLOMALS3D (Pei et al., 2008) and ESPript3 (Robert and Gouet, 2014) servers. Secondary structure elements of ChRmine are shown as coils. Positively and negatively charged residues are highlighted (blue and red, respectively). Stars represent the DTD motif. ECL1 of ChRmine is highlighted (light blue). Counterions are also highlighted (orange). [B] An unrooted phylogenetic tree was drawn for representative microbial rhodopsins using the Neighbor-Joining method (Saitou and Nei, 1987) and 1,000 bootstrap replicates.Evolutionary analysis was performed with MEGA7 (Kumar et al., 2016). White circles represent bootstrap values ​​>85%. [C-F] Five predicted models of ChRmine generated using locally installed AlphaFold2. Ribbon representations are highlighted (colored by pLDDT score (low: red, high: cyan)). [C] Plot of pLDDT scores. [D] Best predicted model superimposed on the cryo-EM structure (yellow). [E-F] Detailed comparison of ECL1 (E) and the Schiff base region (F) between the five predicted models and the cryo-EM structure. Notably, the C-terminal region of ECL1, including D115, has a high pLDDT score, but the conformation of D115 is not correctly predicted. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 8E] See legend to Figure 8A. [Figure 8F] See legend to Figure 8A. [Figure 9A]Figure 9A-9R: Cryo-EM analysis of ChRmine and the ChRmine-Fab02 complex. [A-C] Panels corresponding to ChRmine alone. Representative SEC trace (A), representative cryo-EM micrograph (B), and 2D class average (C), with SDS-PAGE as inset. [D-F] Panels corresponding to the ChRmine-Fab02 complex. Representative SEC trace (D), representative cryo-EM micrograph (E), and 2D class average (F), with SDS-PAGE as inset. [G] Low-resolution reconstruction of ChRmine alone. [H] Data processing workflow of the ChRmine-Fab02 complex. Final cryo-EM map colored at local resolution. [I] Fourier shell correlation (FSC) between two independently refined half-maps. [J] FSC between the model and the map calculated for the model refined against the full reconstruction. [K-N] Cryo-EM densities (FSC-weighted sharpening maps calculated by RELION3.1.1) and models for ChRmine, lipids (K), retinal-binding pocket (L), Schiff base region (M), and twisted ECL1 (N). [O-P] Densities and models near the ECL1 region. FSC-weighted sharpening maps calculated by RELION3.1.1 (blue), and Fo-Fc maps calculated by Servalcat (green). Positive Fo-Fc difference densities (4.3σ, where σ is the standard deviation within the mask) are observed near the nitrogen atoms, suggesting that these densities represent hydrogen atoms. [Q-R] Possible signals of early photointermediates. [Q] Densities and models for the retinal-binding pocket region. Maps (blue and green) are FSC-weighted sharpening maps calculated by RELION3.1.1, and Fo-Fc maps calculated by Servalcat, respectively. Positive and negative Fo-Fc difference density pairings (±3.7σ, where σ is the standard deviation within the mask) are observed around W223, suggesting that this density contains information about a small population of early intermediate states and that W223 moves upward early in the photocycle. [R] Close-up of (Q). [Figure 9B] See legend to Figure 9A. [Figure 9C]See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 9E] See legend to Figure 9A. [Figure 9F] See legend to Figure 9A. [Figure 9G] See legend to Figure 9A. [Figure 9H] See legend to Figure 9A. [Figure 9I] See legend to Figure 9A. [Figure 9J] See legend to Figure 9A. [Figure 9K] See legend to Figure 9A. [Figure 9L] See legend to Figure 9A. [Figure 9M] See legend to Figure 9A. [Figure 9N] See legend to Figure 9A. [Figure 9O] See legend to Figure 9A. [Figure 9P] See legend to Figure 9A. [Figure 9Q] See legend to Figure 9A. [Figure 9R] See legend to Figure 9A. [Figure 10A]Figures 10A-10L: Spectroscopic, structural, and HPLC characterization of ChRmine and ChRmine-Fab02 complex, related to Figure 1. [A] FSEC screening of Fab fragments. [B] Titration of Fab02 fragments against ChRmine. [C and D] Transient absorption spectra of ChRmine WT (C) and ChRmine-Fab02 complex (D) excited at λexc=532 nm. [E] Time traces of absorption changes of ChRmine WT (solid line) and ChRmine-Fab02 complex (right) at probe wavelengths of 363 (blue), 406 (cyan), 520 (green), and 588 nm (red). [F] Photocycle scheme of ChRmine determined by flash photolysis shown in (E). [G] Absorption spectra of ChRmine in the initial state (gray, ChRmine), L1 (orange, K / L1), M1 / L2 (green), and M2 (blue) calculated from the decay-related spectrum of the transient absorption change (Inoue et al., 2013). [H] Cryo-EM map of the ChRmine-Fab02 complex. [I] Interaction between ChRmine and Fab02. [J-K] HPLC analysis of the chromophore structure of ChRmine. [J] Representative HPLC profiles of the chromophore of ChRmine in the dark (top), under illumination (middle), and after light adaptation (bottom). "at", "11", and "13" indicate the peaks of all-trans, 11-cis, and 13-cis-retinal oximes, respectively. [K] Calculated compositions of all-trans and 13-cis-retinal oximes. Data are presented as mean ± sem (n = 3). Purified samples of ChRmine were prepared by supplementing with all-trans-retinal. Illumination was with green light (530±5 nm). Light adaptation was achieved by 1 min of illumination followed by 2 min of incubation in the dark. [L]ChRmine WT and mutants show lifetimes of each intermediate. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 10E] See legend to Figure 10A. [Figure 10F] See legend to Figure 10A. [Figure 10G] See legend to Figure 10A. [Figure 10H] See legend to Figure 10A. [Figure 10I] See legend to Figure 10A. [Figure 10J] See legend to Figure 10A. [Figure 10K] See legend to Figure 10A. [Figure 10L] See legend to Figure 10A. [Figure 11A] 11A-11F: Comparison of Schiff base regions related to FIG. 2. [A] Schiff base counterion and proton acceptor concept. [B] Superimposed Schiff base regions of ChRmine (red) and HsBR (blue). Spheres (red and blue) represent water molecules of ChRmine and HsBR, respectively. Black dashed lines represent hydrogen bonds. [C] Overlaid Schiff base regions of ChRmine (red) and representative microbial rhodopsins (CrChR2 (Volkov et al., 2017), C1Chrimson (Oda et al., 2018), GtACR1 (Kim et al., 2018), schizorhodopsin 4 (SzR4) (Higuchi et al., 2021), KR2 (Kato et al., 2015b), HsHR (Kolbe et al., 2000), NpSRII (Gordeliy et al., 2002), and heliodropsin (Shihoya et al., 2019)) are displayed with high transparency except for ChRmine. [D] List of Schiff base regions of representative microbial rhodopsins. [E-F] pH titration experiments of WT ChRmine. [E] Absorption spectra of WT ChRmine measured at pH 2.2–10.0. [F] λmax at each pH. [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 11D-1] See legend to Figure 11A. [Figure 11D-2]See legend to Figure 11A. [Figure 11E] See legend to Figure 11A. [Figure 11F] See legend to Figure 11A. [Figure 12A] Figures 12A-12D: Electrophysiology related to Figures 2-4. [A] Representative traces of ChRmine WT and 13 mutants expressed in HEK293 cells by lipofectamine transfection measured at a holding potential of -70 mV in voltage clamp. Traces were recorded while cells were stimulated with an irradiance of 1 mW mm-2 at 580 nm for 1.0 s. [B-D] Summary of steady-state to peak ratio of photocurrent (B), τoff of channel closure (C), and τoff of channel desensitization (=τdesen) (D). Mutants are classified as counterion, central constriction site (CCS), intracellular constriction site (ICS), and trimer interface mutants. Data are means ± sem (n = 3-9); one-way ANOVA followed by Dunnett's test. **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. [Figure 13A]Figures 13A-13H: Detailed characterization of rsChRmine, related to Figure 6. [A] Current-voltage dependence of unnormalized (left) and normalized (right) peak photocurrents of ChRmine at various voltages from -75 mV to 45 mV, stimulated with irradiance of 1 mW mm-2 at 580 nm for 1 s. (n=6-10). [B and C] Action spectra of single (B) and double (C) mutants of residues in RBP. (n=4-16). [D] Peak photocurrents of RBP mutants. (n=4-16, Kruskal-Wallis test with Dunn's test, asterisks indicate comparison with WT ChRmine, *p<0.05, **p<0.01, ****p<0.0001). [E] Normalized photocurrent versus light power in cultured neurons using cyan (470 nm), orange (585 nm), and red (650 nm) light (n=5 for WT and 7 for rs). [F] Effective power density (EPD50) of WT and rsChRmine (two-tailed Student's t-test, n=5 for WT and 7 for rs, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [G and H] Spike probability versus light intensity (G) and pulse width (H) in cultured neurons expressing opsin (n=6 for WT and 7 for RS, Kruskal-Wallis test with Dunn's test). All data are mean ± sem, and sample size n indicates number of cells unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 13B] See legend to Figure 13A. [Figure 13C] See legend to Figure 13A. [Figure 13D] See legend to Figure 13A. [Figure 13E] See legend to Figure 13A. [Figure 13F] See legend to Figure 13A. [Figure 13G] See legend to Figure 13A. [Figure 13H] See legend to Figure 13A. [Figure 14A]Figures 14A-14H: Detailed comparison of ChRmine and ChroME variants, related to Figure 6. [A] Confocal images of cultured neurons expressing WT ChRmine, hsChRmine, rsChRmine, ChroME2f, and ChroME2s. Scale bar = 10 μm. [B] Summary of on (left) and off (right) dynamics of ChRmine and ChroME variants (n = 3-23, Kruskal-Wallis test with Dunn's test. Asterisks indicate comparison with WT ChRmine, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [C] Exemplary current-clamp traces of hsChRmine, WT ChRmine, and ChroME2f expressing neurons with light stimulation. [D] Summary of opsin spike detection fidelity (n=5 for all variants, Kruskal-Wallis test with Dunn's test. Asterisks indicate comparison between ChroME2s and WT ChRmine, *p<0.05, **p<0.01, ***p<0.001). [E and F] Normalized photocurrent (E) and peak photocurrent (F) action spectra of ChRmine and ChroME variants (n=4–16). [G and H] Example traces of rsChRmine (G) and normalized action spectra of ChRmine and ChroME variants (H) under two-photon excitation (n=4–6). All data are means ± s.e.m., and sample size n indicates number of cells unless otherwise stated. For the WT ChRmine (compared here under the same conditions with other ChRs in the same preparation for a rigorous and unbiased comparison of performance in terms of photocurrents, action spectra, spike frequency responses, and EPD50), we observed slightly lower spike detection fidelity than observed in (Marshel et al., 2019), which may be due to preparation-to-preparation variation in opsin expression levels in cultured neurons. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 14D] See legend to Figure 14A. [Figure 14E]See legend to Figure 14A. [Figure 14F] See legend to Figure 14A. [Figure 14G] See legend to Figure 14A. [Figure 14H] See legend to Figure 14A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Detailed Description Provided is a pump-like cation-permeable channelrhodopsin. Designed three types of proteins with desired properties in optogenetics. Examples of these proteins include rsChRmine and hsChRmine, which have further red-shifted and fast properties, respectively, and frChRmine, which has faster / accelerated kinetics and larger red-shifting performance compared to rsChRmine. These proteins can be used in neuroscience research, especially using optogenetics.

[0043] Before the present invention is described in more detail, it is to be understood that the invention is not limited to the described embodiments, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0044] Where a range of values ​​is provided, unless the context dictates otherwise, it is understood that each intervening value between the upper and lower limits of that range is also specifically disclosed to the tenth of the unit of the lower limit. Each smaller range between any stated value or intervening value within a stated range and any other stated value or intervening value within that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the range, and each range in which either, neither, or both of the upper and lower limits are included within the invention, subject to any specifically excluded limitations in the stated range. Where a stated range includes one or both limits, ranges excluding either or both of those included limits are likewise included within the invention.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some possible and exemplary methods and materials can be described here. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It should be understood that to the extent of any conflict, the present disclosure supersedes any disclosure of the incorporated publications.

[0046] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, a reference to "a protein" includes a plurality of such proteins, a reference to "a mutation" includes a reference to one or more distinct mutations, and so forth. It is further noted that the claims may be drafted to exclude any element, e.g., any optional element. Thus, this declaration is intended to serve as a predicate basis for using exclusive language, such as "solely," "only," and the like, or for using "negative" limitations in connection with the recitation of claim elements.

[0047] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. In the event that the definition or scope of any term in this specification conflicts with the definition or use of the term in an application or reference incorporated herein by reference, this application controls.

[0048] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other various embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0049] definition The term "kinetics" or "kinetic property / characteristics" as used herein with respect to a ChRmine protein refers to the rate at which the ChRmine protein opens and closes the channelrhodopsin ion channel. A ChRmine protein that has a higher rate at which the channelrhodopsin ion channel opens and closes compared to another ChRmine protein is said to have faster or accelerated kinetics or kinetic property / characteristics compared to the other ChRmine protein.

[0050] A "fast variant ChRmine protein" has accelerated kinetic properties / characteristics, i.e., faster kinetic properties / characteristics compared to the parent ChRmine protein used to produce the fast variant ChRmine protein. Fast variant ChRmine proteins can have kinetic properties that are 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 80% or more faster than the parent ChRmine protein used to produce the fast variant ChRmine protein.

[0051] ChRmine dynamics is measured using the "rise time", the tau off (τ オフ ), or a combination of both.

[0052] "Rise time" (tpeak) is the time to peak from the cessation of light stimulation to the point at which maximum amplitude fluorescence is reached. For example, a wild-type ChRmine protein may have a time to peak of 15-20 milliseconds, while a corresponding fast variant ChRmine protein may have a time to peak of 5-10 milliseconds. Thus, compared to the parent ChRmine protein used to produce the fast variant ChRmine protein, the fast variant ChRmine protein may have a time to peak that is reduced by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[0053] For example, wild-type ChRmine proteins have a τ of 50–150 ms. オフ whereas the corresponding fast variant ChRmine proteins have a τ of 20-50 ms. オフThus, compared to the parent ChRmine protein used to produce the fast variant ChRmine protein, the fast variant ChRmine protein may have a τ that is reduced by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. オフ may have 。

[0054] As used herein with respect to a ChRmine protein, a "red-shifted spectrum" refers to a red-shifted absorption by a ChRmine protein, e.g., a variant ChRmine protein, compared to another ChRmine protein, e.g., a parent ChRmine protein. A ChRmine protein having a red-shifted absorption compared to a parent ChRmine protein is referred to herein as a "red-shifted variant."

[0055] The wavelength that elicits the maximum photocurrent is the same for both opsin proteins, but the maximum photocurrent is different for all other wavelengths in the parent ChRmine protein compared to the corresponding redshifted variant ChRmine protein. For example, photocurrent may be lower at 380, 440, and 480 nm for the redshifted variant ChRmine protein compared to the corresponding parent ChRmine protein, indicating a decrease in photocurrent at blue wavelengths, which represents a redshift, and photocurrent is higher at 650 nm, which also represents a redshift.

[0056] For example, the parent ChRmine protein can have the following maximum photocurrents: 380 nm: 0.49, 440 nm: 0.78, 480 nm: 0.94, 513 nm: 1, 580 nm: 0.82, 650 nm: 0.18. These absorption values ​​are normalized to the maximum photocurrent at 513 nm. The corresponding red-shifted variant ChRmine protein can have the following maximum photocurrents: 380 nm: 0.41, 440 nm: 0.50, 480 nm: 0.80, 513 nm: 1, 580 nm: 0.81, and 650 nm: 0.31. Again, these absorption values ​​are normalized to the maximum photocurrent at 513 nm.

[0057] Thus, at wavelengths shorter than those providing maximum photocurrent, the redshifted variant ChRmine protein may have a maximum photocurrent that is reduced by 10% or more, 20% or more, 30% or more, or 40% or more compared to the maximum photocurrent of the parent ChRmine protein. On the other hand, at wavelengths longer than those providing maximum photocurrent, the redshifted variant ChRmine protein may have a maximum photocurrent that is increased by 10% or more, 20% or more, 30% or more, or 40% or more compared to the maximum photocurrent of the parent ChRmine protein.

[0058] As used herein, "parent ChRmine protein" refers to a wild-type or naturally occurring ChRmine protein. In some examples, the parent ChRmine protein can be mutated to produce a variant ChRmine protein. The parent protein can be a wild-type or naturally occurring ChRmine protein or a homolog thereof. Non-limiting examples of such parent ChRmine proteins are provided in FIG. 8A and SEQ ID NOs: 1-29. Additional examples of ChRmine proteins that can be used as parent ChRmine proteins are well known in the art, and the use of such ChRmine proteins to produce the variant ChRmine proteins disclosed herein is within the scope of the present disclosure. Certain such examples include CrChR1 (GenBank AAL08946.1, SEQ ID NO: 17), VChR1 (GenBank ABZ90900.1, SEQ ID NO: 18), VChR2 (GenBank ABZ90902.1, SEQ ID NO: 19), Chronos (GenBank KF992040.1, SEQ ID NO: 20), GtACR2 (GenBank AKN63095.1, SEQ ID NO: 21), RlACR (GenBank APZ76712.1, SEQ ID NO: 22), MerMAID1 (GenBank QCW06519.1, SEQ ID NO: 23) (Oppermann et al., 2019), PymeACR1 (GenBank QNU12853.1, SEQ ID NO: 24) (Rozenberg et al., 2020), vPyACR_21821 (GenBank QNU12854.1, sequence number 25) (Rozenberg et al., 2020), HsHR (PDB: 1E12, sequence number 26) (Kolbe et al., 2000), BPRMed12 (PDB: 4JQ6, sequence number 27) (Ran et al., 2013), XR (PDB: 3DDL, sequence number 28) (Luecke et al., 2008), and KR2 (PDB: 3X3B, sequence number 29) (Kato et al., 2015b).

[0059] A "homologous protein or protein homolog" of a protein is another protein having a similar or identical function, as well as a similar primary, secondary, and / or tertiary structure. Typically, a homologous protein or protein homolog has substantial sequence identity, e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity. Certain non-limiting examples of ChRmine protein homologs are provided as SEQ ID NOs: 1-29. A sequence alignment of some of these proteins is shown in FIG. 8A. Additional examples of ChRmine protein homologs are well known in the art, and the use of such ChRmine proteins to produce variant ChRmine proteins disclosed herein is within the scope of this disclosure. For example, one of skill in the art can select a wild-type, naturally occurring, or artificial / mutated ChRmine protein and make amino acid substitutions, particularly conservative amino acid substitutions, that do not affect the function or structure of the protein, thereby producing another ChRmine protein homolog. Specific examples of conservative amino acid substitutions, i.e., substitutions within the same class of amino acids, are provided in Table 1 below. The homologs produced after such conservative amino acid substitutions can then be further modified in accordance with the present disclosure to produce fast or red-shifted variant ChRmine proteins.

