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, 482 (7385), 369-74

Crystal Structure of the Channelrhodopsin Light-Gated Cation Channel


Crystal Structure of the Channelrhodopsin Light-Gated Cation Channel

Hideaki E Kato et al. Nature.


Channelrhodopsins (ChRs) are light-gated cation channels derived from algae that have shown experimental utility in optogenetics; for example, neurons expressing ChRs can be optically controlled with high temporal precision within systems as complex as freely moving mammals. Although ChRs have been broadly applied to neuroscience research, little is known about the molecular mechanisms by which these unusual and powerful proteins operate. Here we present the crystal structure of a ChR (a C1C2 chimaera between ChR1 and ChR2 from Chlamydomonas reinhardtii) at 2.3 Å resolution. The structure reveals the essential molecular architecture of ChRs, including the retinal-binding pocket and cation conduction pathway. This integration of structural and electrophysiological analyses provides insight into the molecular basis for the remarkable function of ChRs, and paves the way for the precise and principled design of ChR variants with novel properties.


Figure 1
Figure 1. Structure of C1C2 and comparison with BR
a–c, Crystal structure of the C1C2 dimer, viewed parallel to the membrane from two angles (a, b), and viewed from the extracellular side (c). C1C2 consists of the N domain, the seven transmembrane helices (TM1–TM7) connected by extracellular loops (ECL1–ECL3) and intracellular loops (ICL1–ICL3), and the C domain. Disordered regions are represented as dotted lines. The ATR is coloured pink. d, e, Side view (d) and extracellular view (e) of the superimposed TMs of C1C2 (green) and BR (orange). The yellow double arrows indicate the shifts of the extracellular parts of TM1 and TM2.
Figure 2
Figure 2. Structural comparison of the retinal-binding pocket between C1C2 and BR
a, Structure of the retinal-binding pocket of C1C2, with an omit map of ATR at 3σ and of the surrounding residues (subtract 39 from the C1C2 residue number to obtain ChR2 numbering) at 3.5σ. b, Structure of the retinal-binding pocket of BR.
Figure 3
Figure 3. The protonated Schiff base and its counterions in C1C2 and BR
a, b, Structures of the environment around the Schiff base in C1C2 (a) and BR (b). Numbers indicate the distance between two atoms connected by dashed lines. c, Effects of the mutation of two possible counterions on the photocurrent. Photocurrents on C1C2-expressing HEK293 cells were measured at 16 different holding potentials. WT, wild type. d, The peak amplitudes of the photocurrents, normalized by the cell’s input capacitance. Values are means and s.e.m. of 7–15 experiments. **P < 0.01, ***P < 0.001.
Figure 4
Figure 4. Cation-conducting pathway formed by TM1, 2, 3 and 7
a, Pore-lining surface calculated by the CAVER program, coloured by the electrostatic potential. Dashed red lines indicate putative intracellular vestibules. b, Close-up views of the surface of the pore, with the 17 polar lining residues (subtract 39 from the C1C2 residue number to obtain ChR2 numbering). Hydrogen bonds are shown as black dashed lines. c, Photocurrents of mutants of the five residues within the pathway, measured under the same conditions as in Fig. 3c. d, The peak amplitudes of the photocurrents, as in Fig. 3d. *P < 0.05, ***P < 0.001. Error bars represent s.e.m.
Figure 5
Figure 5. Two constriction sites on the cytoplasmic side of C1C2 in the closed state
a, The first constriction site is formed by Ser 102 (63), Glu 129 (90) and Asn 297 (258). Hydrogen bonds are shown as black dashed lines. The blue dashed line represents a possible proton transfer pathway. b, The second constriction, made by Tyr 109. The cavity formed by TM1, 2 and 7 is occluded by Tyr 109, and the cavity formed by TM2, 3 and 7 is occluded by Glu 122 and His 173.
Figure 6
Figure 6. Distribution of known mutations and possible candidates for future mutations
a, Mutations that affect both the absorption spectrum and the kinetics (Cys 167 (128), Glu 162 (123) and Asp 195 (156); deep red), the conductance (Thr 198 (159); light green), the selectivity (Leu 171 (132); dark blue) and the kinetics (Glu 122 (83), Ile 170 (131) and His 173 (134); dark cyan) of ChR2. b, Polar (Glu 162 (123), Thr 166 (127), Cys 167 (128), Asp 195 (156), Thr 198 (159) and Ser 295 (256); magenta) and non-polar (Ile 170 (131), Ile 199 (160), Gly 202 (163), Leu 221 (182) and Gly 263 (224); orange) residues surrounding ATR.

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