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Inward H+ pump xenorhodopsin: Mechanism and alternative optogenetic approach

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Science Advances  22 Sep 2017:
Vol. 3, no. 9, e1603187
DOI: 10.1126/sciadv.1603187
  • Fig. 1 Electrogenic properties of XeR.

    (A) pH changes upon illumination in E. coli cell suspensions expressing different XeRs. Graphs show the pH changes with and without the addition of CCCP. (B) pH changes upon illumination in liposome suspension with reconstructed NsXeR (with and without CCCP). (C) pH changes upon illumination in liposome suspension measured under different pH values.

  • Fig. 2 Spectroscopic characterization of NsXeR.

    (A) Absorption spectra of representatives of XeR family solubilized in the detergent DDM (n-dodecyl-β-d-maltoside). The corresponding positions of absorption maximum are indicated in the legend. A.U., arbitrary units. (B) Transient absorption changes of NsXeR (pH 7.5, T = 20°C) at three representative wavelengths: 378, 408, and 564 nm. Black lines represent experimental data, and red and blue lines represent the result of global fit using five exponents. The photocycles were measured for the two preparations: NsXeR in nanodiscs (red) and in liposomes (blue). Note that the differences in amplitudes between the samples are due to the approximately two times higher concentration of NsXeR in liposomes than in nanodiscs (see fig. S3). ΔOD, change in absorbance. (C) Proposed model of NsXeR photocycle in nanodiscs.

  • Fig. 3 High-performance liquid chromatography measurements.

    (A) Retinal extraction of light- and dark-adapted solubilized NsXeR reconstituted in liposomes and (B) of light- and dark-adapted solubilized BR (as reference). mOD, milli OD (optical density).

  • Fig. 4 NsXeR structure.

    (A) Comparison of NsXeR (yellow) and BR (magenta) motifs. Residues are shown as an NsXeR motif (WDSAPK) and a BR motif (RDTDDK). Two residues, H48 and D220, in NsXeR are shown as an analog of the D96 residue in BR. (B) Putative proton acceptor region in detail. The distance between the Schiff base and water molecule 2 is shown with a double-arrow red line (8.0 Å). Distances between D76 (D85) and the Schiff base in NsXeR (BR) are 4.9 Å (3.8 Å), respectively. Cavities inside the protein calculated by HOLLOW1.2 are shown transparent pink.

  • Fig. 5 Putative NsXeR ion translocation pathway.

    Cavities (transparent pink) and putative key residues inside protein are shown. Black arrows show the putative proton path. Helices F and G are not shown. The hydrophobic membrane core boundaries were calculated using the PPM (Positioning of Proteins in Membrane) server and are shown by gray lines.

  • Fig. 6 Photocurrents in HEK293 and NG108-15 cells.

    Photocurrents in cells expressing NsXeR at the membrane potentials changed in 20-mV steps from −100 mV and corresponding current-voltage curves. (A) HEK293 cells with pipette solution [110 mM NaCl, 2 mM MgCl2, 10 mM EGTA, and 10 mM Hepes (pH 7.4)] and bath solution [140 mM NaCl, 2 mM MgCl2, and 10 mM Hepes (pH 7.4)]. (B) NG108-15 cells with pipette solution [110 mM Na2SO4, 4 mM MgSO4, 10 mM EGTA, and 10 mM Hepes (pH 7.4)] and bath solution [140 mM N-methyl-d-glucosamine, 4 mM MgSO4, and 10 mM Hepes (pH 7.4)] (control measurements to confirm that protons are responsible for inwardly directed current).

  • Fig. 7 Spiking traces at different light-pulse frequencies.

    Rat hippocampal neurons heterologously expressing NsXeR, C-terminally fused to the Kir2.1 membrane trafficking signal, investigated by current clamp measurements in the whole-cell configuration. Action potentials were triggered by 40 light pulses at indicated frequencies. The light pulses had a pulse width of 3 ms, a wavelength of λ = 532 nm, and an intensity of 23 mW/mm2.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/9/e1603187/DC1

    fig. S1. Phylogenetic tree of microbial rhodopsin proteins.

    fig. S2. Sequence alignment of microbial rhodopsins.

    fig. S3. Photocycles of the NsXeR in nanodisc (ND, upper row) and liposome (LIP, lower row) preparations (20°C, pH 7.5).

    fig. S4. Crystal packing of NsXeR.

    fig. S5. Overall architecture of NsXeR.

    fig. S6. Comparison of NsXeR structure with structures of other microbial rhodopsins.

    fig. S7. Details of the NsXeR proton-translocation pathway.

    fig. S8. Action spectrum of photocurrents in NG108-15 cells expressing NsXeR (black) and NsXeR absorption spectrum (red).

    fig. S9. NsXeR photocurrents in response to ultrashort light pulses.

    fig. S10. Light intensity dependence of NsXeR.

    fig. S11. Variability of spike latency.

    table S1. Crystallographic data collection and refinement statistics.

    table S2. List of mutations to NsXeR and their implications on proton pumping.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Phylogenetic tree of microbial rhodopsin proteins.
    • fig. S2. Sequence alignment of microbial rhodopsins.
    • fig. S3. Photocycles of the NsXeR in nanodisc (ND, upper row) and liposome (LIP, lower row) preparations (20°C, pH 7.5).
    • fig. S4. Crystal packing of NsXeR.
    • fig. S5. Overall architecture of NsXeR.
    • fig. S6. Comparison of NsXeR structure with structures of other microbial rhodopsins.
    • fig. S7. Details of the NsXeR proton-translocation pathway.
    • fig. S8. Action spectrum of photocurrents in NG108-15 cells expressing NsXeR (black) and NsXeR absorption spectrum (red).
    • fig. S9. NsXeR photocurrents in response to ultrashort light pulses.
    • fig. S10. Light intensity dependence of NsXeR.
    • fig. S11. Variability of spike latency.
    • table S1. Crystallographic data collection and refinement statistics.
    • table S2. List of mutations to NsXeR and their implications on proton pumping.

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