[0060] (Table 1) TIFF2025508610000002.tif95166

[0061] The phrase "corresponding residue in a homolog of the ChRmine protein" refers to a residue in a homolog of the ChRmine protein that aligns with a reference residue in the ChRmine protein, for example, as shown in FIG. 8A. For example, the histidine at position 33 of the ChRmine protein shown in FIG. 8A corresponds to a serine residue in the HcKCR1 protein, as shown in this figure. Similarly, position 33 of the ChRmine protein shown in FIG. 8A corresponds to aspartic acid, asparagine, alanine, leucine, or glutamine in certain other ChRmine homologs. One skilled in the art can readily identify residues in a homolog of the ChRmine protein that correspond to a reference residue in the ChRmine protein by generating and analyzing sequence alignments of amino acid sequences, such as those provided in FIG. 8A.

[0062] Variant ChRmine proteins Variants of ChRmine proteins, which are pump-like cation-permeable channelrhodopsins, are provided.

[0063] In certain embodiments, a variant ChRmine protein is provided, which is a fast variant ChRmine protein having faster kinetic properties compared to the parent ChRmine protein, and which has one or more amino acid substitutions compared to the parent ChRmine protein.

[0064] In some cases, the fast variant ChRmine protein may have one or more amino acid substitutions in the counterion of the Schiff base. Certain such amino acids are identified in Figures 8A and 11A.

[0065] The fast variant ChRmine protein can also have one or more amino acid substitutions that change the pore electrostatic potential of the parent protein. Certain such amino acid substitutions include substitutions at one or more of histidine at position 33 or corresponding, aspartic acid at position 92 or corresponding, glutamic acid at position 154 or corresponding, glutamic acid at position 158 or corresponding, aspartic acid at position 242 or corresponding, and glutamic acid at position 246 or corresponding. Each of these positions can be substituted with any other amino acid. The substitution at histidine at position 33 or corresponding can be with histidine (if the corresponding amino acid is not histidine), arginine, or lysine. The substitutions at aspartic acid or a corresponding position 92, glutamic acid or a corresponding position 154, glutamic acid or a corresponding position 158, aspartic acid or a corresponding position 242, and glutamic acid or a corresponding position 246 may be with aspartic acid, glutamic acid, asparagine, or glutamine.

[0066] For example, the substitution at histidine or a corresponding position at position 33 may be with arginine. The substitution at histidine or a corresponding position at position 33 may also be with lysine. If the amino acid corresponding to histidine at position 33 is not histidine, such amino acid may be substituted with histidine.

[0067] The substitution at aspartic acid at position 92 or a corresponding position may be with aspartic acid. The substitution at aspartic acid at position 92 or a corresponding position may also be with glutamic acid. The substitution at aspartic acid at position 92 or a corresponding position may also be with asparagine. The substitution at aspartic acid at position 92 or a corresponding position may also be with glutamine.

[0068] The substitution at glutamic acid or a corresponding position at position 154 may be with aspartic acid. The substitution at glutamic acid or a corresponding position at position 154 may also be with glutamic acid. The substitution at glutamic acid or a corresponding position at position 154 may also be with asparagine. The substitution at glutamic acid or a corresponding position at position 154 may also be with glutamine.

[0069] The substitution at glutamic acid or a corresponding position at position 158 may be with aspartic acid. The substitution at glutamic acid or a corresponding position at position 158 may also be with glutamic acid. The substitution at glutamic acid or a corresponding position at position 158 may also be with asparagine. The substitution at glutamic acid or a corresponding position at position 158 may also be with glutamine.

[0070] The substitution at aspartic acid at position 242 or a corresponding position may be with aspartic acid. The substitution at aspartic acid at position 242 or a corresponding position may also be with glutamic acid. The substitution at aspartic acid at position 242 or a corresponding position may also be with asparagine. The substitution at aspartic acid at position 242 or a corresponding position may also be with glutamine.

[0071] The substitution at glutamic acid or a corresponding position at position 246 may be with aspartic acid. The substitution at glutamic acid or a corresponding position at position 246 may be with glutamic acid. The substitution at glutamic acid or a corresponding position at position 246 may be with asparagine. The substitution at glutamic acid or a corresponding position at position 246 may also be with glutamine.

[0072] A fast variant ChRmine protein may be produced from a parent ChRmine protein selected from the proteins shown in FIG. 8A and SEQ ID NOs: 1-29. In some cases, compared to the parent ChRmine protein or a homolog thereof, the fast variant ChRmine protein has a substitution at the histidine residue at position 33 of the ChRmine protein as shown in FIG. 8A or the corresponding residue in the first transmembrane domain of a homolog of the ChRmine protein. For example, the fast variant ChRmine protein has an arginine substitution at the histidine residue at position 33 of the ChRmine protein as shown in FIG. 8A or the corresponding residue in the first transmembrane domain of a homolog of the ChRmine protein.

[0073] The parent ChRmine protein can have a sequence selected from SEQ ID NOs: 1-29, or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1-29. A sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to a sequence selected from SEQ ID NOs: 1-29 can have conservative amino acid substitutions compared to the sequence from which it is derived. For example,

[0074] In one embodiment, compared to the parent ChRmine protein, the fast variant ChRmine protein has a substitution at the histidine residue at position 33 or the corresponding residue in the first transmembrane domain of the parent ChRmine protein. The histidine amino acid may be substituted with any other amino acid, for example, histidine (if the corresponding amino acid is not histidine), arginine or lysine, i.e., a basic amino acid.

[0075] For example, the substitution at histidine or a corresponding position at position 33 may be with arginine. The substitution at histidine or a corresponding position at position 33 may be with lysine. If the amino acid corresponding to histidine at position 33 is not histidine, such amino acid may be substituted with histidine.

[0076] In certain embodiments, the fast variant ChRmine protein has an arginine substitution at the histidine residue at position 33 or the corresponding residue in the first transmembrane domain of the parent ChRmine protein. Thus, the fast variant ChRmine protein can have a sequence of SEQ ID NO: 30, or a sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO: 30, and the difference in the sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO: 30 excludes the amino acid substitution used to produce the fast variant ChRmine protein. In certain embodiments, the fast variant ChRmine protein has a sequence of SEQ ID NO: 30.

[0077] Additional embodiments of the present disclosure provide red-shifted variant ChRmine proteins having a red-shifted spectrum compared to the parent ChRmine protein, which red-shifted variant ChRmine proteins have one or more amino acid substitutions compared to the parent ChRmine protein.

[0078] The red-shifted variant ChRmine protein can have one or more amino acid substitutions in the retinal binding pocket (RBP) of the parent ChRmine protein. Certain such amino acid substitutions include substitutions at one or more of isoleucine 146 or the corresponding position, glycine 174 or the corresponding position, and phenylalanine 178 or the corresponding position. Each of these positions can be substituted with any other amino acid. The substitution at isoleucine 146 or the corresponding position can be with serine, cysteine, threonine, or methionine, i.e., a hydroxyl or sulfur / selenium-containing amino acid. The substitution at glycine 174 or the corresponding position can be with serine, cysteine, threonine, or methionine, i.e., a hydroxyl or sulfur / selenium-containing amino acid. The substitution at phenylalanine 178 or the corresponding position can be with phenylalanine (if the corresponding amino acid is not phenylalanine), tyrosine, or tryptophan, i.e., an aromatic amino acid.

[0079] For example, the substitution at isoleucine at position 146 or a corresponding position may be with serine. The substitution at isoleucine at position 146 or a corresponding position may also be with cysteine. The substitution at isoleucine at position 146 or a corresponding position may also be with threonine. The substitution at isoleucine at position 146 or a corresponding position may also be with methionine.

[0080] The substitution at glycine at position 174, or a corresponding position, may be with serine. The substitution at glycine at position 174, or a corresponding position, may also be with cysteine. The substitution at glycine at position 174, or a corresponding position, may also be with threonine. The substitution at glycine at position 174, or a corresponding position, may also be with methionine.

[0081] The substitution at phenylalanine at position 178 or a corresponding position may be with tyrosine. The substitution at phenylalanine at position 178 or a corresponding position may also be with tryptophan. If the corresponding amino acid at phenylalanine at position 178 is not phenylalanine, it may be substituted with phenylalanine.

[0082] A red-shifted variant ChRmine protein may be produced from a parent ChRmine protein selected from the proteins shown in Figure 8A or SEQ ID NOs: 1 to 29. In some cases, compared to the parent ChRmine protein, the red-shifted variant ChRmine protein has one or both of (i) a substitution at an isoleucine residue at position 146 of the ChRmine protein as shown in Figure 8A or the sequence of SEQ ID NOs: 1 to 29, or a corresponding residue in the fourth transmembrane domain of a homologue of the ChRmine protein, and (ii) a substitution at a glycine residue at position 174 of the ChRmine protein as shown in Figure 8A, or a corresponding residue in the fifth transmembrane domain of a homologue of the ChRmine protein. For example, compared to the parent ChRmine protein, the red-shifted variant ChRmine protein has one or both of: (i) a methionine substitution at the isoleucine residue at position 146 of the ChRmine protein as shown in Figure 8A or the corresponding residue in the fourth transmembrane domain of a homolog of the ChRmine protein, and (ii) a serine substitution at the glycine residue at position 174 of the ChRmine protein as shown in Figure 8A or the corresponding residue in the fifth transmembrane domain of a homolog of the ChRmine protein.

[0083] The parent ChRmine protein can have a sequence selected from SEQ ID NOs: 1-29 or a sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to a sequence selected from SEQ ID NOs: 1-29. A sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to a sequence selected from SEQ ID NOs: 1-29 can have conservative amino acid substitutions compared to the sequence from which it is derived.

[0084] In certain embodiments, the red-shifted variant ChRmine protein has one or both of (i) a substitution at the isoleucine residue at position 146 or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein, and (ii) a substitution at the glycine residue at position 174 or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein. The isoleucine residue at position 146 or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein may be substituted with any other amino acid, for example, serine, cysteine, threonine, or methionine, i.e., a hydroxyl or sulfur / selenium-containing amino acid. The glycine residue at position 174 or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein may be substituted with any other amino acid, for example, serine, cysteine, threonine, or methionine, i.e., a hydroxyl or sulfur / selenium-containing amino acid.

[0085] For example, the isoleucine residue at position 146 or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein may be substituted with serine. The isoleucine residue at position 146 or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein may also be substituted with cysteine. The isoleucine residue at position 146 or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein may also be substituted with threonine. The isoleucine residue at position 146 or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein may also be substituted with methionine.

[0086] The glycine residue at position 174 or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein may be substituted with serine. The glycine residue at position 174 or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein may also be substituted with cysteine. The glycine residue at position 174 or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein may also be substituted with threonine. The glycine residue at position 174 or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein may also be substituted with methionine.

[0087] Thus, a redshifted variant ChRmine protein can have the sequence of SEQ ID NO:31, or a sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:31, where the difference in the sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:31 excludes the amino acid substitutions used to produce the redshifted variant ChRmine protein. In a specific embodiment, the redshifted variant ChRmine protein has the sequence of SEQ ID NO:31.

[0088] A further embodiment of the present disclosure provides a fast and red-shifted variant ChRmine protein having faster kinetics and a red-shifted spectrum compared to the parent ChRmine protein, the fast and red-shifted variant ChRmine protein having one or more amino acid substitutions compared to the parent ChRmine protein.

[0089] The fast and red-shifted variant ChRmine protein may comprise (i) one or more amino acid substitutions in the counterion of the Schiff base of the parent ChRmine protein, or one or more amino acid substitutions that alter the pore electrostatic potential of the parent ChRmine protein, and (ii) one or more amino acid substitutions in the retinal binding pocket (RBP) of the parent ChRmine protein. In certain cases, the fast and red-shifted variant ChRmine protein comprises (i) one or more amino acid substitutions that alter the pore electrostatic potential of the parent ChRmine protein, and (ii) one or more amino acid substitutions in the retinal binding pocket (RBP) of the parent ChRmine protein.

[0090] The one or more amino acid substitutions that alter the pore electrostatic potential may be at histidine or corresponding position 33, aspartic acid or corresponding position 92, glutamic acid or corresponding position 154, glutamic acid or corresponding position 158, aspartic acid or corresponding position 242, or glutamic acid or corresponding position 246. Histidine or corresponding position 33 may be substituted with histidine if the corresponding amino acid is not histidine, arginine, or lysine. Each of aspartic acid or corresponding position 92, glutamic acid or corresponding position 154, glutamic acid or corresponding position 158, aspartic acid or corresponding position 242, and glutamic acid or corresponding position 246 may be substituted independently with aspartic acid, glutamic acid, asparagine, or glutamine.

[0091] For example, the substitution at histidine or a corresponding position at position 33 may be with arginine. The substitution at histidine or a corresponding position at position 33 may also be with lysine. If the amino acid corresponding to histidine at position 33 is not histidine, such amino acid may be substituted with histidine.

[0092] The substitution at aspartic acid at position 92 or a corresponding position may be with aspartic acid. The substitution at aspartic acid at position 92 or a corresponding position may also be with glutamic acid. The substitution at aspartic acid at position 92 or a corresponding position may also be with asparagine. The substitution at aspartic acid at position 92 or a corresponding position may also be with glutamine.

[0093] The substitution at glutamic acid or a corresponding position at position 154 may be with aspartic acid. The substitution at glutamic acid or a corresponding position at position 154 may also be with glutamic acid. The substitution at glutamic acid or a corresponding position at position 154 may also be with asparagine. The substitution at glutamic acid or a corresponding position at position 154 may also be with glutamine.

[0094] The substitution at glutamic acid or a corresponding position at position 158 may be with aspartic acid. The substitution at glutamic acid or a corresponding position at position 158 may also be with glutamic acid. The substitution at glutamic acid or a corresponding position at position 158 may also be with asparagine. The substitution at glutamic acid or a corresponding position at position 158 may also be with glutamine.

[0095] The substitution at aspartic acid at position 242 or a corresponding position may be with aspartic acid. The substitution at aspartic acid at position 242 or a corresponding position may also be with glutamic acid. The substitution at aspartic acid at position 242 or a corresponding position may also be with asparagine. The substitution at aspartic acid at position 242 or a corresponding position may also be with glutamine.

[0096] The substitution at glutamic acid or a corresponding position at position 246 may be with aspartic acid. The substitution at glutamic acid or a corresponding position at position 246 may also be with glutamic acid. The substitution at glutamic acid or a corresponding position at position 246 may also be with asparagine. The substitution at glutamic acid or a corresponding position at position 246 may also be with glutamine.

[0097] The one or more amino acid substitutions in the RBP of the parent ChRmine protein may include substitutions at one or more of isoleucine 146 or the corresponding position, glycine 174 or the corresponding position, and phenylalanine 178 or the corresponding position. The substitution at isoleucine 146 or the corresponding position may be with serine, cysteine, threonine, or methionine. The substitution at glycine 174 or the corresponding position may be with serine, cysteine, threonine, or methionine. The substitution at phenylalanine 178 or the corresponding position may be with phenylalanine if the corresponding amino acid is not phenylalanine, tyrosine, or tryptophan. Any combination of these substitutions may be generated.

[0098] For example, the substitution at isoleucine at position 146 or a corresponding position may be with serine. The substitution at isoleucine at position 146 or a corresponding position may also be with cysteine. The substitution at isoleucine at position 146 or a corresponding position may also be with threonine. The substitution at isoleucine at position 146 or a corresponding position may also be with methionine.

[0099] The substitution at glycine at position 174, or a corresponding position, may be with serine. The substitution at glycine at position 174, or a corresponding position, may also be with cysteine. The substitution at glycine at position 174, or a corresponding position, may also be with threonine. The substitution at glycine at position 174, or a corresponding position, may also be with methionine.

[0100] The substitution at phenylalanine at position 178 or a corresponding position may be with tyrosine. The substitution at phenylalanine at position 178 or a corresponding position may also be with tryptophan. If the corresponding amino acid at phenylalanine at position 178 is not phenylalanine, it may be substituted with phenylalanine.

[0101] A fast and red-shifted variant ChRmine can be produced from a parent ChRmine protein selected from the proteins provided in Figure 8A or a ChRmine protein having a sequence selected from SEQ ID NOs: 1-29. In some cases, compared to the parent ChRmine protein, the fast and red-shifted variant ChRmine protein has one or more of: (i) a substitution at a histidine residue at position 33 of the ChRmine protein as shown in Figure 8A or a corresponding residue in the first transmembrane domain of a homolog of the ChRmine protein, (ii) a substitution at an isoleucine residue at position 146 of the ChRmine protein as shown in Figure 8A or a corresponding residue in the fourth transmembrane domain of a homolog of the ChRmine protein, and (iii) a substitution at a glycine residue at position 174 of the ChRmine protein as shown in Figure 8A or a corresponding residue in the fifth transmembrane domain of a homolog of the ChRmine protein. For example, compared to the parent ChRmine protein, the fast and red-shifted variant ChRmine protein has (i) an arginine substitution at the histidine residue at position 33 of the ChRmine protein as shown in Figure 8A or the corresponding residue in the first transmembrane domain of a homolog of the ChRmine protein, (ii) a methionine substitution at the isoleucine residue at position 146 of the ChRmine protein as shown in Figure 8A or the corresponding residue in the fourth transmembrane domain of a homolog of the ChRmine protein, and (iii) a serine substitution at the glycine residue at position 174 of the ChRmine protein as shown in Figure 8A or the corresponding residue in the fifth transmembrane domain of a homolog of the ChRmine protein.

[0102] The parent ChRmine protein can have a sequence selected from SEQ ID NOs: 1 to 29, or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1 to 29. The sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1 to 29 can have conservative amino acid substitutions compared to the sequence from which it is derived.

[0103] In certain embodiments, the fast and red-shifted variant ChRmine protein has one or more of: (i) a substitution with a histidine residue at position 33 or the corresponding residue in the first transmembrane domain of the parent ChRmine protein; (ii) a substitution with an isoleucine residue at position 146 or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein; and (iii) a substitution with a glycine residue at position 174 or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein. The histidine amino acid at position 33 or the corresponding residue in the first transmembrane domain of the parent ChRmine protein may be substituted with any other amino acid, for example, histidine (if the corresponding amino acid is not histidine), arginine or lysine, i.e., a basic amino acid. The isoleucine residue at position 146 or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein may be substituted with any other amino acid, for example, serine, cysteine, threonine, or methionine, i.e., a hydroxyl or sulfur / selenium-containing amino acid. The glycine residue at position 174 or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein may be substituted with any other amino acid, e.g., serine, cysteine, threonine, or methionine, i.e., a hydroxyl or sulfur / selenium-containing amino acid.

[0104] For example, the substitution at histidine or a corresponding position at position 33 may be with arginine. The substitution at histidine or a corresponding position at position 33 may also be with lysine. If the amino acid corresponding to histidine at position 33 is not histidine, such amino acid may be substituted with histidine.

