Abstract

The naturally occurring channelrhodopsin variant anion channelrhodopsin-1 (ACR1), discovered in the cryptophyte algae Guillardia theta, exhibits large light-gated anion conductance and high anion selectivity when expressed in heterologous settings, properties that support its use as an optogenetic tool to inhibit neuronal firing with light. However, molecular insight into ACR1 is lacking owing to the absence of structural information underlying light-gated anion conductance. Here we present the crystal structure of G. theta ACR1 at 2.9 Å resolution. The structure reveals unusual architectural features that span the extracellular domain, retinal-binding pocket, Schiff-base region, and anion-conduction pathway. Together with electrophysiological and spectroscopic analyses, these findings reveal the fundamental molecular basis of naturally occurring light-gated anion conductance, and provide a framework for designing the next generation of optogenetic tools.

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Data availability

The protein coordinate and atomic structure factor have been deposited in the Protein Data Bank (PDB) under accession number 6CSM. The raw diffraction images have been deposited in the SBGrid Data Bank repository (ID: 569). All other data are available from the corresponding authors upon reasonable request.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

We thank C. Lee, M. Lo, K. Geiselhart and M. Lima for technical support; K. K. Kumar, N. R. Latorraca, M. Inoue and K. Katayama for critical comments; and the APS beamline staff at 23ID-B and 23ID-D for assistance in data collection. We acknowledge support by the Stanford Bio-X and the Kwanjeong Foundation (Y.S.K.), JST PRESTO (JPMJPR1782 to H.E.K., JPMJPR15P2 to K.I.), the US Department of Energy, Scientific Discovery through Advanced Computing (SciDAC) program (R.O.D.), MEXT (17H03007 to K.I., 25104009/15H02391 for H.K.), J.S.T. CREST (JPMJCR1753, H.K.) and Mathers Charitable Foundation (B.K.K.). K.D. was supported by a grant for channelrhodopsin crystal structure determination from the NIMH (R01MH075957 to K.D.).

Reviewer information

Nature thanks P. Scheerer, L. Tian and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Yoon Seok Kim, Hideaki E. Kato

Affiliations

  1. Department of Bioengineering, Department of Psychiatry and Behavioral Sciences, and Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA

    • Yoon Seok Kim
    • , Charu Ramakrishnan
    • , Lief E. Fenno
    • , Kathryn E. Evans
    •  & Karl Deisseroth
  2. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA

    • Hideaki E. Kato
    •  & Brian K. Kobilka
  3. PRESTO, Japan Science and Technology Agency, Honcho, Kawaguchi, Japan

    • Hideaki E. Kato
    •  & Keiichi Inoue
  4. RIKEN SPring-8 Center, Hyogo, Japan

    • Keitaro Yamashita
  5. Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya, Japan

    • Shota Ito
    • , Keiichi Inoue
    •  & Hideki Kandori
  6. OptoBioTechnology Research Center, Nagoya Institute of Technology, Showa-ku, Nagoya, Japan

    • Keiichi Inoue
    •  & Hideki Kandori
  7. Department of Computer Science, Stanford University, Stanford, CA, USA

    • Joseph M. Paggi
    •  & Ron O. Dror
  8. Institute for Computational and Mathematical Engineering, Stanford University, Stanford, CA, USA

    • Joseph M. Paggi
    •  & Ron O. Dror

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Contributions

Y.S.K. and H.E.K. contributed equally and either has the right to list himself first in bibliographic documents. Y.S.K. and H.E.K. expressed, purified and crystallized GtACR1, harvested crystals, and collected diffraction data. H.E.K. and K.Y. processed the diffraction data and solved the structure. Y.S.K. and L.E.F. performed electrophysiology. Y.S.K. measured UV-vis spectra. S.I. performed FTIR experiments under the guidance of K.I. and H.K. J.M.P. and R.O.D. provided input on structural considerations. C.R. and K.E.E. performed cell cultures and molecular cloning for electrophysiology. K.D. initiated and supervised this ChR structure/function project; Y.S.K., H.E.K., B.K.K. and K.D. planned and guided the work, and interpreted the data. Y.S.K., H.E.K. and K.D. prepared the manuscript and wrote the paper with input from all the authors.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Hideaki E. Kato or Karl Deisseroth.