[0105] Also, the substitution at isoleucine at position 146 or a corresponding position may be with serine. The substitution at isoleucine at position 146 or a corresponding position may also be with cysteine. The substitution at isoleucine at position 146 or a corresponding position may also be with threonine. The substitution at isoleucine at position 146 or a corresponding position may also be with methionine.

[0106] The substitution at glycine at position 174, or a corresponding position, may be with serine. The substitution at glycine at position 174, or a corresponding position, may also be with cysteine. The substitution at glycine at position 174, or a corresponding position, may also be with threonine. The substitution at glycine at position 174, or a corresponding position, may also be with methionine.

[0107] Thus, the fast and red-shifted variant ChRmine protein can have a sequence of SEQ ID NO: 32, or a sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO: 32, where the difference in the sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO: 32 excludes the amino acid substitutions used to produce the fast and red-shifted variant ChRmine protein. In a specific embodiment, the fast and red-shifted variant ChRmine protein has a sequence of SEQ ID NO: 32. A further embodiment of the present disclosure provides a nucleic acid encoding the variant ChRmine protein disclosed herein. Based on the sequence of the variant ChRmine protein and the known codon usage, one skilled in the art can design a nucleic acid encoding a particular variant ChRmine protein. The nucleic acids can be optimized for expression in a particular cell, such as a mammalian cell or an insect cell. Methods for such codon optimization are well known in the art and are within the scope of the present disclosure.

[0108] The nucleic acid encoding such a variant ChRmine protein can be incorporated into an expression cassette, e.g., an expression vector, for expressing the variant ChRmine protein in a cell, non-limiting examples of which include a bacterial cell, a fungal cell, an insect cell, a plant cell, or a mammalian cell.

[0109] Thus, a further embodiment of the present disclosure provides a genetically modified cell comprising a nucleic acid encoding a variant ChRmine protein. Methods for introducing a nucleic acid, e.g., a nucleic acid in an expression construct, into a target cell and expressing and purifying a protein are well known in the art, and such embodiments are within the scope of the present disclosure.

[0110] method Optogenetics involves genetically modifying neurons, followed by contacting the modified neurons with light, which causes the neurons to express light-sensitive ion channels, and contacting the neurons with light activates these channels, influencing neuronal activity.

[0111] As discussed above, certain embodiments of the present disclosure provide variant ChRmine proteins that exhibit faster kinetics and / or red-shifted spectra compared to the parent ChRmine protein, and when used in optogenetic methods, such variant ChRmine proteins offer certain advantages over the parent ChRmine protein.

[0112] Accordingly, certain embodiments of the present disclosure provide optogenetic methods comprising genetically modifying a subject to express a variant ChRmine protein disclosed herein in brain cells of the subject, applying stimulating light to the subject's brain, and imaging the subject's brain.

[0113] The subject can be a human, non-human primate, bovine, porcine, feline, or canine animal.

[0114] Details of optogenetic methods are well known in the art, and generally, it is within the scope of this disclosure to apply such methods using the variant ChRmine proteins disclosed herein.

[0115] For example, in some cases, the methods involve electrical stimulation of a brain region using one or more electrodes, which can be placed in the brain region either temporarily or permanently.

[0116] In some cases, the brain region genetically modified for the optogenetic method is selected from the group consisting of hippocampus, septo-hippocampus, anterior cingulate cortex (ACC), basolateral amygdala (BLA), midline thalamus, insulating region, medial septum, and fimbria-fornix. In some cases, the brain region is the hippocampus. In some cases, the brain region is the septo-hippocampus. In some cases, the brain region is the ACC. In some cases, the brain region is the BLA. In some cases, the brain region is the medial septum. In some cases, the brain region is the fimbria-fornix. In some cases, more than one of the listed brain regions is genetically modified.

[0117] Additional embodiments of the present disclosure provide a method comprising genetically modifying a subject to express a variant ChRmine protein disclosed herein in a cell and / or organ. The method can further comprise applying a stimulating light to the modified cell and / or organ and imaging the cell and / or organ of the subject. The cell and / or organ can belong to the cardiovascular system, the digestive system, the urinary system, the respiratory system, the reproductive system, the musculoskeletal system, or the pancreatic / endocrine system.

[0118] Regardless of the scope of the appended claims, the present disclosure is also clarified by the following embodiments. Embodiment 1. A high-speed variant ChRmine protein having faster kinetic properties compared to the parent ChRmine protein, the high-speed variant ChRmine protein having one or more amino acid substitutions compared to the parent ChRmine protein. Embodiment 2. A fast variant ChRmine protein described in embodiment 1, comprising one or more amino acid substitutions in the counterion of the Schiff base of the parent ChRmine protein. Embodiment 3. A high-rate variant ChRmine protein described in embodiment 1, comprising one or more amino acid substitutions that alter the pore electrostatic potential of the parent ChRmine protein. Embodiment 4. A high-speed variant ChRmine protein described in embodiment 3, wherein one or more amino acid substitutions that change the pore electrostatic potential of the parent ChRmine protein are selected from histidine at position 33 or a corresponding position, aspartic acid at position 92 or a corresponding position, glutamic acid at position 154 or a corresponding position, glutamic acid at position 158 or a corresponding position, aspartic acid at position 242 or a corresponding position, and glutamic acid at position 246 or a corresponding position. Embodiment 5. A high-rate variant ChRmine protein described in embodiment 4, in which the 33rd histidine or corresponding position is substituted with histidine if the corresponding amino acid is not histidine, arginine, or lysine. Embodiment 6. A high-speed variant of the ChRmine protein described in embodiment 4, in which the 92nd aspartic acid or corresponding position, the 154th glutamic acid or corresponding position, the 158th glutamic acid or corresponding position, the 242nd aspartic acid or corresponding position, and the 246th glutamic acid or corresponding position are each independently substituted with aspartic acid, glutamic acid, asparagine, or glutamine. Embodiment 7. A fast variant ChRmine protein of any one of embodiments 1 to 6, wherein the parent ChRmine protein has an array selected from SEQ ID NOs: 1 to 29, or an array having at least 80% sequence identity to an array selected from SEQ ID NOs: 1 to 29. Embodiment 8. A high-speed variant ChRmine protein described in any one of embodiments 1 to 7, having a substitution at a histidine residue at position 33 or the corresponding residue in the first transmembrane domain of the parent ChRmine protein compared to the parent ChRmine protein. Embodiment 9. A high-rate variant ChRmine protein described in embodiment 8, having an arginine substitution at the histidine residue at position 33 or the corresponding residue in the first transmembrane domain of the parent ChRmine protein. Embodiment 10. A nucleic acid sequence having the sequence of SEQ ID NO: 30 or a sequence having at least 80% sequence identity to SEQ ID NO: 30, Differences in sequences having at least 80% sequence identity to SEQ ID NO: 30 exclude amino acid substitutions used to produce high-rate variant ChRmine proteins; A high-speed variant ChRmine protein described in any one of embodiments 1 to 9. Embodiment 11. A high-speed variant ChRmine protein described in any one of embodiments 1 to 10, having the sequence of SEQ ID NO: 30. Embodiment 12. A red-shifted variant ChRmine protein having a red-shifted spectrum compared to the parent ChRmine protein, the red-shifted variant ChRmine protein having one or more amino acid substitutions compared to the parent ChRmine protein. Embodiment 13. A red-shifted variant ChRmine protein as described in embodiment 12, comprising one or more amino acid substitutions in the retinal binding pocket (RBP) of the parent ChRmine protein. Embodiment 14. A red-shifted variant ChRmine protein as described in embodiment 13, wherein the one or more amino acid substitutions in the RBP of the parent ChRmine protein include substitutions at one or more of isoleucine at position 146 or a corresponding position, glycine at position 174 or a corresponding position, and phenylalanine at position 178 or a corresponding position. Embodiment 15. A red-shifted variant ChRmine protein as described in embodiment 13, wherein the substitution at isoleucine at position 146 or the corresponding position is with serine, cysteine, threonine, or methionine; the substitution at glycine at position 174 or the corresponding position is with serine, cysteine, threonine, or methionine; or, if the corresponding amino acid is not phenylalanine, tyrosine, or tryptophan, the substitution at phenylalanine at position 178 or the corresponding position is with phenylalanine. Embodiment 16. A red-shifted variant ChRmine protein described in any one of embodiments 12 to 15, wherein the parent ChRmine protein has an array selected from SEQ ID NOs: 1 to 29 or an array having at least 80% sequence identity to an array selected from SEQ ID NOs: 1 to 29. Embodiment 17. A red-shifted variant ChRmine protein described in any one of embodiments 12 to 16, which has, compared to the parent ChRmine protein, one or both of: (i) a substitution at an isoleucine residue at position 146 or a corresponding residue in the fourth transmembrane domain of the parent ChRmine protein, and (ii) a substitution at a glycine residue at position 174 or a corresponding residue in the fifth transmembrane domain of the parent ChRmine protein. Embodiment 18. A red-shifted variant ChRmine protein described in embodiment 17, which has, compared to the parent ChRmine protein, one or both of: (i) a methionine substitution at the isoleucine residue at position 146 or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein, and (ii) a serine substitution at the glycine residue at position 174 or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein. Embodiment 19. A nucleic acid sequence having the sequence of SEQ ID NO: 31 or a sequence having at least 80% sequence identity to SEQ ID NO: 31, Differences in sequences having at least 80% sequence identity to SEQ ID NO: 31 exclude amino acid substitutions used to produce red-shifted variant ChRmine proteins; A red-shifted variant ChRmine protein described in any one of embodiments 12 to 18. Embodiment 20. A red-shifted variant ChRmine protein described in any one of embodiments 12 to 18, having the sequence of SEQ ID NO: 31. Embodiment 21. A fast and red-shifted variant ChRmine protein having faster kinetics and a red-shifted spectrum compared to the parent ChRmine protein, the fast and red-shifted variant ChRmine protein having one or more amino acid substitutions compared to the parent ChRmine protein. Embodiment 22. A fast and red-shifted variant ChRmine protein described in embodiment 21, comprising (i) one or more amino acid substitutions in the counterion of the Schiff base of the parent ChRmine protein or one or more amino acid substitutions that change the pore electrostatic potential of the parent ChRmine protein, and (ii) one or more amino acid substitutions in the retinal binding pocket (RBP) of the parent ChRmine protein. Embodiment 23. A fast and red-shifted variant ChRmine protein described in embodiment 21 or 22, comprising (i) one or more amino acid substitutions that alter the pore electrostatic potential of the parent ChRmine protein, and (ii) one or more amino acid substitutions in the retinal binding pocket (RBP) of the parent ChRmine protein. Embodiment 24. A fast and red-shifted variant ChRmine protein as described in embodiment 23, wherein the one or more amino acid substitutions that change the pore electrostatic potential are selected from histidine at position 33 or a corresponding position, aspartic acid at position 92 or a corresponding position, glutamic acid at position 154 or a corresponding position, glutamic acid at position 158 or a corresponding position, aspartic acid at position 242 or a corresponding position, and glutamic acid at position 246 or a corresponding position. Embodiment 25. A fast and red-shifted variant ChRmine protein as described in embodiment 24, wherein the 33rd histidine or corresponding position is substituted with histidine if the corresponding amino acid is not histidine, arginine, or lysine. Embodiment 26. A fast and red-shifted variant ChRmine protein as described in embodiment 24, wherein each of aspartic acid or a corresponding position 92, glutamic acid or a corresponding position 154, glutamic acid or a corresponding position 158, aspartic acid or a corresponding position 242, and glutamic acid or a corresponding position 246 is independently substituted with aspartic acid, glutamic acid, asparagine, or glutamine. Embodiment 27. A fast and red-shifted variant ChRmine protein described in any one of embodiments 22 to 26, wherein the one or more amino acid substitutions in the RBP of the parent ChRmine protein include substitutions at one or more of isoleucine at position 146 or a corresponding position, glycine at position 174 or a corresponding position, and phenylalanine at position 178 or a corresponding position. Embodiment 28. A fast and red-shifted variant ChRmine protein as described in embodiment 27, wherein the substitution at isoleucine at position 146 or the corresponding position is with serine, cysteine, threonine, or methionine; the substitution at glycine at position 174 or the corresponding position is with serine, cysteine, threonine, or methionine; or, if the corresponding amino acid is not phenylalanine, tyrosine, or tryptophan, the substitution at phenylalanine at position 178 or the corresponding position is with phenylalanine. Embodiment 29. A fast and red-shifted variant ChRmine protein described in any one of embodiments 21 to 28, wherein the parent ChRmine protein has an array selected from SEQ ID NOs: 1 to 29 or an array having at least 80% sequence identity to an array selected from SEQ ID NOs: 1 to 29. Embodiment 30. A fast and red-shifted variant ChRmine protein described in any one of embodiments 21 to 29, having, compared to the parent ChRmine protein, one or more of the following: (i) a substitution at a histidine residue at position 33 or the corresponding residue in the first transmembrane domain of the parent ChRmine protein, (ii) a substitution at an isoleucine residue at position 146 or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein, and (iii) a substitution at a glycine residue at position 174 or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein. Embodiment 31. A fast and red-shifted variant ChRmine protein described in embodiment 30, having, compared to the parent ChRmine protein, one or more of the following: (i) an arginine substitution at the histidine residue at position 33 or the corresponding residue in the first transmembrane domain of the parent ChRmine protein, (ii) a methionine substitution at the isoleucine residue at position 146 or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein, and (iii) a serine substitution at the glycine residue at position 174 or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein. Embodiment 32. A nucleic acid sequence having a sequence of SEQ ID NO: 32 or a sequence having at least 80% sequence identity to SEQ ID NO: 32, Differences in sequences having at least 80% sequence identity to SEQ ID NO: 32 exclude amino acid substitutions used to produce fast and red-shifted variant ChRmine proteins; A fast and red-shifted variant ChRmine protein according to any one of embodiments 21 to 31. Embodiment 33. A fast and red-shifted variant ChRmine protein described in any one of embodiments 21 to 31, having the sequence of SEQ ID NO: 32. Embodiment 34 A nucleic acid encoding a variant ChRmine protein of any one of the preceding embodiments. Embodiment 35. A genetically modified cell comprising the nucleic acid of embodiment 34. 36. Genetically modifying a subject to express in brain cells of the subject a variant ChRmine protein according to any one of embodiments 1 to 33; applying a stimulating light to the subject's brain; Imaging the subject's brain An optogenetic method comprising: Embodiment 37 The optogenetic method of embodiment 36, wherein the subject is a mammal. Embodiment 38. The optogenetic method of embodiment 37, wherein the mammal is a rodent, primate, bovine, porcine, feline, or canine. Embodiment 39. A method comprising genetically modifying a subject to express a variant ChRmine protein described in any one of embodiments 1 to 33 in cells and / or organs. Embodiment 40 The method of embodiment 39, further comprising applying stimulating light to the modified cells and / or organs and imaging the cells and / or organs of the subject. Embodiment 41. The method of embodiment 40, wherein the cells and / or organs may belong to the cardiovascular system, the digestive system, the urinary system, the respiratory system, the reproductive system, the musculoskeletal system, or the pancreatic / endocrine system. EXAMPLES

[0119] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for.

[0120] Example 1 - Determination of the crystal structure of rhodopsin protein Light, an important source of energy and environmental signal, is typically captured by motile organisms using rhodopsins, which are mainly classified into two groups, microbial and animal, both of which consist of a seven-transmembrane (7TM) protein (opsin) and a covalently bound chromophore (retinal). Light absorption induces the isomerization of retinal, which then induces a series of photochemical reactions, the photocycle (Zhang et al., 2011; Ernst et al., 2014; Deisseroth and Hegemann, 2017), which ultimately exerts direct biochemical effects in microbial rhodopsins (examples include pumps, channels, sensors, and enzymes (Kandori, 2020; Kato, 2021)). Targeted expression of these proteins (especially channel and pump types) in specific cell types, when applied with precise light delivery, enables the causal study of cellular activity (optogenetics) in behaving organisms (Deisseroth, 2015; Kurihara and Sudo, 2015; Deisseroth, 2021).

[0121] In optogenetics, cation-permeable channelrhodopsins (cation ChRs or CCRs) are typically used to activate target cells (Deisseroth and Hegemann, 2017). The first description of a CCR (CrChR1 from the green alga C. reinhardtii, Nagel et al., 2002) was followed by the characterization of discovered or engineered variants with novel functions spanning ion selectivity, photocurrent amplitude, absorption, sensitivity, and kinetics (Deisseroth and Hegemann, 2017). Natural CCRs include CrChR2 (ChR2 from C. reinhardtii) (Nagel et al., 2003), VChR1 (ChR1 from V. carteri) (Zhang et al., 2008), and Chrimson (from C. noctigama) (Klapoetke et al., 2014), which were initially described primarily from green algae, but the identification of ChRs from other species has further expanded the toolkit. In 2016-17, a subfamily of microbial rhodopsins was reported from the geophyte G. theta (Govorunova et al., 2016; Yamauchi et al., 2017) and identified as CCRs, but more homologous to archaeal ion pumps such as H. salinarum bacteriorhodopsin (HsBR). Furthermore, unlike the green algae CCR, the geophyte CCR does not accept outward protons (H +) share three amino acids on TM3 essential for pumping [DTD motif (Inoue et al., 2013); D85, T89, and D96 in HsBR] and have been termed bacteriorhodopsin-like cation ChRs or BCCRs (Sineshchekov et al., 2017) (Figures 8A and 8B). ChRmine, a member of this subfamily discovered through structural information mining (Marshel et al., 2019), exhibits extremely high current and light sensitivity as well as a remarkable red-shifted spectrum; these properties have enabled all-optical interrogation of hundreds of individually addressed single neurons (Marshel et al., 2019) and fully noninvasive high-speed control of deep brain circuits (Chen et al., 2021).

[0122] Experimental Method and Results The high-resolution structure of this protein family will help understand the structure-function relationship between pump- and channel-type rhodopsins and design the next generation of optogenetic tools. This has previously led to the generation of the first anion-permeable ChR (ACR, Berndt et al., 2014, 2016; Kato et al., 2012; Wietek et al., 2014). To that end, the present disclosure provides a cryo-electron microscopy (cryo-EM) structure of ChRmine at 2.0 Å resolution. The structural information was also used to create variants with faster kinetics and larger red shifts while maintaining high current and light sensitivity. These variant channelrhodopsins disclosed herein can be used for optical neuroscience studies and targeted functional analysis in diverse systems.

[0123] Structure determination Our initial efforts to crystallize ChRmine resulted in low-resolution crystals, and therefore we turned to single-particle cryo-EM (Figures 9A-9R). A fundamental limitation of single-particle cryo-EM is that images of small membrane proteins in detergent micelles do not have sufficient features for image alignment in data processing. Indeed, due to the compactness of ChRmine (~35 kDa without extracellular or intracellular domains), particles from the initial cryo-EM data set were not well aligned to yield a 3D reconstruction (Figures 9A-C, 9G). To provide defined features for image alignment, we generated a conformation-specific antibody against ChRmine, Fab02 (STAR ​​Methods, Figures 10A, 10B). We spectroscopically analyzed the ChRmine-Fab02 complex along with ChRmine to confirm that Fab02 binding does not affect the photocycle, and both ChRmine and ChRmine-Fab02 complexes show similar K, L1, L2, and L3 binding properties with similar lifetimes. 2、 The M1 and M2 intermediates are shown (Fig. 10C-10G, 10L). Using Fab02, the structure of the ChRmine-Fab02 complex in the dark was determined at an overall resolution of 2.0 Å (Fig. 9D-9F, 9H-9J, 10H, 10I, Table 2). The density was of excellent quality, allowing accurate modeling of ChRmine continuously from residues 10-279, except for the disordered N-terminal 9 residues and C-terminal 25 residues (Fig. 9K-9R, STAR method), and clearly resolving several lipids, water molecules, and retinal (using a specific all-trans retinal conformer confirmed by HPLC; Fig. 9K-9N, 10J, and 10K). The signals of the putative hydrogen atoms were calculated using the difference (F o -F c ) maps (Figures 9O, 9P) (Yamashita et al., 2021) - something that had not been achieved for rhodopsin before but is now made possible by this high resolution.

[0124] Table 2. Cryo-EM data collection and refinement statistics related to the STAR method. TIFF2025508610000003.tif172128

[0125] Overall structure and comparison of HsBR and C1C2 Cryo-EM density maps revealed that the quaternary structure of ChRmine is significantly different from that of other structurally resolved ChRs (Kato et al., 2012) (Figures 1A-1B). Instead of the classical ChR dimer, ChRmine forms a trimer, in which TM2 interacts with TM4 of the adjacent protomer, as observed in archaeal ion pump rhodopsins, including HsBR (Pebay-Peyroula et al., 1997) and HsHR (Kolbe et al., 2000) (Figure 1C). To confirm this result under more physiological conditions, we reconstituted ChRmine in lipid bilayers and performed high-speed atomic force microscopy (HS-AFM), which similarly revealed a trimeric structure (Figure 1D).

[0126] The ChRmine monomer consists of an extracellular N-terminal domain (residues 10–26), an intracellular C-terminal domain (residues 271–279), and seven TM domains (residues 27–270) connected by three intracellular loops (ICL1–3) and three extracellular loops (ECL1–3) (Figure 1E). TM1–7 adopt a typical rhodopsin-like topology with retinal covalently bound at K257 on TM7, whereas TM3 deviates significantly from the classical framework, exhibiting an unwound configuration in the middle of the transmembrane region, resulting in a C-shaped structure stabilized by a long twisted ECL1 (residues 95–115) and an extensive H-bond network (Figure 9N).

[0127] To investigate how ChRmine might structurally resemble ion pump rhodopsin and function as a channel, we compared ChRmine to the archaeal ion pump rhodopsin (HsBR) and the green algal CCR (C1C2, a chimera derived from CrChR1 and CrChR2). Consistent with the sequence similarity (Figures ​(Figures8A-8B),8), ChRmine could be better superimposed onto HsBR, with root mean square deviation (rmsd) values ​​for ChRmine versus HsBR and C1C2 measured to be 1.83 Å and 2.14 Å, respectively (Figures1F-1G). Although previous structural studies have revealed that TM1, 2, 3, and 7 of CCR form the ion permeation pathway within each monomer and that the positioning of TM1 / 2 structurally distinguishes ChR from pump-type rhodopsins (Kato et al., 2012), TM1 of ChRmine is positioned more similarly to TM1 of HsBR, shifted by 1.5 Å in its entirety in ChRmine compared to C1C2 (Figure 1G). The overall positioning (and central region) of TM2 is also similar between ChRmine and HsBR, except that both the intracellular and extracellular regions of TM2 are tilted outward in ChRmine (Figure 1F), and these features in TM2 may enlarge the cavity within the monomer and enable ChRmine to function as a CCR.

[0128] Schiff base region In all microbial rhodopsins, retinal is covalently bound to a lysine in TM7 to form a protonated Schiff base, whose positive charge is stabilized by one or two carboxylates on the extracellular side (Figure 11A). After photon absorption, a proton is transferred to the carboxylate (a key step in the operation of most ion-transporting rhodopsins); the carboxylate(s) that stabilize the positive charge and accept the proton (forming the M intermediate) have historically been called the counterion(s) of the Schiff base and the proton acceptor, respectively (Zhang et al., 2011). To gain structural insight into the ChRmine channel gating mechanism and kinetics, we next focused on the counterion and proton acceptor.

[0129] Although the primary sequence, oligomerization number, and overall monomer structure of ChRmine are similar to those of HsBR, the Schiff base region of ChRmine is significantly different from those of both types of rhodopsin (HsBR and C1C2, Fig. 2A) (Figs. 8A, 8B, 1E-G). In HsBR, the protonated Schiff base nitrogen forms a hydrogen (H) bond with a water molecule between the counterions D85 and D212. D212 is fixed by H-bonds with Y57 and Y185 on TM2 and TM6, respectively, while D85, which functions as a proton acceptor from the Schiff base in the M intermediate (Braiman et al., 1988; Gerwert et al., 1990), interacts with R82 through a water molecule (Fig. 2A). In C1C2, D85, Y57, and Y185 of HsBR are replaced by E162, F133, and F265, respectively, and the nitrogen of the Schiff base H-bonds with D292 and no longer interacts with F133 and F265 (Figure 2A). D292 functions as a proton acceptor in the M intermediate (Kato et al., 2012; Lorenz-Fonfria et al., 2013), and E162 is not essential for channel function (Gunaydin et al., 2010; Kato et al., 2012). In contrast, residues corresponding to Y57, R82, D85, and D212 in HsBR are conserved in ChRmine (Y85, R112, D115, D253), but R112 and D115 are moved farther away from the Schiff base upon unwinding of TM3 (Figure 2A); the distances from the Schiff base to R112 and D115 are 13.9 Å and 6.9 Å, respectively, and such long distances have never been observed in microbial rhodopsin structures (Figures S4B-S4D).

[0130] Three water molecules (w1, w2, w3) occupy the space between the Schiff base and D115 created by the unwinding of TM3. In particular, w2 and w3 are well stacked on the carboxyl oxygen of D85 in HsBR, suggesting that these waters structurally mimic D85 (Figure 11B) and participate in counterion complexes with D115 and D253. In addition to these structural changes in the TM3 region, the substitutions of Y185 (in HsBR) to F226 and W86 (in HsBR) to Y116 also rearrange the structure in the TM7 region of ChRmine, with D253 switching H-bonds from F226 to Y116, and unlike HsBR, D253 in ChRmine is anchored by two tyrosines (Y85 and Y116) on TM2 and 3 (Figure 2A).

[0131] To investigate the function of the counterion and proton acceptor candidates D115 and D253, we measured the photocurrent amplitudes of wild-type (WT), D115N, and D253N ChRmine in HEK293 cells, and both D115N and D253N abolished the photocurrent (Figures 2B, 12A). Spectroscopy revealed that these mutants exhibited significantly blue-shifted absorption spectra (lambda at pH 7.5) consistent with loss of function resulting from baseline deprotonation of the Schiff base. max We found that the λ shifts from 520 nm (WT) to 385 nm (D115N) and 363 nm (D253N) (Figure 2C). This is the expected pattern if both D115 and D253 act as counterions to the Schiff base, which must be deprotonated at baseline and physiological pH to stabilize the positive charge of the protonated Schiff base. This idea was supported by the λ shifts, possibly due to the protonation of D115 and D253. max This is also supported by pH titration of WT ChRmine, where it is shifted by decreasing pH (Figures 11E-11F).

[0132] Next, to identify which carboxylate acts as the primary proton acceptor in the M intermediate, we performed flash photolysis of D115N and D253N (Figure 2D). Since rhodopsin with a deprotonated Schiff base cannot respond to light, the photocycle was measured under acidic conditions (pH 4.0) to reprotonate the Schiff base (Figure 2C). Spectroscopy revealed that both mutants have similar photointermediates compared to the WT, but only D115N shows an additional accumulation of a K-like intermediate (long-lived, up to 1 s), accompanied by a lower accumulation of M intermediates (Figure 2D). Indeed, the M2 intermediate decay is delayed by D115N (τ M2 = 190 ± 40 ms) for WT(τ M2 = 1.09 ± 0.06 s) (Figure 10L), which was consistent with D115 acting as the primary proton acceptor. Although D253 is closer to the Schiff base than D115, D253 strongly interacts with Y85 and Y116, making it difficult for D253 to accept a proton from the Schiff base. D212 of HsBR interacts similarly with two tyrosine residues (Y57 and Y185) and does not function as a proton acceptor. D115 is further away from the Schiff base, but several waters are located between them, and water rearrangement is thought to allow a proton to move from the Schiff base to D115 in the M intermediate.

[0133] Ion-permeable pores in the monomer To investigate the location and shape of the ion permeation pathway, we first analyzed the composition of the pore within the monomer. ChRmine exhibits significantly larger intra- and extracellular pores compared to C1C2 and HsBR (Figure 3A). Similar to C1C2, both pores are mainly formed by TM1, 2, 3, and 7 and are occluded by the intracellular and central constriction sites (ICS and CCS), but several important differences in the pore pathways of ChRmine and C1C2 were noted. First, although the electrostatic surface potentials calculated for both ChRmine and C1C2 revealed electronegative pores (Figure 3B-3C), the distribution of negatively charged residues was found to be significantly different. In C1C2 and several other chlorophyte CCRs, five conserved glutamates (E121, E122, E129, E136, and E140 in C1C2) cooperatively create an electronegative surface potential along the pore, but four of these five residues are replaced with neutral or basic residues in ChRmine (Figures ​(Figures8A,3A,3B–3D). Instead, ChRmine displays a set of distinct carboxylates, including E50, E70, D100, D126, E154, E158, D242, E246, and D272, to create a cavity suitable for anion exclusion and cation selectivity (Berndt and Deisseroth, 2015; Berndt et al., 2014, 2016) (Figure3D, top left).

[0134] Second, ChRmine exhibits two intracellular vestibules (IVs) with different electrostatic potentials (Figures 3A-3B). In particular, the location of IV1 of ChRmine is more similar to the IV of CCR C1C2, and the location of IV2 of ChRmine is more similar to the IV of ACR GtACR1 (Kato et al., 2012; Kim et al., 2018) (Figures 3A-3D), in agreement with the fact that ChRmine is phylogenetically closer to GtACR1 than to the chlorophyte CCRs that contain C1C2 (Figure 8B). The corresponding electrostatic surface potentials favor the role of IV2 as a cation-permeable pore in the open state (IV1 and IV2 could be further connected to create a larger intracellular cavity in the open state, as for the extracellular vestibules in other ChRs (Kato et al., 2018; Takemoto et al., 2015)).

[0135] Third, the ICS architectures of ChRmine and C1C2 are different. In C1C2, the ICS is mainly formed by Y109, E122, and H173 (E122 and H173 are H-bonded to each other). In ChRmine, the corresponding residues are L47, A74, and D126, which are involved in the formation of the ICS, respectively, but D126 forms a more extensive H-bond network with Q71, Q130, Y260, and water (Figure 3E). Although the mutation of Y260 does not impair the channel activity, the D126 mutant showed a significantly reduced photocurrent, suggesting that the effect of the loss of a single H-bond in the ICS is minimal, whereas even a small change to D126 as an H-bond network hub can greatly affect the channel activity (Figure 3F, 12).

[0136] Fourth, the size and pathway of the extracellular cavity are significantly different between ChRmine and C1C2. C1C2 has two extracellular vestibules (EV1 and EV2), whereas ChRmine lacks a vestibule corresponding to EV1, and at the same time, the volume of the single EV of ChRmine is significantly enlarged (mostly due to the unwinding of TM3, Fig. 3A). In addition, the EV2 of C1C2 is well away from the Schiff base and terminates in a CCS formed by S102, E129, and N297; in contrast, the EV of ChRmine extends significantly into the Schiff base region (Fig. 3E), and the three residues of ChRmine (L40, A81, S258) that correspond to the CCS of C1C2 do not form a constriction. Instead, an extensive H-bond network formed by counterion complexes (including D115, D253, Y85, Y116, T119, and structured water molecules) occludes the pore and defines the ChRmine CCS. The importance of this H-bond network is supported by the loss-of-function electrophysiological properties of Y85F, Y116F, and T119V mutant photocurrents (Figure 3F).

[0137] ChRmine is similar to HsBR in several respects (primary sequence, overall arrangement of secondary structural elements in the monomer, and quaternary structure of the trimer; Figures 1A-1G and 8A), but the size and shape of the cavity within the monomer clearly show a higher similarity to the C1C2 cavity (Figure 3A), consistent with the cation channel functionality of ChRmine. Next, we sought to understand which structural elements contribute to the formation of these large cavities that constitute the majority of the channel pore in ChRmine by comparing ChRmine and HsBR in more detail. At least two prominent features contribute to this formation of the pore structure. First, as mentioned above, both ends of TM2 are tilted outward in ChRmine, and the cytoplasmic end of TM2, in particular, is tilted by about 50 degrees, which significantly enlarges the intracellular cavity (Figures 1F and 3G). In addition, numerous hydrophilic residues (including S54, E70, Q71, D126, Q130, R268, and D272) face the pore interior, which, together with the structural water, create a favorable environment for water and ion permeation. In contrast, in HsBR, TM2 remains straight all the way to the end, and six of the seven hydrophilic residues mentioned above are replaced by tightly packed hydrophobic residues with no water-accessible voids (Figures 1F, 3A right, and 3H).

[0138] In the second major feature that enables the channel, the unwinding of TM3 and the resulting long ECL1 contribute to creating a large extracellular cavity in ChRmine. The helical structure of the extracellular TM3 is unfolded starting from Y116, and the C-shaped structure of ECL1 protrudes into the center of the trimer interface. This is in contrast to the ECL1 of HsBR, which is in a position to form a β-sheet and half-occlude the extracellular pore (Fig. 3I, left). In addition to the overall position of ECL1, R82 on TM3 and Y79 on ECL1 protrude into and occlude the extracellular cavity of HsBR (Fig. 3I, right). However, Y79 is replaced by G109 in ChRmine, and because TM3 is unfolded, R112 (R82 in HsBR) and G109 are displaced by 4.0 Å and 6.0 Å, respectively, from the corresponding residues in HsBR. As a result, these residues do not block the cavity within ChRmine (Figure 3I, right).

[0139] Notably, ECL1 in C1C2 also forms a β-sheet structure like HsBR, moderately narrowing the pore entrance, which is one of the reasons why the extracellular cavity of C1C2 is smaller than that of ChRmine (Fig. 3I, left). Furthermore, in C1C2, the residues corresponding to R82 and Y79 in HsBR are similarly arranged, but Y79 is replaced by V156, and R159 (R82 in HsBR) adopts a conformation like R112 in ChRmine, pointing toward the extracellular solvent rather than parallel to the membrane (Fig. 3I, right). The outward-facing Arg conformation observed in ChRmine and C1C2 is conserved in other channel-type rhodopsins, including CrChR2, C1Chrimson, and GtACR1, and the parallel Arg conformation observed in HsBR is similar to that observed in halorhodopsin (inward-facing Cl - Pump type), KR2 (outward Na + pump type), and schizorhodopsin (inward H +The parallel conformation of Arg narrows or blocks the extracellular space of the ion transport pathway. This conformation would therefore contribute to preventing large ion fluxes in ion pump rhodopsins. In particular, the outward H from CsR (C. subellipsoidea) is conserved in other pump rhodopsins such as ArgR (C. subellipsoidea) (Figure 11D; STAR methods). + Pump rhodopsin also has a parallel conformation of Arg (R83) in the dark state (Fudim et al., 2019), and mutation of R83Q or the adjacent Tyr (Y57K) disrupts the function of the protein. + H from the pump + channel (Vogt et al., 2015). Furthermore, computational analysis of HsBR with R82Q or Y57K mutations reveals that these mutations significantly alter the conformation of R82Q or R82, respectively, and most notably, R82 faces outward in the Y57K simulations (Vogt et al., 2015). These results suggest that the outward-facing Arg conformation in the dark state, as well as the overall size of the monomer cavity, are key structural elements that define the function of ion-transporting rhodopsin. Interestingly, previous studies have reported that the Arg of some ion pump rhodopsins is maintained in a parallel conformation during the photoperiod (Kouyama et al., 2015; Kovalev et al., 2020), while the corresponding arginine in HsBR transiently changes from parallel to outward to facilitate proton release into the extracellular solvent (Kuehlbrandt, 2000; Nango et al., 2016). Because ChRs likely evolved from ion pump rhodopsins (Inoue et al., 2015), these studies suggest that mutations accumulating near the arginines in ion pump rhodopsins gradually stabilized the outward conformation, and these rearrangements enlarged the extracellular cavity, allowing for a large ion flux of ChRs.

[0140] Functional importance of trimer assembly Like HsBR, ChRmine forms a trimer, where we find that ChRmine has an unexpected additional opening at the trimer interface (Figure 4A, left). Whereas the corresponding region in HsBR is hydrophobic and filled with several lipid molecules, in the case of ChRmine, this region is relatively hydrophilic and negatively charged (Figure 4A, right and 4B). The cup-shaped opening is formed by TM2-4 and ECL1 of the protomers, and the narrowest region is created by ECL1, where the main chain carbonyl oxygens of F104 and I106 face the center of the trimer, forming a central constriction (Figure 4C and 4D).

[0141] ChRmine displays three intermolecular H-bond interactions between adjacent protomers: S138 with E69, the backbone amide of R136 with E69, and Y156 with H96 (Figures 4E and 4F). To analyze the functional importance of trimeric assembly, we introduced mutations into each of these residues to destabilize the trimer. We used fluorescence-detected size-exclusion chromatography (FSEC) (Kawate and Gouaux, 2006) to assess oligomerization and found that the S138W or Y156F mutation shifted the equilibrium in favor of the monomeric state, while the S138W / Y156F double mutation almost completely dissociated the trimer to the monomeric state (Figure 4G). Subsequent electrophysiology revealed that these single and double mutations moderately and severely reduced channel activity, respectively (Figures 4H and 12), and the S138W mutation further reduced cation selectivity (Figure 4I). Because S138 and Y156 are clearly located away from the canonical ion permeation pathway in the monomer and the R136H mutation (which does not affect interprotomer interactions) does not alter channel activity or cation selectivity, these results support the importance of trimer assembly for cation permeation and provide the first evidence of the importance of oligomerization in ChR channel function.