Extended data figures and tables

  1. Extended Data Fig. 1 Crystallography.

    a, Size exclusion chromatogram of the purified GtACR1 protein used for crystallography. Similar results were seen in more than 20 independent experiments. b, Electrophysiology of full-length GtACR1 (left) and the final crystallization construct (right); whole-cell voltage-clamp recordings in five cells held at −70 mV, with 513 nm light at 1.0 mW mm−2 irradiance delivered with timing as shown with green-coloured bars, while cells were held at resting potentials from −95 mV (lowest trace) to +5 mV (uppermost trace) in steps of 10 mV. Similar results were seen in 3–5 cells from each group, and no significant difference was seen in resting potential, input resistance, reversal potential or photocurrent magnitude. c, Confocal images of cultured hippocampal neurons expressing full-length GtACR1 (left) and the final crystallization construct (right). Similar results were seen in more than five cells from 3–5 coverslips. Note the markedly reduced aggregation of the truncated construct. d, Crystals of GtACR1. Similar crystals were generated in more than 200 experiments. e, Lattice packing of GtACR1 crystals, viewed parallel to the x axis (left) and the y axis (right). f, Different amino acid configurations at different chains within the asymmetric unit of GtACR1. g, C-terminal interactions among different chains within the asymmetric unit of GtACR1.

  2. Extended Data Fig. 2 Structural analysis of GtACR1.

    a, 2Fo − Fc maps (blue mesh, contoured at 1σ) for the retinal-binding pockets of chains A–D. b, 2Fo − Fc maps (blue mesh, contoured at 1σ) for the lipid molecules. c, 2Fo − Fc maps (blue mesh, contoured at 1σ) and Fo − Fc maps (green and red meshes, contoured at 3.0σ and 3.0σ, respectively) for the Schiff base region of chains A–D. Water molecules are shown as red spheres. d, Table describing data collection and refinement statistics of GtACR1. Dataset was collected from 80 crystals. Values in parentheses are for the highest-resolution shell.

  3. Extended Data Fig. 3 Structure-based sequence alignment of microbial opsin genes.

    The sequences are GtACR1 (GenBank accession AKN63094.1), GtACR2 (GenBank AKN63095.1), ZipACR (GenBank APZ76709.1), PsuACR1 (GenBank ID: KF992074.1), the chimaeric channelrhodopsin between CrChR1 and CrChR2 (C1C2, PDB code 3UG9)34, CrChR1 (GenBank 15811379), CrChR2 (GenBank 158280944), ChR1 from Volvox carteri (VcChR1, UniProtKB B4Y103), ChR1 from V. carteri (VcChR2, UniProtKB ID: B4Y105), Chrimson (GenBank ID: AHH02126.1), ChR from Tetraselmis striata (TsChR, GenBank ID: KF992089.1), HsBR (PDB code 1C3W)59, HsHR (PDB code 1E12)48, and Krokinobacter eikastus rhodopsin 2 (KR2, PDB code 3X3B)60. The sequence alignment was created using PROMALS3D61 and ESPript 362 servers. Secondary structure elements for GtACR1 are shown as coils and arrows. ‘TT’ represents turns. Cysteine residues forming intermolecular and intramolecular disulfide bridges are highlighted in green and yellow, respectively. The residues of retinal-binding pockets are coloured pink. The residues in the Schiff base region are coloured cyan. The residues forming the ECS2 and CCS are coloured orange and blue, respectively.

  4. Extended Data Fig. 4 Structural comparison among GtACR1, HsBR, HsHR, C1C2 and CrChR2.

    a, b, Side view and extracellular view of the superimposed transmembrane regions of GtACR1 (blue) and HsBR (cyan) (a), GtACR1 (blue) and HsHR (beige) (b), C1C2 (green) and CrChR2 (yellow) (c). The ATRs are shown as stick models, and are coloured orange (GtACR1), salmon (HsBR), light-yellow (HsHR), green (C1C2) and yellow (CrChR2).

  5. Extended Data Fig. 5 Interactions between N- and C-terminal regions and the 7-TM domain.

    a, Interactions between the C-terminal region and the 7-TM domain. Hydrogen bonds are shown by dashed lines. b, Fluorescent size-exclusion chromatography traces of the full-length GtACR1 (1–295), the crystallized construct (1–282), and the C-terminal truncated construct (∆C: 1–253), showing possible importance of the C terminus in proper folding and/or stability. Similar results were observed in three independent experiments. c, Interactions between the N-terminal region and the ECL1. Hydrogen bonds are shown by dashed lines. d, Fluorescent size-exclusion chromatography traces of wild-type and C-to-S mutants of GtACR1. Labels indicate estimated elution positions of the aggregate, GtACR1–eGFP, and free eGFP; C-to-S mutants show decreased (<1/3) expression compared to the wild type. Similar results were observed in three independent experiments. e, Stained SDS–PAGE gel image of wild-type and N-terminal 6-amino-acid-truncated GtACR1 in the presence and absence of reducing reagent (β-mercaptoethanol); the wild type runs as a mixer of monomer and dimer in β-mercaptoethanol,whereas N-terminal-truncated GtACR1 stays monomeric even in the absence of β-mercaptoethanol. This experiment was performed once, but similar experiments with different concentrations of β-mercaptoethanol were performed three times, all with similar results. fh, Dimer interfaces of GtACR1 (f), C1C2 (g) and CrChR2 (h) viewed at two angles from the side; note reduced interface area (outlined) for GtACR1. For gel source data, see Supplementary Fig. 1.