[0142] Computational analysis of pore dynamics To further test this hypothesis, we performed all-atom molecular dynamics simulations of ChRmine in either the dark state or the M intermediate (light state) (Figures 5A-5F). In these simulations of the dark state, the retinal is left in the all-trans configuration and the Schiff base is protonated; in the light state simulations, the retinal is isomerized to the 13 cis configuration and the Schiff base proton is transferred to the putative proton acceptor D115. Although the simulations were not long enough to span the full activation of the channel, we observed significant early conformational changes along the pathway toward activation in the light state simulations, where the trimeric pore alternates between a wider "open" state and a narrower "closed" state. In contrast, in the dark state simulations, the trimeric pore remained in the closed state. The trimer pore radius (the radius of the constriction site formed by backbone interactions between the three F104 residues on each of the monomers) was significantly increased in the light-state simulations compared to the dark-state simulations (Figures 5A and 5B), which was sufficient to allow multiple water molecules to pass through the pore (Figures 5C and 5D). Although the pore did not yet achieve a radius sufficient for ion transmission over the timescale of our simulations, these results suggest that the trimer pore is cooperatively coupled to retinal isomerization and (consistent with the observed ion selectivity changes resulting from mutations in the trimer pore (Figure 4K)) support the idea that the trimer pore may function as a novel second channel via a structural mechanism that is not available to either dimeric green algal channelrhodopsins or trimeric pump rhodopsins (Note S2).

[0143] Example 2 - Structure-guided engineering of ChRmine variants with novel properties Next, we attempted to improve the speed and spectral response of ChRmine for all-optical experiments (Figures 6A-6B). We started with a speed where possible of 2-3x acceleration of ChRmine kinetics (Marshel et al., 2019), noting that this would approach the time constant of principal cells in the brain. One strategy to accelerate closing kinetics is via mutation of the Schiff base to a counterion (Gunaydin et al., 2010; Rajasethupathy et al., 2015), but counterion mutations in ChRmine impair channel function (Figure 2B).

[0144] In another series of studies, we found that other mutations predicted to alter the pore electrostatic potential may also affect the rate (Kato et al., 2018; Kim et al., 2018). However, we did not find that these mutations along the ion permeation pathway of the dimeric ChRs altered the rate of ion permeation in structurally distinct ChRmines with unique properties [e.g., we found that ChRmines have high monovalent cation selectivity (Ca 2+ and Mg 2+ Eliminate Na + K than + We found that the pore exhibits a striking preference for τ (Fig. 13A), which is an extraordinary property of this pump-like ChR. Nevertheless, to test this alternative pathway, we introduced six mutations (Fig. 6A; H33R, D92N, E154Q, E158Q, D242N, E246Q) into the pore and tested the kinetics. Of these, three (H33R, D92N, and E154Q) showed strong effects, most notably the τ of H33R. オフ and τ オン were more than two-fold faster (30 ms and 10 ms, respectively) than their wild-type (WT) counterparts (70 ms and 16 ms, respectively; Figure 6C ), further validating this structure-informed design approach, previously successful for CCRs and ACRs ( Deisseroth and Hegemann, 2017 ), here for pump-like ChRs.

[0145] Next, we modified the spectral properties, particularly the red light activation, to produce a genetically encoded Ca2+ gene that is activated by blue light. 2+ We attempted to improve the fit of ChRmine RBP to the retinal binding pocket (GECI). Previous studies have shown that mutations in the retinal binding pocket (RBP) can change the spectral properties, including the peak and shape of the action spectrum (Kato et al., 2015a; Oda et al., 2018; Pan et al., 2014), but the sequence identity between ChRmine and structurally solved CCRs is low (~20%) (Marshel et al., 2019), which precluded effective homology modeling of ChRmine RBP before the structure was solved (Figure 6B). Because the tight packing of RBP residues relative to the retinal polyene chain and polar interactions with the retinal β-ionone ring have been reported to contribute to the red-shifted spectra (Kamiya et al., 2013; Oda et al., 2018; Prigge et al., 2012), we designed seven constructs with RBP mutations (I146M, G174S, F178Y, I146M / G174S, I146M / F178Y, G174S / F178Y, and I146M / G174S / F178Y) for testing in cultured neurons (Figures S13B-S13D). Under one-photon (1P) irradiation, the double mutant I146M / G174S showed a surprisingly large reduction and red shift in the blue shoulder (Figures ​(Figures6D-6G) (a 2.6-fold decrease in cyan light sensitivity and a 1.3-fold increase in red light sensitivity compared to WT ChRmine; Figures S3E-6F).

[0146] We designated the faster or accelerated kinetics variant (H33R) as hsChRmine (for fast) and the optimal redshifted variant (I146M / G174S) as rsChRmine (for redshift) (Figures 6F and 14). Both variants showed robust expression (Figure 14A), whereas many strategies to accelerate channel kinetics and shift the action spectrum reduce photocurrents (Gunaydin et al., 2010, Kato et al., 2018; Mager et al., 2018, Oda et al., 2018), and both ChRmine variants showed peak photocurrent amplitudes similar to those of the WT (Figures 6F and 14F). Consistent with their kinetics (Figure 14B), we observed that hsChRmine enabled high-fidelity spiking up to 40 Hz (Figures 14C-14D, STAR methodology). Combining all three mutations (H33R / I146M / G174S) resulted in a further accelerated opening (FIG. S4B) and a larger redshift (FIGS. S4E-F), albeit with a reduced photocurrent (still >500 pA, FIG. S4F); we call this variant frChRmine (combining high speed with further redshift capability).

[0147] Finally, we compared the two-photon (2P) spectra of ChRmine variants with ChroME2 (Sridharan et al., 2021). We found that the spectral shift of rsChRmine was even larger with 2P, and at 825 nm, faint rsChRmine currents were detected, whereas WT ChRmine at 825 nm showed 40% of the maximum photocurrent evoked at 1050 nm. Furthermore, the 2P spectrum of ChroME2f / 2s was found to be blue-shifted compared to ChRmine, similar to 1P stimulation (Figures 6G-6H, 14E-14H). Together, these ChRmine variants offer significant practical advantages compared to opsins, which are otherwise good candidates for all-optical interrogation of intact neural circuits.

[0148] Minimal crosstalk for all-optical experiments with rsChRmine The blue shoulder persists in all published action spectra of ChRs, thus raising the prospect that the unique spectral properties of rsChRmine will minimize optical crosstalk, which is problematic for all-optical neuroscience. We characterized the spike fidelity of rsChRmine and WT ChRmine as a function of pulse width and irradiance in brain slices (Figure 6I) and cultured neurons (Figures S13G-13H); in both settings, rsChRmine exhibited the desired properties. Orange light (580 nm) stimulation of rsChRmine reliably evoked action potentials in brain slices, whereas blue light (440 nm) did not (pulse width 1 ms, irradiance 0.3 mW / mm). 2 , Figures 6J-6K). In contrast, spike fidelity of WT ChRmine under orange and blue light was similar at all pulse widths and irradiance levels. Thus, the reduction in the blue shoulder of rsChRmine minimized optical crosstalk, which is assessed in a critical readout of neural spikes.

[0149] To test the potential utility of all-optical experiments, we characterized its compatibility with the green GECI XCaMP-G (Inoue et al., 2019) in cultured neurons using 1P stimulation (Figure 7A). rsChRmine indeed significantly increased the response to red (635 nm) light, but not orange (585 nm) light (Figures 7B-7C). Furthermore, in testing the readout from the blue GECI XCaMP-B (Inoue et al., 2019) using an excitation wavelength of 385 nm, we found that rsChRmine showed reduced sensitivity to blue (435 nm) and cyan (488 nm) light, but no difference was observed for green (570 nm) light (Figure 7D), revealing that the structurally informed design of rsChRmine indeed resulted in properties appropriately optimized for all-optical experiments. Also noteworthy, we observed faster kinetics of hsChRmine-induced XCaMP-G responses compared to those of WT ChRmine, although no differences in response amplitudes were measured for the same light exposure times (Figures 7E-7F).

[0150] Example 3 - Simultaneous in vivo activity recording and optogenetic control in mice These results indicated that rsChRmine may be useful for a new kind of simultaneous optical imaging and control in vivo. To explicitly test this, we first applied frame projection independent fiber photometry (FIP) for simultaneous recording (Kim et al., 2016) and perturbation of activity in pyramidal (Pyr) neurons of the medial prefrontal cortex (mPFC) of mice (Figure 7G-7H), co-expressing GCaMP6m and opsins to compare rsChRmine to other opsins (WT ChRmine and ChrimsonR, Klapoetke et al., 2014; Marshel et al., 2019; Figure 7I). We first measured neural responses using interleaved 2.5 μW of 470 nm imaging light to measure GCaMP6m responses evoked by rsChRmine activation at 594 nm. Consistent with previous findings ( Marshel et al., 2019 ) and our results in cultured neurons ( Figures 7A–7D ), 594 nm light produced significantly larger GCaMP6m response amplitudes (4-fold larger ΔF / F ) that were 2-fold more sensitive to irradiance levels in cells expressing rsChRmine or WT ChRmine compared to cells expressing ChrimsonR ( Figures 7J–7L ).

[0151] To quantify the independence of the optical information channels in a practical setting, we tested the concomitant stimulation of target cells with 470 nm blue light pulses intended for GCaMP imaging, rather than red-shifted opsin stimulation. rsChRmine- and ChrimsonR-expressing cells showed little evoked change in fluorescence even with up to 20 μW of 470 nm light, whereas WT ChRmine showed significant fluorescence changes from 3 μW (Figure 7M). Consistent with this improvement, we also detected a side effect, fluorescence ramping, at the beginning of recording with 470 nm imaging light, but only in WT ChRmine (Figure 7N). Thus, rsChRmine is characteristic in jointly maximizing the redshift and the size of the photocurrent for a given light level, prompting us to further investigate the sensitivity and efficacy in vivo at longer wavelengths. rsCHRmine-expressing neurons responded to 720 nm and 750 nm light stimuli, albeit at higher powers than red light, whereas neurons expressing WT ChRmine and ChrimsonR did not (Figures 7O-7P); thus, rsChRmine represents the first ChR reported to drive neural responses in the near-infrared (740-1400 nm) illumination band.

[0152] Finally, we investigated whether the shifted spectrum of rsChRmine would allow stimulation of activity in a target neural population during simultaneous recording of activity in both stimulated and downstream neural populations. Thus, we expressed both rsChRmine and XCaMP-B in Pyr neurons and GCaMP6f in parvalbumin-expressing (PV) interneurons in the mPFC, thereby combining presynaptic rsChRmine stimulation with recording of XCaMP-B in parallel with recording of postsynaptic GCaMP6f (Figure 7Q). 1P light was delivered at 380 nm to excite XCaMP-B, 470 nm to excite GCaMP6, and 590 nm to stimulate rsChRmine. After a burst of light stimulation of the Pyr neurons, we could track the temporal evolution of excitation in the stimulated Pyr population while observing the downstream activity response of the PV neurons. Conversely, when the targeting strategy was reversed (XCaMP-B for Pyr neurons and GCaMP6m / rsChRmine for PV neurons), the opposite result was obtained: upon photostimulation of PV neurons that produced an increase in activity in the PV cell population, there was a strong decrease in the detected activity of Pyr neurons (Figure 7R). The use of rsChRmine to measure such inter-type impulse responses between cell populations in alert animals (thus obtaining a measure of the instantaneous influence of one cell type on another) is an important step towards a more controlled and realistic analysis of the complexities of intact brain dynamics.

[0153] Testing Method: Cloning, protein expression, and purification Wild-type ChRmine (M1-R304, with the C-terminal five amino acids truncated from the previous construct (Marshel et al., 2019)) was modified to contain an N-terminal influenza hemagglutinin (HA) signal sequence and a FLAG tag epitope, as well as a C-terminal enhanced green fluorescent protein (eGFP) and 10x histidine tag; the N- and C-terminal tags are removable by human rhinovirus 3C protease cleavage. Constructs were expressed in Spodoptera frugiperda (Sf9) insect cells using the pFastBac baculovirus system. Sf9 insect cells were cultured at 3.5x10 6Cells were grown in suspension to a density of 1000 cells / mL, infected with ChRmine baculovirus, and shaken at 27.5°C for 24 h. Cultures were then supplemented with 10 μM all-trans retinal (ATR) (Sigma-Aldrich) and shaken for an additional 24 h. Cell pellets were lysed in hypotonic lysis buffer (20 mM HEPES-NaOH pH 7.5, 20 mM NaCl, 10 mM MgCl2, 1 mM benzamidine, 1 μg / ml leupeptin, 10 μM ATR) and cell pellets were collected by centrifugation at 10,000 × g for 30 min. After repeating the above process twice, the cell pellet was disrupted by homogenization with a glass Dounce homogenizer in hypertonic lysis buffer (20 mM HEPES-NaOH pH 7.5, 1 M NaCl, 10 mM MgCl2, 1 mM benzamidine, 1 μg / ml leupeptin, 10 μM ATR) and the crude membrane fraction was collected by ultracentrifugation (45Ti rotor, 125,000 × g for 1 h). After repeating the above process twice, the membrane fraction was homogenized by glass douncer in solubilization buffer (1% n-dodecyl-β-D-maltoside (DDM) (EMD Millipore), 0.2% cholesteryl hemisuccinate (CHS) (Sigma-Aldrich), 20 mM HEPES-NaOH (pH 7.5), 500 mM NaCl, 20% glycerol, 5 mM imidazole, 1 mM benzamidine, 1 μg / ml leupeptin) and solubilized for 2 h at 4 °C. Insoluble cell debris was removed by centrifugation (125,000 × g, 1 h), and the supernatant was mixed with Ni-NTA Superflow resin (QIAGEN) for 1 h at 4 °C.The Ni-NTA resin was collected in a glass chromatography column, washed with 2.5 CV of wash 1 buffer (0.05% DDM, 0.01% CHS, 20 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 50 mM imidazole), 2.5 CV of wash 2 buffer (0.05% DDM, 0.06% GDN (glycodiosgenin), 0.016% CHS, 20 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 50 mM imidazole), and 2.5 CV of wash 3 buffer (0.06% GDN, 0.006% CHS, 20 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 50 mM imidazole) and eluted with wash 3 buffer supplemented with 300 mM imidazole. FLAG tag and eGFP-His. 10 After His-tagged 3C protease cleavage of the tag, the sample was reloaded onto the Ni-NTA column to isolate the cleaved eGFP-His 10 The flow-through containing ChRmine in the final buffer (20 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 0.03% GDN, 0.003% CHS) was collected, concentrated and purified by gel filtration chromatography.

[0154] antibody generation Mouse monoclonal antibodies against ChRmine were produced according to a previously described method (Jaenecke et al., 2018). Briefly, proteoliposome antigen was prepared by reconstituting purified functional ChRmine into phospholipid vesicles consisting of a 10:1 mixture of chicken egg yolk phosphatidylcholine (egg PC, Avanti Polar Lipids) and adjuvant lipid A (Sigma-Aldrich) at high density to promote immune responses. BALB / c mice were immunized with proteoliposome antigen using three injections at 2-week intervals. Antibody-producing hybridoma cell lines were generated using a conventional fusion protocol. Biotinylated proteoliposomes were prepared by reconstituting ChRmine with a mixture of egg PC and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (16:0 biotinyl Cap-PE, Avanti) and used as a binding target for conformation-specific antibody selection. Targets were immobilized on streptavidin-coated microplates (Nunc). Hybridoma clones producing antibodies recognizing conformational epitopes in ChRmine were selected by enzyme-linked immunosorbent assay (liposome ELISA) on immobilized biotinylated proteoliposomes, allowing positive selection of antibodies recognizing the native conformation of ChRmine. Additional screening for reduced antibody binding to SDS-denatured ChRmine was used for negative selection against linear epitope-recognizing antibodies. Stable complex formation between ChRmine and each antibody clone was checked using fluorescence-detected size-exclusion chromatography. The sequence of the Fab from antibody clone number YN7002_7 (designated as Fab02) was determined by standard 5'-RACE using total RNA isolated from hybridoma cells.

[0155] Formation and purification of ChRmine-Fab02 complex Purified ChRmine was mixed with a 4-fold molar excess of Fab and the coupling reaction was carried out overnight at 4°C. The ChRmine-Fab02 complex was purified by size-exclusion chromatography on a Superdex200 Increased 10 / 300GL column (Cytiva) in 20 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 0.03% GDN, 0.003% CHS. Peak fractions were concentrated to approximately 15 mg / mL for electron microscopy studies.

[0156] Cryo-EM data acquisition and image processing Cryo-EM images were acquired on a Krios G3i microscope (Thermo Fisher Scientific) equipped with a Gatan BioQuantum energy filter and a K3 direct detection camera at 300 kV in electron counting mode. Movie data sets were collected in correlated double sampling (CDS) mode using a nine-hole image shift strategy in SerialEM software (Mastronarde, 2005b), with a nominal defocus range of 0.8–1.6 μm. 3,528 movies were analyzed using a 6.3e - / pixel / sec dose rate, with a pixel size of 0.83 Å, and 46 e - / Å 2 The total dose was 100 μg / kg.

[0157] Image processing was performed in RELION-3.1 (Zivanov et al., 2018). Beam-induced motion correction and dose weighting were performed with RELION's implementation of the MotionCor2 algorithm (Zheng et al., 2017), and CTF parameters were estimated with CTFFIND-4.1.13 (Rohou and Grigorieff, 2015). Particles were first picked using a Laplacian-of-Gaussian algorithm, and a 2D class average image was generated as a template for reference-based automatic picking. The 2,958,159 reference-based picked particles were subjected to several rounds of 2D and 3D classification. The selected 555,801 particles were subjected to 3D automatic refinement, resulting in a 2.8 Å map. Subsequent Bayesian polishing (Zivanov et al., 2019) and CTF refinement (Zivanov et al., 2020), followed by 3D automatic refinement, resulted in a 2.6 Å map. The density of the constant regions of the micelles and Fab fragments was subtracted from the particle images, and the subtracted particles were subjected to masked 3D classification without alignment. After 3D automatic refinement of the selected 185,895 particles, three rounds of CTF refinement were performed in the following order: refinement of magnification anisotropy; refinement of optical aberrations; refinement of defocus per particle and astigmatism per micrograph. Another round of 3D automatic refinement resulted in a map of 2.13 Å. These particles were subjected to a second round of Bayesian polishing, CTF refinement, and 3D automatic refinement focused on the transmembrane regions using the reconstruction algorithm SIDESPLITTER (Ramlaul et al., 2020), resulting in a final map with a global resolution of 2.02 Å.

[0158] Model building and refinement An initial model was formed by rigid-body fitting of C1C2 (PDB:3UG9) (Kato et al., 2012). This starting model was then subjected to iterative rounds of manual and automated refinement in the Servalcat pipeline (Yamashita et al., 2021) of Coot (Emsley and Cowtan, 2004) and Refmac5 (Murshudov et al., 2011), respectively. Refmac5 refinement was performed using C3 symmetry constraints. The final model was visually inspected for overall fit to the map, and the geometry was further assessed using Molprobity (Chen et al., 2010). The final refinement statistics are summarized in Table 1. All molecular graphic figures were prepared using UCSF Chimera (Pettersen et al., 2004), UCSF Chimera X (Goddard et al., 2018), and Cuemol (see World Wide Website: cuelmol.org).

[0159] Analysis of structure The ion-permeable pore was calculated by the software HOLLOW using a grid spacing of 1.0 Å. The electrostatic potential of the pore was calculated by the PDB2PQR server (Baker et al., 2001; Dolinsky et al., 2004). The trimer opening radius of ChRmine was calculated with HOLE.

[0160] High-performance liquid chromatographic (HPLC) analysis of retinal isomers. Retinal isomers were analyzed with an HPLC system equipped with a silica column (particle size 3 μm, 150 × 6.0 mm; Pack SIL, YMC, Japan), a pump (PU-4580, JASCO, Japan), and a UV-visible detector (UV-4570, JASCO, Japan). Purified samples in a buffer containing 20 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 0.035% GDN, and 0.0035% CHS (GDN:CHS = 10:1) were dark-adapted for 2 days at 4 °C. 75 μL of sample and 280 μL of 90% (v / v) aqueous methanol were mixed on ice, and then 25 μL of 2 M hydroxylamine (NH2OH) was added to convert the retinal chromophore to retinal oxime, which was extracted with 800 μL of n-hexane. 200 μL of the extract was injected into the HPLC system. A solvent containing 15% ethyl acetate and 0.15% ethanol in hexane was added in 1.0 mL portions. -1 The mobile phase was used at a flow rate of 1000 nm. Illumination was performed on ice with green light (530 ± 5 nm) for 20 s for illuminated samples and 60 s for light adaptation. The molar composition of the samples was calculated from the areas of the peaks at 360 nm and the molar extinction coefficients (all-trans-15-syn: 54,900 M -1 cm -1 ;All-trans-15-anti: 51,600M -1 cm -1 ;13-cis-15-syn, 49,000M -1 cm -1 ;13-cis-15-anti:52,100M -1 cm -1 ;11-cis-15-syn:35,000M -1 cm -1 ;11-cis-15-anti:29,600M -1 cm -1 )(Trehan et al., 1990).

[0161] Preparation of lipid-reconstituted ChRmine for high-speed AFM imaging For HS-AFM imaging of lipid-reconstituted ChRmine, we applied membrane scaffolding proteins (MSPs) developed for nanodisc technology (Bayburt et al., 2002, Denisov and Sligar, 2016). As previously described (Shibata et al., 2018), we followed the manufacturer's protocol for nanodiscs (Sigma-Aldrich, St. Louis, MO, USA) with minor modifications. Briefly, for the reconstituted lipids, a mixture of phospholipids, soybean-derived asolectin, was used (Sigma-Aldrich, No. 11145). Asolectin (120 μg) was dissolved in chloroform and then evaporated under N2 gas to completely remove the solvent. The lipids were then suspended in 50 μL of buffer A (20 mM HEPES-KOH, pH 7.4, 100 mM NaCl, and 4% DDM) and sonicated with a tip sonicator for about 1 min. Then, dissolved membrane protein (1 nmol) and MSP (50 μL, 1 mg / mL) (MSP1E3D1, Sigma-Aldrich, No. M7074) were added to the lipid suspension and mixed for about 1 h with rotation in the dark at 4 °C. Finally, 60 mg of Bio-Beads SM-2 (Bio-Rad, Hercules, CA, USA, No. 1523920) were added and the sample was dialyzed overnight in detergent at 4 °C. According to the manufacturer's protocol, the nanodisc sample should be fractionated on a column to purify the nanodiscs based on size (diameter about 10 nm). Here, we did not purify the reconstituted sample, but obtained flat membranes with limited size, less than 30 nm in diameter.

[0162] High-speed AFM measurements A homemade HS-AFM operated in tapping mode was used (Shibata et al., 2017, 2018). The deflection of the cantilever (Olympus, Tokyo, Japan: BL-AC10DS-A2) was detected using an infrared (IR) laser at 780 nm and 0.7 mW by an optical beam deflection detector. The IR beam was focused on the backside of the cantilever covered with a gold film through a 60x objective lens (Nikon, Tokyo, Japan: CFI S Plan Fluor ELWD 60x). The reflected IR beam was detected by a two-segment PIN photodiode. The free vibration amplitude of the cantilever was about 1 nm, and the setpoint amplitude was about 90% of the free amplitude for feedback control of the HS-AFM observation. An amorphous carbon tip (about 500 nm long) grown by electron beam deposition with a scanning electron microscope was used as the AFM probe. As HS-AFM substrates, we used mica surfaces treated with 0.01% (3-aminopropyl)triethoxysilane (Shin-Etsu Silicone, Tokyo, Japan). All HS-AFM experiments were carried out in a buffer solution containing 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl at room temperature (24–26 °C), and data analysis was performed using laboratory-developed software based on IgorPro 8 software (WaveMetrics, USA). Typically, the image was 43 × 32 nm with 130 × 95 pixels. 2 A scan area of ​​100 nm was used. HS-AFM images were acquired at a frame rate of 2 fps. All HS-AFM images were processed by a Gaussian noise reduction filter.

[0163] Measurement of UV absorption spectrum For pH titration, the final purified product (20 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 0.03% GDN, 0.003% CHS) was diluted with 100 mM of each pH buffer (StockOptions pH buffer kit) and UV-Vis spectra were measured.

[0164] Laser flash photolysis For laser flash photolysis spectroscopy, wild-type ChRmine was solubilized in 20 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 0.035% GDN, 0.0035% CHS (GDN:CHS = 10:1) or in 20 mM sodium acetate (pH 4.0), 100 mM NaCl, 0.03% GDN, 0.003% CHS (GDN:CHS = 10:1), and ChRmine D115N and D253N mutants were solubilized in 20 mM sodium acetate (pH 4.0), 100 mM NaCl, 0.03% GDN, 0.003% CHS (GDN:CHS = 10:1). The optical density of the protein solution was adjusted to about 0.4 at the absorption maximum wavelength (protein concentration about 0.28 mg / mL). Laser flash photolysis measurements were performed as previously described (Inoue et al., 2013). ChRmine wild type at pH 7.5 was stimulated with nanosecond pulsed Nd 3+ -YAG laser (excitation wavelength (λ exc ) = 532 nm, 4.5 mJ / pulse, 1.4-0.5 Hz, INDI40, Spectra-Physics, CA) and excited by the second harmonic of Nd 3+ Nanosecond pulses from an optical parametric oscillator (4.5 mJ / pulse, basiScan, Spectra-Physics, CA) pumped by the third harmonic of a -YAG laser (λ = 355 nm, INDI40, Spectra-Physics, CA) stimulated ChRmine wild-type (λ exc =505 nm), ChRmine D115N (λ exc = 488 nm) and D253N (λ excA 1000 nm wavelength was used for excitation at 100 nm (=500 nm). Transient absorption spectra were obtained by monitoring the intensity change of white light from a Xe-arc lamp (L9289-01, Hamamatsu Photonics, Japan) passing through the sample using an ICCD linear array detector (C8808-01, Hamamatsu, Japan). To increase the signal-to-noise (S / N) ratio, 45–60 spectra were averaged and singular value decomposition (SVD) analysis was applied. To measure the time evolution of the transient absorption change at a specific wavelength, the light from the Xe-arc lamp (L9289-01, Hamamatsu Photonics, Japan) was monochromatized by a monochromator (S-10, SOMA OPTICS, Japan), and the change in intensity after photoexcitation was monitored by a photomultiplier tube (R10699, Hamamatsu Photonics, Japan). To increase the S / N ratio, 100–200 signals were averaged. The time evolution of the transient absorption changes was analyzed by global multi-exponential fitting to determine the time constants of each reaction step and the absorption spectra of the photointermediates. Some reaction steps were reproduced by double or triple exponential functions. In this case, the average time constant was Calculated by TIFF2025508610000004.tif8128, where A i and k i is the amplitude of the wavelength representing the M intermediate and the rate constant of the i-th exponential function (i = 1 to 3).

[0165] Molecular cloning All ChRmine mutant plasmids were constructed in AAV-CaMKIIa or pcDNA3.1 backbones using overlap PCR as previously described (Fenno et al., 2020; Marshel et al., 2019). ChRmine-Oscarlet-Kv2.1, WT, rs, and hs mutants were transferred into the Elav3 backbone using AgeI and MluI sites to generate transgenic fish lines. All plasmids were sequence verified.

[0166] Primary cell transfection For neuronal transfection, 2.0 µg of plasmid DNA was added to 1.875 µL of 2 M CaCl in 15 µL of HO (final Ca 2+ The transfection was allowed to proceed for 45-60 min at 37 °C, after which each well was washed with 3 × 1 ml of warm MEM before adding back the original growth medium. The transfected DNA was allowed to express in neurons for 6-8 days before the experiment.

[0167] For HEK cell transfection, 0.8 µg of plasmid DNA was mixed with 2 µL of Lipofectamine 2000 (Invitrogen) in 100 µL of Opti-MEM (Invitrogen, incubated for 20 min at room temperature (20-22 °C)) and the mixture was added dropwise into each well (wells from which growth medium was removed and replaced with 400 µL of pre-warmed Opti-MEM). Transfection was allowed to proceed for 2 h at 37 °C, after which the transfection medium was replaced with regular HEK cell growth medium. Cells were allowed to express the transfected DNA for 2-3 days before the experiment.

[0168] Virus production AAV-8(Y733F) was produced by the Stanford Neuroscience Gene Vector and Virus Core. Briefly, AAV8 was produced by standard triple transfection of AAV293 cells (Agilent). 72 hours after transfection, cells were harvested and lysed by a freeze-thaw procedure. Viral particles were then purified by iodixanol step gradient ultracentrifugation. Iodixanol was diluted and AAV was concentrated using a 100 kDa molecular weight cutoff ultrafiltration device. Genomic titers were determined by quantitative PCR. All viruses were tested in cultured neurons for expected expression patterns before in vivo use.

[0169] In vitro electrophysiology in HEK293 cells HEK293 cells transfected with the pcDNA3.1(+) plasmid were placed in extracellular Tyrode's medium (150 mM NaCl, 4 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES pH 7.4, and 10 mM glucose). Borosilicate patch pipettes (Harvard Apparatus) with resistances of 4-6 Mohm were filled with intracellular medium (140 mM potassium gluconate, 10 mM EGTA, 2 mM MgCl2, and 10 mM HEPES pH 7.2). Light was provided by a Spectra X light engine (Lumencor) connected to the fluorescence port of a Leica DM LFSA microscope equipped with a 580 nm filter for orange light generation.

[0170] Channel kinetics and photocurrent amplitude were measured in voltage clamp mode at a holding potential of -70 mV. To determine channel kinetics and photocurrent amplitude, traces were first smoothed at 1,000 Hz using a low-pass Gaussian filter with a -3 dB cutoff for signal attenuation and noise reduction, and then analyzed with Clampfit software (Axon Instruments). Fluid-phase interface potentials were corrected using a Clampex-embedded fluid-phase interface potential calculator as previously described. Statistical analyses were performed using t-tests or one-way ANOVA, and Kruskal-Wallis tests for nonparametric data, using Prism7 (GraphPad) software. Data collection for opsins was randomized and distributed to minimize between-group differences in expression time, room temperature, and related experimental factors.

[0171] Ion selectivity assay in HEK293 cells HEK293 cells and devices for measurements were prepared as described in the previous section. For high sodium extracellular / high potassium intracellular conditions, a sodium bath solution containing 120 mM NaCl, 4 mM KCl, 2 mM CaCl2, 2 mM MgCl2, and 10 mM HEPES pH 7.2 (with glucose added to an osm of 310 mOsm) and a potassium pipette solution containing 120 mM KCl, 10 mM EGTA, 4 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, and 10 mM HEPES pH 7.2 (with glucose added to an osm of approximately 290) were used. For high potassium extracellular / high sodium intracellular conditions, NaCl and KCl concentrations were reversed and all other ion concentrations were kept constant. For ion selectivity measurements, ions in both bath and pipette solutions were replaced with either 120 mM NaCl, 120 mM KCl, 80 mM CaCl2, 80 mM MgCl2, or 120 mM NMDG-Cl, and all other components were at low concentrations (4 mM NaCl, 4 mM KCl, 2 mM CaCl2, 2 mM MgCl2, and 10 mM HEPES). Glucose was added to increase the intracellular solution to 310 mOsm and the extracellular solution to 290 osm. Photocurrent amplitudes were measured at a holding membrane potential of -70 mV. Equilibrium potentials were measured by holding the membrane potential from -75 mV to +45 mV in 10 mV increments.

[0172] In vitro one-photon electrophysiology in cultured hippocampal neurons Primary rat hippocampal cultured neurons were transfected with pAAV ChRmine carrying plasmid and measured in the same setup as described in the HEK293 electrophysiology section. Voltage clamp recordings were performed in the presence of tetrodotoxin (TTX, 1 µM, Tocris) added to the bath. For action spectrum screening, cells were exposed to 1.0 mW / mm for 1 s at wavelengths (nm) of 390, 438, 485, 513, 585 and 650 generated using filters with corresponding peak wavelengths and bandwidths of 15-30 nm. 2The cells were held at a resting potential of -70 mV with light delivery of 100 µM. Channel kinetics and photocurrent amplitude were measured at -70 mV holding membrane potential. The liquid-phase interface potential was corrected using a liquid-phase interface potential calculator incorporating Clampex as previously described. Current clamp measurements were performed in the presence of glutamatergic synaptic transmission blockers: 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM, Tocris) and D(-)-2-amino-5-phosphonovaleric acid (APV; 25 µM, Tocris).

[0173] For the light pulse duration experiments, a frequency of 5 Hz and a power of 0.7 mW / mm were used. 2 585 nm light with an intensity of 0.003, 0.01, 0.03, 0.1, 0.3, 0.7, and 1.0 was used at various pulse width values ​​(mW / mm2) of 0.5, 1, 2, 5, and 10. For the photosensitivity experiments, 585 nm light with a frequency of 5 Hz and a pulse width of 5 msec was used at various light power densities (mW / mm2) of 0.003, 0.01, 0.03, 0.1, 0.3, 0.7, and 1.0. 2 For spike fidelity experiments, a pulse width of 1 ms was used for the ChRmine variant, with a power of 0.7 mW / mm 2 A 585 nm light with a power density of 100 Hz was used. Data collection for opsins was randomized and distributed to minimize between-group differences in expression time, room temperature, and related experimental factors. Statistical analyses were performed using t-tests or one-way ANOVA, and Kruskal-Wallis tests with Dunn's test for multiple comparisons of nonparametric data, using Python and Prism7 (GraphPad) software.

[0174] In vitro two-photon electrophysiology All two-photon electrophysiology experiments were performed with hippocampal neurons cultured in the same intracellular and extracellular solutions as the one-photon electrophysiological characterization. Experiments were performed on a commercial microscope (Bruker Ultima running PrairieView v5.4) using a Nikon 16x / 0.8NA (CFI75) long working distance objective for light delivery. For two-photon stimulation, spiral scanning was performed through a defined spiral ROI with a diameter of 15 μm, 10 rotations per spiral, and a total exposure duration of 1.3 ms with a laser repetition rate of 80 MHz (Coherent Discovery). The axial point spread function FWHM of the two-photon stimulation beam was measured to be 6.9 ± 0.2 μm at 920 nm using 1 μm diameter beads (Invitrogen Focal Check Slide #1, F36909).

[0175] For two-photon action spectrum characterization, recordings were performed in voltage clamp mode with a holding voltage of -75 mV. Action spectra were measured at wavelengths (nm) of 825, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, and 1300 in a randomized test order with a laser power of 20 mW. 10 turns / spiral, 15 mm diameter spiral, 1.3 ms duration, and 80 MHz laser repetition rate. We measured the focus shift as we systematically varied the wavelength from 825 to 1300 nm and found that there was a difference of about 25 μm in focus between 825 nm and 1300 nm. Therefore, the z-focus was adjusted to compensate for the empirically measured focus shift during randomized wavelength delivery.

[0176] No measures were taken to compensate for the potential effects of pulse broadening due to spectral dispersion. All measurements were normalized by the maximum value of a single recording session and then averaged across cells.

[0177] Stereotactic surgery A midline incision was made to expose the skull, and a small cranial incision was made over the injection site using a Meisinger Carbide Burr size 1 / 4. All virus dilutions were performed in ice-cold PBS, and all viruses were produced at the Stanford Gene and Viral Vector Core. Virus was infused at 100 nL per minute using a 10 μL syringe (World Precision Instruments) and a 33-gauge beveled needle (World Precision Instruments) using an injection pump (World Precision Instruments). -1For slice physiology experiments, mice were injected with either AAV8-CaMKIIα-ChRmine-p2A-oscarlet (2.0e13vg / mL) or AAV8-CaMKIIα-rsChRmine-p2A-oscarlet (7.30e12vg / mL). One microliter of virus was stereotaxically injected bilaterally into the motor cortex of 8-12 week old mice at 1.7mm AP, 0.75mm ML, and 1.5mm DV from bregma. For fiber photometry experiments, mice were injected with either AAV8-CaMKIIα-GcaMP6m-2A-opsin (where "opsin" is one of the three opsins shown in Figures 7G-7R): rsChRmine (1.0e12 vg / ml), WT ChRmine (1.0e12 vg / ml), or ChrimsonR (1.0e12 vg / ml). For cross-type experiments, PV-2a-Cre mice were injected with either a mixture of AAV8-CaMKIIα-rsChRmine-oScarlett-Kv2.1 (3.0e12 vg / ml), AAV8-CaMKIIα-XcaMP-B (8.0e12 vg / ml), and AAVdj-EF1α-DIO-GcaMP6f (3.0e12 vg / ml), or a mixture of AAV8-CaMKIIα-XcaMP-B (8.0e12 vg / ml) and AAV8-EF1α-DIO-GcaMP6m-2A-rsChRmine (5.0e11 vg / ml). 0.7 μl of virus was stereotaxically injected unidirectionally into the mPFC of 8–12 week-old mice at 1.8 mm AP, 0.35 mm ML, and 2.4 mm DV from the bregma. After injection, the needle was held at the injection site for 10 min and then slowly withdrawn. For postoperative pain management, mice were administered 0.5–1.0 mg kg -1 Subcutaneous buprenorphine-SR (ZooPharma) was administered.

[0178] Acute slice electrophysiology Recordings of rsChRmine and ChRmine-expressing pyramidal cells were performed in acute slices from wild-type C57BL / 6 mice 4–5 weeks after virus injection. Coronal slices 300 μm thick were prepared after intracardiac perfusion with ice-cold N-methyl-d-glutamine (NMDG) containing cutting solution: 93 mM NMDG, 2.5 mM KCl, 25 mM glucose, 1.2 mM NaH2PO4, 10 mM MgSO4, 0.5 mM CaCl2, 30 mM NaHCO3, 5 mM Na ascorbate, 3 mM Na pyruvate, 2 mM thiourea, and 20 mM HEPES (pH 7.3–7.4). Sections were incubated at 34 °C for 12 min and then transferred to room temperature oxygenated artificial cerebrospinal fluid (ACSF) solution: 124 mM NaCl, 2.5 mM KCl, 24 mM NaHCO3, 2 mM CaCl2, 2 mM MgSO4, 1.2 mM NaH2PO4, 12.5 mM glucose, and 5 mM HEPES pH 7.3-7.4.