  6. Extended Data Fig. 6 Conductances, reversal potentials, absorption spectra and kinetics of wild-type GtACR1 and mutants.

    ac, Photocurrents (a), reversal potentials (b) and absorption spectra (c) of wild-type GtACR1 and ten mutants of the retinal-binding pocket. λmax values are listed in the table (c, bottom). Photocurrents are measured in whole-cell voltage-clamp recordings held at −70 mV, with 513 nm light at 1.0 mW mm−2 irradiance. Data are mean and s.e.m.; n = 9 for WT, 6 for E163Q, 5 for C102A, M105A, C133A, C133R, C153A, E163A and C237A, and 4 for the rest. *P < 0.05, **P < 0.01, one-way ANOVA followed by Dunnett’s test. Reversal potentials are measured with identical light stimulation while cells were held at resting potentials from −95 mV to +15 mV in steps of 10 mV. Data are mean and s.e.m. n = 10 for WT and C237A, 6 for E163A and E163Q, 5 for C102A, M105A, C133A and C153A, and 4 for the rest. **P = 0.0022, one-way ANOVA followed by Dunnett’s test. Spectra measurement was performed in two independent trials, with wild type as a positive control. d, Comparison of fast closing (left) and slow closing (right) coefficients of wild-type and Y72F mutant GtACR1. Data are mean and s.e.m. n = 10 for WT and 5 for Y72F. P = 0.7 for both graphs, two-tailed t-test.

  7. Extended Data Fig. 7 Current–voltage (IV) relationships of wild-type GtACR1 and mutants.

    The IV relationship between −95 mV and +15 mV was determined from the single current amplitude at the indicated potentials. Each measurement is normalized to the current amplitude measured at −25 mV. Data are mean and s.e.m. n = 10 for WT and C237A, 8 for E223A, 6 for Q46C, E163A and E163Q, 4 for E68S, E68T, C102S and M105I, and 5 for the rest.

  8. Extended Data Fig. 8 Representative traces of the IV measurement of wild-type GtACR1 and mutants.

    Voltage clamp traces corresponding to the IV relationships in Extended Data Fig. 7 between −95 mV and +15 mV.

  9. Extended Data Fig. 9 Spectroscopic characterization of wild-type GtACR1 and the D234N mutant.

    a, Absorption spectra of wild-type GtACR1 (top left) and the D234N mutant (top right) measured from pH 3.0 to 10.0. The λmax value at each pH is listed in the table (bottom). b, Difference FTIR spectra of wild-type GtACR1 and the D234N mutant measured at 77 K, 170 K and 200 K. c, Difference FTIR spectra of wild-type GtACR1 in the 1,690–1,770 cm−1 region measured at pH 5.0, 7.0 and 9.0. Forty identical recordings at 77 K and seven identical recordings at 170 K and 200 K were averaged.

  10. Extended Data Fig. 10 Comparison of surface electrostatic potential of GtACR1 and C1C2.

    a, b, Electrostatic potential surfaces of GtACR1 (a) and C1C2 (b) viewed from four angles. The surface is coloured on the basis of the electrostatic potential contoured from −15 kT (red) to +15 kT (blue). c, d, Representation of positively charged amino acids (lysine and arginine residues) in GtACR1 (c), and negatively charged amino acids (aspartate and glutamate residues) in C1C2 (d).

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Further reading

  • Structural mechanisms of selectivity and gating in anion channelrhodopsins

    • Hideaki E. Kato
    • , Yoon Seok Kim
    • , Joseph M. Paggi
    • , Kathryn E. Evans
    • , William E. Allen
    • , Claire Richardson
    • , Keiichi Inoue
    • , Shota Ito
    • , Charu Ramakrishnan
    • , Lief E. Fenno
    • , Keitaro Yamashita
    • , Daniel Hilger
    • , Soo Yeun Lee
    • , Andre Berndt
    • , Kang Shen
    • , Hideki Kandori
    • , Ron O. Dror
    • , Brian K. Kobilka
    •  & Karl Deisseroth

    Nature (2018)

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