[0179] Current clamp measurements were performed as described in the in vitro electrophysiology section. Briefly, a current of 5 Hz and 0.7 mW / mm 2 Pulse width was tested using 585 nm light with an intensity of 0.025, 0.1, 0.25, 0.3, 0.4, 0.5, and 10 at various pulse width values ​​(milliseconds), and 585 nm light with a frequency of 5 Hz and a pulse width of 5 milliseconds was used at various optical power densities (mW / mm 2 For spike fidelity experiments, a pulse width of 1 ms was used for the ChRmine variant, with a power of 0.7 mW / mm 2 A 585 nm light with a power density of 100 Hz was used. Data collection for opsins was randomized and distributed to minimize between-group differences in onset time, room temperature, and related experimental factors.

[0180] In vitro characterization of the preparation of the all-optical setup Dissociated hippocampal neurons were cultured and infected with both red-shifted opsin variants and XcaMP-G or XcaMP-B as previously described ( Marshel et al., 2019 ). One microliter of virus suspension of WT ChRmine (AAV8-CaMKIIα-ChRmine-oScarlet-Kv2.1, 1.3e13vg / mL), rsChRmine (AAV8-CaMKIIα-rsChRmine-oScarlet-Kv2.1, 8.8e12vg / mL), or hsChRmine (AAV8-CaMKIIα-hsChRmine-oScarlet-Kv2.1, 1.8e13vg / mL) combined with 1 µL of XcaMP-G (AAV8-CaMKIIα-XcaMP-G, 6.9e12vg / mL) or 1 µL of XcaMP-B (AAV8-CaMKIIα-XcaMP-B, 2.4e13vg / mL) was added after 5 DIV. Cultured neurons were used between 12 and 14 DIV for experiments. Coverslips of cultured neurons were transferred from the culture medium into a recording bath filled with Tyrode's solution containing (129 mM NaCl, 5 mM KCl, 30 mM glucose, 25 mM HEPES-NaOH pH 7.4, 1 mM MgCl2, and 3 mM CaCl2) supplemented with 10 μM CNQX and 25 μM APV to prevent contamination from spontaneous and recurrent synaptic activity. Photostimulation and imaging were performed using a 40× / 0.6-NA objective (Leica), a sCMOS camera (Hamamatsu, ORCA-Flash4.0), and an LED light source (Spectra X light engine, Lumencor), all coupled to a Leica DMI 6000 B microscope. XcaMP-B or XcaMP-G were excited at 390 nm (Semrock, FF01-390 / 18) or 488 nm (Semrock, LL01-488-12.5), respectively, with a Spectra X light engine. XcaMP-B emission was reflected off a four-wavelength dichroic mirror (Semrock, FF409 / 493 / 573 / 652-Di02) and passed through a triple-band fluorescence filter (Semrock, FF01-432 / 523 / 702-25) for various color photostimulation.The emission of XcaMP-G was reflected off a two-wavelength dichroic mirror (Chroma, ZT488 / 594rpc) for orange light stimulation or another mirror (ZT488 / 640rpc) for red light stimulation and passed through a 535-30 nm emission filter (Chroma, ET535 / 30m). Red-responsive opsins were stimulated with 585 nm orange light (Semrock, FF01-585 / 29-25, 2.0 mW / mm). 2 ) or 635 nm red light (Semrock, FF01-635 / 18-25, 2.0 mW / mm 2 For light sensitivity experiments, the cells were activated with a Spectra X light engine filtered with 434 nm blue light (Semrock, 434 / 17), 488 nm cyan light (Semrock, LL01-488-12.5), or 570 nm green light (Chroma, HQ570 / 20m) at various light power densities (mW / mm) of 0.013, 0.066, 0.30, and 1.0 with a pulse duration of 400 ms. 2 ) was used.

[0181] The fluorescence of XcaMP-B or XcaMP-G was observed at low intensity of 385 nm (10 μW / mm ) without substantially activating red-responsive opsins, respectively. 2 ) or 488 nm (8 μW / mm 2 ) laser light. Images were acquired at 20 Hz using MicroManager (http: / / micro-manager.org). The stimulation light was controlled by LabVIEW (National Instruments) and was illuminated every 10 s with exposure times of 10, 50, 200, and 800 ms. The imaging data were analyzed in MATLAB (MathWorks). Circular regions of interest (ROIs) were manually drawn based on the averaged images. We used Ca 2+Background subtraction was performed before calculating the signal. The ΔF / F response was calculated by dividing the signal in each ROI by its mean total fluorescence intensity and subtracting 1 to normalize the signal. Noise was calculated as the standard deviation of the total ΔF / F fluctuations during the 3 s before stimulation. The signal-to-noise ratio (SNR) was then calculated as the ΔF / F response divided by the noise. The peak amplitude was calculated from the maximum value during the 2 s after the cessation of stimulation. To compare red-responsive opsin with triggered XcaMP-G kinetics, we performed a 200 ms exposure triggered Ca 2+ Transients were calculated. The "rise time" (tpeak) was defined as the time from the cessation of light stimulation to the peak at which maximum amplitude fluorescence was reached. The decay constant (tau) was determined by single exponential fitting from the peak of the fluorescence response 2 seconds after stimulation.

[0182] FIP design and analysis We used a single optical fiber to collect bulk fluorescence from the target brain region while delivering excitation light for fiber photometry, as previously described (Inoue et al., 2019; Kim et al., 2016). We extended these methods to the case of dual excitation wavelengths (380 and 470 nm) with stimulation wavelengths (590, 720, or 750 nm) delivered through the same fiber to allow tracking of activity in different cell populations (transmitters and receivers) during optogenetic stimulation of the transmitter population. Low-fluorescence 400 mm diameter 0.66 NA single fiber optic cannulae (Doric Lenses) were implanted above the mPFC for fiber photometry. The cannulae were fixed to the skull using a base layer of adhesive dental cement (C&B-Metabond, Parkell), followed by a second layer of cranioplastic cement (Ortho-Jet, Lang). Experiments were performed for FIP recordings 4–6 weeks later to allow for sufficient virus expression and post-operative recovery. One end of the patch cord was terminated with an SMA connector (Thorlabs, SM1SMA) attached at the working distance of the objective, and the other end was terminated with a 2.5 mm diameter stainless steel ferrule. These ferrules were coupled to ferrules implanted in the mouse via a bronze sleeve (Doric, SLEEVE_BR_2.5). The fiber surface was imaged through a 20× / 0.75NA objective (Nikon, CFI Plan Apo Lambda 20×) via a series of reconfigurable dichroic mirrors.

[0183] In the standard configuration, three LEDs (M385F1, M470F3, and M595F2, Thorlabs) were filtered with 380-14 nm, 473 nm, and 586-20 nm bandpass filters (FF01-380 / 14-25, LL01-473-25, and FF01-586 / 20-25, Semrock). Excitation and optogenetic stimulation light from two light sources (470 and 590 nm) was passed through a 525 nm longpass dichroic mirror (T525lpxr, Chroma) and then combined with 380 nm light using a second 425 nm longpass dichroic (T425lpxr, Chroma) before finally being coupled into a fiber optic patch cord using a triple multiband dichroic (69013bs, Chroma). For XcaMP-B and GcaMP6 recordings, the fluorescence emission was passed through a multi-bandpass fluorescence emission filter (Semrock, FF01-425 / 527 / 685-25). A 575 nm short-pass filter (Edmund, 575 nm 25 mm diameter, OD4.0 short-pass filter) was directed towards the tube lens to minimize the direct LED emission detected by the camera. Imaging optical powers of 380 nm and 470 nm were used at the distal end of the patch cord at 5 μW and 2.5 μW, respectively. The fluorescence image was focused onto the sensor of an sCMOS camera (Hamamatsu, ORCA-Flash4.0) via a tube lens (Thorlabs, AC254-035-A-ML).

[0184] A custom MATLAB (Mathworks, Natick, MA) GUI was created to control the sample illumination protocol and provide power modulation pulses to the LEDs (National Instruments, NI PCIe-6343-X) that temporally align each LED illumination with the camera frame acquisition (HCImage, Hamamatsu). A typical illumination protocol repeats a sequence of three frame sampling periods: one isobestic at 380 nm, one signal at 470 nm, and one optogenetic above 470 nm (Figure 7G). Maintaining a dedicated frame for optogenetic excitation faithfully removes potential cross-excitation artifacts from the isobestic and signal sampling windows.

[0185] To quantify the spectral cross-excitation of opsins from the signal illumination, the 470 nm LED was additionally pulsed during the optogenetic sampling period. The pulse duration of this additional illumination matched the signal pulse width (23 ms). The minimum excitation power for the sweep was equivalent to that used for the signal pulse (2.5 µW). The digital camera acquired data at a total of 30 Hz. Thus, with a consecutive three-frame sampling protocol, isobestic and signal samples were acquired at 10 Hz each, and it is assumed that all optogenetic stimulations occurred at a similar 10 Hz rate. The duration of this 10 Hz optogenetic stimulation was 2 s. To quantify the excitation efficiency of opsins to 594 nm orange light, the associated LED was pulsed during the optogenetic sampling period (10 ms pulse width). For the light intensity sweep, four samples of each power were randomly interleaved with random ITIs of 20-30 s. Optogenetic excitation in the NIR window at 720 nm and 750 nm was characterized separately using this same protocol (Inoue et al., 2019; Kim et al., 2016). For 720 nm optogenetic stimulation, the 594 nm LED was replaced with a 730 nm LED (M730L5, Thorlabs). The 730 nm laser was filtered with a 716-43 nm bandpass filter (Semrock, FF01-716 / 43-25). For 750 nm optogenetic stimulation, the 594 nm LED was replaced with a 750 nm laser (CivilLaser). The 750 nm laser was filtered with a 750-10 nm bandpass filter (Thorlabs, FB750-10).

[0186] Pyr-PV impulse response data were acquired using the same optical configuration. A 594 nm LED was delivered using a 10 ms pulse width and 1 mW power. Pulse frequency (1, 2, 5, 10, 20 Hz) and pulse number (10, 20, 30, 40, 60, 80, 120) were controlled by TTL signals delivered by a microcontroller (Arduino, Uno) communicating with MATLAB (MathWorks). Four samples at each frequency and number were randomly interleaved with an ITI of 30 s.

[0187] Fluorescence signals were calculated using a custom written MATLAB script. We fitted a double exponential function to the thresholded version of the fluorescence time series and subtracted the best fit from the unthresholded signal to account for slow photobleaching artifacts. Fluorescence signals were normalized within each mouse by calculating ΔF / F as (F-baseline (F)) / baseline (F), where the baseline was taken from the average for 5 s before optogenetic stimulation. Peak ΔF / F amplitude was calculated from the maximum value for 2 s after stimulation cessation. Noise was calculated as the standard deviation of ΔF / F fluctuations for 5 s before optogenetic stimulation. Signal-to-noise ratio (SNR) responses were then calculated as the ΔF / F response divided by the noise. Each measurement point (light intensity and wavelength) represents the average of four trials at 20-30 s intervals. Optical EPD50 in Figure 7K was quantified by dividing the ΔF / F amplitude at each light intensity by the ΔF / F amplitude at 1 mW.

[0188] Histology and confocal microscopy To analyze the expression patterns of opsins and GcaMP, immunohistochemistry was performed on brain tissues removed from virus-injected mice. Animals were anesthetized and perfused transcardially with ice-cold 1x PBS followed by 4% paraformaldehyde (PFA) in PBS. Brains were dissected and fixed overnight at 4°C in the same fixative. Tissues were cut into 60 μm-thick slices with a vibratome (Leica, VT1000) and floated in PBS. For immunohistochemistry, brain slices were blocked with 3% normal donkey serum / 0.3% Triton X-100 / PBS and incubated overnight at 4°C on a shaker with primary antibodies diluted in blocking buffer. The antibody used was mouse monoclonal anti-HA tag (1:500, Fisher Scientific A26183). After washing with 0.3% Triton X-100 / PBS, tissue sections were incubated with secondary antibody, Alexa Fluor 647-conjugated donkey anti-mouse antibody (1:500, A-31571, Thermo Fisher Scientific) and DAPI for 2 h at room temperature and mounted on slides in tissue mounting medium containing antifade polyvinyl alcohol mounting medium with DABCO (Millipore Sigma). Confocal imaging of GcaMP fluorescence, HA antibody staining for opsin localization, and DAPI staining for cytoarchitecture were performed using a Leica TCS SP8 or TCS SP5 confocal scanning laser microscope with a 10× / NA-0.4 or 25× / NA-0.95 water immersion objective. Colocalization was performed using 25× images by annotating GcaMP6m-expressing cell body positions, overlaying these annotations, and verifying expression in the anti-HA images. Quantitative analysis of GcaMP expression levels in individual mice was performed using 10x images (5-6 z-slices spaced 3 μm apart through each section) by annotating GcaMP6m expression. GcaMP6m fluorescence intensity was quantified from slices with the highest fluorescence intensity by setting a 400 μm square ROI directly beneath the fiber tract using ImageJ (NIH).

[0189] Quantification and statistical analysis For electrophysiology experiments, data were recorded and analyzed using pClamp10.6 (Molecular Devices), Python, and Prism7 (GraphPad) software. Nonparametric tests (Wilcoxon rank sum test and signed rank test) were used for single comparisons. For multiple comparisons, Kruskal-Wallis tests were performed, followed by Dunn's test for post-hoc comparisons. Peak photocurrent was identified as the maximum difference in current in the interval from laser onset to laser offset. Tau-off was calculated by fitting a single exponential curve to the waveform from laser offset to baseline. Time to peak was calculated by measuring the time difference between laser onset and peak current.

[0190] To calculate the action spectra, photocurrents were first normalized to the peak photocurrent of each cell. These normalized spectra were then averaged across cells to generate action spectra for each opsin variant in both one-photon and two-photon measurements. To calculate EPD50, photocurrents were first normalized to the photocurrent evoked at the highest light power. Linear interpolation was then used to estimate the light power level that produced 50% of the maximum photocurrent.

[0191] Example 4 - Crystal structure determination and structure-based mutagenesis The extraordinary properties of ChRmine open new avenues for optogenetic research in the study of cell-specific activity within biological systems ( Chen et al., 2021 ; Marshel et al., 2019 ), along with extremely large photocurrents, red-shifted actuation, and photosensitivity ( Marshel et al., 2019 ), making ChRmine a novel Ca 2+ It showed virtually no conductance (Figure 13A) and Ca 2+This is a valuable property in long-term optogenetic applications to avoid the accidental induction of photodependent plasticity. Herein, we uncover the structure-function relationships underlying these remarkable properties along with insights into the evolution of microbial opsins. First, despite its fundamentally different channel-based mechanism, ChRmine is strikingly similar to the HsBR pump in terms of oligomerization number (Fig. 1C), overall monomeric structure (Fig. 1E), and proton acceptor (Fig. 2D), suggesting that ChRmine evolved from the archaeal ion pump rhodopsin. In HsBR, DTD motif D85 and D96 play a critical function as a relay to pump a single proton from the inside to the outside of the cell in response to an absorbed photon. In ChRmine, these two Asps are conserved (D115 and D126), but H + Rather than being the pumping relay, they form the two constriction sites (ICS and CCS) of the passive ion permeable pore within the monomer. Thus, these two residues continue to play important roles, but the H + Not as a transport relay point, but in opening and closing channels.

[0192] Arginine Conformation in Dark State and Function of Ion Transport Rhodopsin. R82 in HsBR is highly conserved among microbial rhodopsins, but in dark state, it is found in two different conformations: outward and parallel. In all structurally solved channelrhodopsins (C1C2 (PDB ID: 3UG9) (Kato et al., 2012), CrChR2 (PDB ID: 6EID) (Volkov et al., 2017), C1Chrimson (PDB ID: 5ZIH) (Oda et al., 2018), GtACR1 (PDB ID: 6CSM) (Kim et al., 2018), and ChRmine), the arginine residue faces outward. In contrast, HsBR (PDB ID:5ZIM) (Hasegawa et al., 2018), HwBR (PDB ID:4QID), crixrhodopsin-3 (PDB ID:4JR8) (Chan et al., 2014), deltarhodopsin (PDB ID:4FBZ) (Zhang et al., 2013), GR (PDB ID:6NWD) (Morizumi et al., 2019), archerhodopsin-1 (PDB ID:1UAZ) (Enami et al., 2006), archerhodopsin-2 (PDB ID:2EI4) (Yoshimura and Kouyama, 2008), PR from a depth of 12 m in the Mediterranean Sea (Med12BPR, PDB ID:4JQ6) (Ran et al., 2013), and PR from a depth of 75 m in the Pacific Ocean near Hawaii (HOT75BPR, PDB ID:4JQ7) (Ran et al., 2013). In many ion pump rhodopsins, including TM1 (PDB ID: 4KLY) (Ran et al., 2013), CsR (6GYH) (Fudim et al., 2019), HsHR (PDB ID: 1E12) (Kolbe et al., 2000), NpHR (PDB ID: 3A7K) (Kouyama et al., 2010), ClR (PDB ID: 5ZTK) (Yun et al., 2021), KR2 (PDB ID: 3X3C) (Kato et al., 2015), and schizorhodopsin 4 (PDB ID: 7E4G) (Higuchi et al., 2021), the arginine tip runs parallel to the membrane and faces TM1.Arginines in the parallel conformation narrow or block the extracellular space of the ion transport pathway and thus this conformation may contribute to preventing large ion fluxes in the ion pump rhodopsin.

[0193] In particular, CsR (an outward proton pumping rhodopsin from Coccomyxa subellipsoidea) also has an arginine (R83) in a parallel conformation in the dark state (Fudim et al., 2019), and the R83Q mutation or mutation of the adjacent tyrosine (Y57K) converts the function from a proton pump to a proton channel (Vogt et al., 2015). Furthermore, computational analysis of HsBR with

[0194] Mutation of R82Q or Y57K revealed that these mutations significantly change the conformation of R82Q or R82, respectively, and most notably, R82 faces outward in the Y57K simulation (Vogt et al., 2015). These observations suggest that the conformation of arginine in the dark state is one of the structural elements that distinguish channel-type rhodopsins from pump-type rhodopsins. Interestingly, previous studies have reported that arginines in some ion pump rhodopsins are maintained in a parallel conformation during the photoperiod (Kouyama et al., 2015; Kovalev et al., 2020), whereas the corresponding arginine in HsBR transiently changes from parallel to outward, facilitating proton release into the extracellular solvent (Kuehlbrandt, 2000; Nango et al., 2016). Because channelrhodopsins likely evolved from ion pump rhodopsins ( Inoue et al., 2015 ), these studies suggest that mutations accumulated near arginines in ion pump rhodopsins gradually stabilized their outward-facing conformations, and these rearrangements enlarged the extracellular cavity, allowing channelrhodopsins to have a large ion flux.

[0195] Conformational changes of the monomer pore during simulation: In addition to the opening of the trimer pore, we also observed that the size of the monomer pore increased during the light-state simulation (Figure 5E). Upon isomerization and proton transfer, both retinal and D115 rotate away from the inner monomer pore, increasing the space within the monomer (Figure 5F). On the timescale of our simulation, the pore radius does not become large enough to allow the movement of ions through the monomer (as with the trimer pore), but the changes in the monomer are in accordance with the opening that is expected to occur upon light activation.

[0196] Despite its similarity to HsBR, ChRmine exhibits some atypical characteristics in its high-resolution structure, including its long, twisted ECL1. The ECL1 not only significantly distorts the structure around the Schiff base, but also expands the extracellular cavity within the monomer. Interestingly, the length and sequence of the ECL1 are not highly conserved in this subfamily, and it has been reported that cation selectivity differs between pump-like ChRs that we predict to have long ECL1 domains and pump-like ChRs that we predict to have short ECL1 domains (Sineshchekov et al., 2020). Furthermore, we show that ChRmine (with its long ECL1) preferentially binds monovalent cations, especially K + 13A), whereas HcKCR1 and HcKCR2 (recently discovered members of the same pump-like subfamily as ChRmine, which possess a shorter ECL1 motif) similarly exclude divalent cations and selectively reject Na + K +It has been reported that ECL1 further shows selectivity for HcKCR1 (Govorunova et al., 2021). Several molecular features within ECL1 may be relevant (e.g., the conserved Arg residue (R112 in ChRmine) on ECL1 is replaced by Trp in HcKCR1 and HcKCR2), and ECL1 may be explored for its role in cation permeation and selectivity. Indeed, given the knowledge of the extraordinary structural and electrophysiological properties of ChRmine reported herein (in particular the unique ECL1 feature shared by ChRmine relatives but not found in other rhodopsins (Figures 8A and 11B) and high monovalent cation selectivity (Govorunova et al., 2021; Shigemura et al., 2019; Figure 13A)), an updated comprehensive name for this growing ChR family would be pump-like ChRs (PLCRs; Figures 8A-8B), which are not particularly similar to bacterial rhodopsin over other pumps, nor are they generally permeable to all cations as currently known).

[0197] Proton donors and acceptors: In HsBR, D85 accepts a proton from the protonated Schiff base and releases it to the extracellular bulk solvent. D96 accepts a proton from the intracellular bulk solvent and donates it to the deprotonated Schiff base. These proton transfers generate a net flow of protons from the inside to the outside of the cell, and these two functionally important residues, together with T89, are called the DTD motif.

[0198] In GtCCR2, a ChRmine homologue of the BCCR family, both D85 and D96 are conserved (D87 and D98, respectively), but the proposed proton transfer pathway is completely different: GtCCR2 does not exhibit outward proton pump activity (Sineshchekov et al., 2017), and protons shuttle between the Schiff base and D85. Deprotonation and reprotonation of D98 is hypothesized to occur, and although deprotonation is necessary for channel gating, D98 does not donate a proton to the deprotonated Schiff base (Sineshchekov et al., 2017). If some channelrhodopsins retain residual pump activity (Feldbauer et al., 2009), the D85 homologue can presumably release a proton into the extracellular bulk solvent after accepting a proton from the Schiff base. However, it remains unclear which residues function as proton donors to the deprotonated Schiff base, as opposed to proton acceptors. D96 in HsBR is also conserved in ChRmine (D126), but is exposed to the intracellular bulk solvent in our structure, and the calculated pKa of D126 is low, 6.28. D126 may not function as the sole proton donor (Figure 3G), but the deprotonated Schiff base may directly accept a proton from a water molecule. Future studies are needed to clarify the molecular nature of the proton donor and understand how proton transfer is involved in the channel gating of ChRmine.

[0199] Indeed, our current ChRmine structural information already provides a framework for further development of ChRmine-based optogenetic tools: hs, rs, and frChRmine (Figures 6A-6K and 7A-7R). It was a surprise to see the reduction of the blue shoulder, which has been very difficult to address despite more than 15 years of intensive efforts in many laboratories (Deisseroth and Hegemann, 2017). Thus, in the future, the structures of rs or frChRmine may prove even more valuable in elucidating the mechanism of this effect and in transferring these properties to other microbial opsins. Other design goals may include combining the key properties of rs, hs, and frChRmine with other ideas that have arisen during opsin engineering. For example, we previously found it productive to combine mutations that enabled chloride flux (via a switch in cation-to-anion selectivity) with mutations that conferred a large increase in light sensitivity (via slowed kinetics) to yield a single chloride-permeable step-function ChR that exhibits a bistable inhibitory current (Berndt et al., 2016). The high-resolution ChRmine structures and new variants described herein may point the way toward such a combination.

[0200] The published structures of ChRs were determined experimentally using crystallography alone. However, we found that the combination of antibodies and single particle cryo-EM techniques (Wu et al., 2012) is powerful enough to determine high-resolution structures of small proteins such as ChRmine, and thus represents a new and promising option for the structural analysis of microbial rhodopsins in parallel with X-ray crystallography. The two techniques, as well as structure prediction methods, complement each other and may thus facilitate the structural biology of microbial rhodopsins, and the resulting information will lead both to further developments in optogenetics and to an understanding of the fundamental mechanisms of these remarkable photoreceptor proteins.

[0201] A small population of early intermediates: Early structural changes in ChRmine: Our 2.0 Å cryo-EM map allowed accurate modeling of the ATR and surrounding residues, but the C13 and C14 atoms of ATR and W223 showed weaker density in that region. Furthermore, positive and negative Fo-Fc difference density was observed around W223, suggesting that this cryo-EM density map contains information on a small population of early intermediate states (likely K intermediates) (Figures 9Q and 9R). Although we could not detect further structural changes propagated from W223, the extent of conformational change in ChRmine W223 was significantly greater than that in C1C2 and similar to that of HsBR (Oda et al., 2021, Weinert et al., 2019). Thus, we conclude that the early conformational changes in the ChRmine photocycle may resemble those of ion pump rhodopsins more than those of canonical green algal channelrhodopsins.

[0202] Valuable further structural studies include molecular dynamics simulations and structures of intermediate states. Investigating the cooperativity / allosteric properties linking different monomers within the trimer can not only help tune the light-responsive properties of optogenetic tools, but can also illuminate the fundamental science of ChR origins, including the conversion from a trimeric to a dimeric assembly logic, possibly during the evolutionary separation of non-pump-like ChRs from their pump-like ancestors. Recent discoveries and applications using optogenetics can be extended to a wide range of opportunities with these ChRmine variants, taking advantage of the significantly higher photocurrents and photosensitivity of the parent opsins, as well as the improved properties of the variants (Bansal et al., 2021, Sahel et al., 2021). Structural insights and structure-informed design of microbial opsins continue to open pathways for discovery and understanding, expanding upon the third of the three major known ChR types: cation-permeable ( Kato et al., 2012 ), anion-permeable ( Kim et al., 2018 , Kato et al., 2018 ), and pump-like channelrhodopsins.

[0203] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art that, in view of the teachings of this invention, certain changes and modifications can be made without departing from the spirit or scope of the appended claims.

[0204] Thus, the above merely describes the principles of the invention. It will be appreciated that those skilled in the art may devise various modifications that embody the principles of the invention and are within the spirit and scope of the invention, although not expressly described or shown herein. Furthermore, all examples and conditional terms recited herein are intended primarily to aid the reader in understanding the principles of the invention and the concepts provided by the inventors to further the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein that recite principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Moreover, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed to perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be dedicated to the public, regardless of whether such disclosure is expressly recited in the claims.

[0205] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. In the claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) are expressly defined as applying a limitation in a claim only when an exact phrase "means for" or "step for" is recited at the beginning of the limitation in the claim, and if such an exact phrase is not used in the limitation in the claim, neither 35 U.S.C. 112(f) nor 35 U.S.C. 112(6) will apply.

Claims

1. A fast variant ChRmine protein having faster kinetic properties compared to a parent ChRmine protein, the fast variant ChRmine protein having one or more amino acid substitutions compared to the parent ChRmine.

2. The fast variant ChRmine protein of claim 1, comprising one or more amino acid substitutions in the counterion of the Schiff base of the parent ChRmine protein.

3. The fast variant ChRmine protein of claim 1, comprising one or more amino acid substitutions that alter the pore electrostatic potential of the parent ChRmine protein.

4. The high-speed variant ChRmine protein of claim 3, wherein the one or more amino acid substitutions that alter the pore electrostatic potential of the parent ChRmine protein are selected from histidine at position 33 or a corresponding position, aspartic acid at position 92 or a corresponding position, glutamic acid at position 154 or a corresponding position, glutamic acid at position 158 or a corresponding position, aspartic acid at position 242 or a corresponding position, and glutamic acid at position 246 or a corresponding position.

5. The high-speed variant ChRmine protein of claim 4, wherein the 33rd histidine or a corresponding position is substituted with histidine if the corresponding amino acid is not histidine, arginine, or lysine.

6. A high-speed variant of the ChRmine protein described in claim 4, wherein the 92nd aspartic acid or corresponding position, the 154th glutamic acid or corresponding position, the 158th glutamic acid or corresponding position, the 242nd aspartic acid or corresponding position, and the 246th glutamic acid or corresponding position are each independently substituted with aspartic acid, glutamic acid, asparagine, or glutamine.

7. The high-speed variant ChRmine protein of claim 1, wherein the parent ChRmine protein has a sequence selected from SEQ ID NOs: 1 to 29, or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1 to 29.

8. The high-speed variant ChRmine protein of claim 1, which has a substitution at the histidine residue at position 33 or the corresponding residue in the first transmembrane domain of the parent ChRmine protein compared to the parent ChRmine protein.

9. The high-speed variant ChRmine protein of claim 8, having an arginine substitution for the histidine residue at position 33 or the corresponding residue in the first transmembrane domain of the parent ChRmine protein.

10. having the sequence of SEQ ID NO: 30 or a sequence having at least 80% sequence identity to SEQ ID NO: 30; The differences in the sequence having at least 80% sequence identity to SEQ ID NO: 30 exclude the amino acid substitutions used to produce the high-rate variant ChRmine protein. The high-speed variant ChRmine protein of claim 1.

11. The high-speed variant ChRmine protein of claim 1, having the sequence of SEQ ID NO:

30.

12. A red-shifted variant ChRmine protein having a red-shifted spectrum compared to a parent ChRmine protein, the red-shifted variant ChRmine protein having one or more amino acid substitutions compared to the parent ChRmine protein.

13. 13. The red-shifted variant ChRmine protein of claim 12, comprising one or more amino acid substitutions in the retinal binding pocket (RBP) of the parent ChRmine protein.

14. The red-shifted variant ChRmine protein of claim 13, wherein the one or more amino acid substitutions in the RBP of the parent ChRmine protein include substitutions at one or more of isoleucine at position 146 or a corresponding position, glycine at position 174 or a corresponding position, and phenylalanine at position 178 or a corresponding position.

15. 14. The red-shifted variant ChRmine protein of claim 13, wherein the substitution at the isoleucine at position 146 or a corresponding position is with serine, cysteine, threonine, or methionine; the substitution at the glycine at position 174 or a corresponding position is with serine, cysteine, threonine, or methionine; or, if the corresponding amino acid is not phenylalanine, tyrosine, or tryptophan, the substitution at the phenylalanine at position 178 or a corresponding position is with phenylalanine.

16. 13. The red-shifted variant ChRmine protein of claim 12, wherein the parent ChRmine protein has a sequence selected from SEQ ID NOs: 1 to 29, or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1 to 29.

17. 13. The red-shifted variant ChRmine protein of claim 12, which, compared to the parent ChRmine protein, has one or both of: (i) a substitution at position 146 for an isoleucine residue or a corresponding residue in the fourth transmembrane domain of the parent ChRmine protein; and (ii) a substitution at position 174 for a glycine residue or a corresponding residue in the fifth transmembrane domain of the parent ChRmine protein.

18. 18. A red-shifted variant ChRmine protein as described in claim 17, which, compared to the parent ChRmine protein, has one or both of: (i) a methionine substitution at the isoleucine residue at position 146 or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein, and (ii) a serine substitution at the glycine residue at position 174 or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein.

19. having the sequence of SEQ ID NO: 31 or a sequence having at least 80% sequence identity to SEQ ID NO: 31; The differences in the sequence having at least 80% sequence identity to SEQ ID NO: 31 exclude the amino acid substitutions used to produce the red-shifted variant ChRmine protein.

13. The redshifted variant ChRmine protein of claim 12.

20. 13. The red-shifted variant ChRmine protein of claim 12, having the sequence of SEQ ID NO:

31.

21. A fast and red-shifted variant ChRmine protein having faster kinetics and a red-shifted spectrum compared to the parent ChRmine protein, wherein the fast and red-shifted variant ChRmine protein has one or more amino acid substitutions compared to the parent ChRmine protein.

22. 22. The fast and red-shifted variant ChRmine protein of claim 21, comprising (i) one or more amino acid substitutions in the counterion of the Schiff base of the parent ChRmine protein or one or more amino acid substitutions that alter the pore electrostatic potential of the parent ChRmine protein, and (ii) one or more amino acid substitutions in the retinal binding pocket (RBP) of the parent ChRmine protein.

23. 22. The fast and red-shifted variant ChRmine protein of claim 21, comprising (i) one or more amino acid substitutions that alter the pore electrostatic potential of the parent ChRmine protein, and (ii) one or more amino acid substitutions in the retinal binding pocket (RBP) of the parent ChRmine protein.

24. 24. The fast and red-shifted variant ChRmine protein of claim 23, wherein the one or more amino acid substitutions that alter the pore electrostatic potential are selected from histidine at position 33 or a corresponding position, aspartic acid at position 92 or a corresponding position, glutamic acid at position 154 or a corresponding position, glutamic acid at position 158 or a corresponding position, aspartic acid at position 242 or a corresponding position, and glutamic acid at position 246 or a corresponding position.

25. 25. The fast and red-shifted variant ChRmine protein of claim 24, wherein the histidine at position 33 or a corresponding position is substituted with histidine if the corresponding amino acid is not histidine, arginine, or lysine.

26. 25. The fast and red-shifted variant ChRmine protein of claim 24, wherein each of the following positions is independently substituted: aspartic acid or a corresponding position 92, glutamic acid or a corresponding position 154, glutamic acid or a corresponding position 158, aspartic acid or a corresponding position 242, and glutamic acid or a corresponding position 246 with aspartic acid, glutamic acid, asparagine, or glutamine.

27. The fast and red-shifted variant ChRmine protein of claim 22, wherein the one or more amino acid substitutions in the RBP of the parent ChRmine protein include substitutions at one or more of isoleucine at position 146 or a corresponding position, glycine at position 174 or a corresponding position, and phenylalanine at position 178 or a corresponding position.

28. 28. The fast and red-shifted variant ChRmine protein of claim 27, wherein the substitution at the isoleucine at position 146 or a corresponding position is with serine, cysteine, threonine, or methionine; the substitution at the glycine at position 174 or a corresponding position is with serine, cysteine, threonine, or methionine; or, if the corresponding amino acid is not phenylalanine, tyrosine, or tryptophan, the substitution at the phenylalanine at position 178 or a corresponding position is with phenylalanine.

29. 22. The fast and red-shifted variant ChRmine protein of claim 21, wherein the parent ChRmine protein has a sequence selected from SEQ ID NOs: 1-29 or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1-29.

30. 22. The fast and red-shifted variant ChRmine protein of claim 21, which has, compared to the parent ChRmine protein, one or more of the following: (i) a substitution of a histidine residue at position 33 or a corresponding residue in the first transmembrane domain of the parent ChRmine protein; (ii) a substitution of an isoleucine residue at position 146 or a corresponding residue in the fourth transmembrane domain of the parent ChRmine protein; and (iii) a substitution of a glycine residue at position 174 or a corresponding residue in the fifth transmembrane domain of the parent ChRmine protein.

31. 31. The fast and red-shifted variant ChRmine protein of claim 30, which, compared to the parent ChRmine protein, has one or more of the following: (i) an arginine substitution at the histidine residue at position 33 or the corresponding residue in the first transmembrane domain of the parent ChRmine protein; (ii) a methionine substitution at the isoleucine residue at position 146 or the corresponding residue in the fourth transmembrane domain of the parent ChRmine protein; and (iii) a serine substitution at the glycine residue at position 174 or the corresponding residue in the fifth transmembrane domain of the parent ChRmine protein.

32. having the sequence of SEQ ID NO: 32 or a sequence having at least 80% sequence identity to SEQ ID NO: 32; The differences in the sequence having at least 80% sequence identity to SEQ ID NO: 32 exclude the amino acid substitutions used to generate the fast and red-shifted variant ChRmine protein.

22. The fast and red-shifted variant ChRmine protein of claim 21.

33. 22. The fast and red-shifted variant ChRmine protein of claim 21, having the sequence of SEQ ID NO:

32.

34. A nucleic acid encoding a variant ChRmine protein described in any one of claims 1 to 33.

35. A genetically modified cell comprising the nucleic acid of claim 34.

36. An expression construct comprising a nucleic acid encoding a variant ChRmine protein according to any one of claims 1 to 33 for use in an optogenetic method for imaging the brain of a subject, comprising: The method comprises: introducing the expression construct into a subject so as to express the variant ChRmine protein in brain cells of the subject; applying a stimulating light to the brain of the subject; imaging the brain of the subject; Including, The expression construct.

37. 37. The expression construct of claim 36, wherein the subject is a mammal.

38. 38. The expression construct of claim 37, wherein the mammal is a rodent, primate, bovine, porcine, feline, or canine.

39. An expression construct comprising a nucleic acid encoding a variant ChRmine protein according to any one of claims 1 to 33 for use in a method of genetically modifying cells and / or organs of a subject, comprising: The method comprises: introducing the expression construct into the subject so as to express the variant ChRmine protein in cells and / or organs of the subject; The expression construct.

40. An expression construct comprising a nucleic acid encoding a variant ChRmine protein according to any one of claims 1 to 33 for use in an optogenetic method for imaging cells and / or organs of a subject, comprising: The method comprises: introducing the expression construct into a subject so as to express the variant ChRmine protein in cells and / or organs of the subject; applying a stimulating light to cells and / or organs of the subject; imaging the cells and / or organs of said subject; Including, The expression construct.

41. 41. The expression construct of claim 40, wherein the cells and / or organs may belong to the cardiovascular system, the digestive system, the urinary system, the respiratory system, the reproductive system, the musculoskeletal system, or the pancreatic / endocrine system.

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