Figures and data
Phylogeny, spectral sensitivity, and light dependence of ancyromonad ChRs.
(A) A maximum-likelihood phylogenetic tree of selected ChRs. The circles show bootstrap support from 40 to 100. (B) The photocurrent action spectra. The data points are the mean ± SEM values (n = 14 cells for AnsACR, and 9 cells each for FtACR and NlCCR). (C) The absorption spectra of detergent-purified proteins.
(D) The action spectra of photocurrents at −60 mV upon co-expression of AnsACR and Chrimson, compared to each gene expressed alone. The data points are the mean ± SEM values (n = 14 cells each for the co-expression and AnsACR, and 4 cells for Chrimson). (E, F) The dependence of the peak current amplitude (E) and reciprocal time to the peak (F) on the photon flux density for the indicated ChRs activated at their respective λmax. The data points are the mean ± SEM values (n = 8 cells for each variant).
The online version of this article includes the following source data for Figure 1:
Source data 1. Source data for the protein names and accession numbers used to construct the tree in (A); numerical values for the data shown in (B-F).
Characterization of ancyromonad ChRs by automated patch clamping.
(A-C) Photocurrent traces recorded in response to 200-ms light pulses (470 nm) at voltages varied in 20-mV steps from −80 mV using the KF-based internal and NaCl-based external solutions. The dashed lines show the zero-current level. (D-F) The IV curves of the peak photocurrent (filled circles) and the current at the end of illumination (empty circles). The numbers in the parentheses are the numbers of cells sampled. (G) Comparison of the Vr at the photocurrent peak time (filled circles) and the end of illumination (empty circles). The p-values were determined by the two-tailed Wilcoxon signed-rank test; the number of cells sampled for each variant was the same as in panels D-F. (H-J) The Vr values in the indicated external solutions. The circles are the data from individual cells; the lines are the mean and SEM values.
The online version of this article includes the following source data for Figure 2:
Source data 1. Source data for the numbers of cells sampled and numerical values shown in (D-J).
Photocurrent and transient absorption changes under single-turnover conditions in AnsACR and FtACR.
(A, D) Photocurrent traces of AnsACR (A) and FtACR (D) evoked by 6-ns laser flashes recorded by manual patch clamping at the holding voltages increased in 30-mV steps from −60 mV. (B, E) Transient absorption changes recorded at the indicated wavelengths from detergent-purified proteins. (C, F) Comparison of the photocurrent kinetics (red, left axis) and the M intermediate kinetics (black, left axis). In all panels, the thin, solid lines are experimental recordings, and the thick, dashed lines are multiexponential approximations. The numbers are the τ values of the individual kinetic components.
Probing the residues in the counterion positions.
(A) pH titration of λmax (black filled circles, left axis) and maximal absorption changes (black empty circles, right axis) in wild-type AnsACR. (B, C) pH titration of λmax in FtACR (B) and NlCCR (C). (D) The photocurrent action spectrum of the AnsACR_D226N mutant (red) compared to the WT (black). The data points are the mean ± SEM values (n = 8 cells). (E) Photocurrent traces of AnsACR_D226N mutant evoked by 6-ns laser flashes recorded by manual patch clamping at the holding voltages increased in 30-mV steps from −60 mV. The wild-type photocurrent trace recorded at −60 mV is shown in black for comparison. The thin lines are experimental recordings, and the thick dashed lines are multiexponential approximations. The numbers are the τ values of the individual kinetic components. (F) The voltage dependence of the decay components τ in the AnsACR_D226N mutant (red) and the WT (black). (G) pH titration of λmax in AnsACR_G86E mutant. (H) Transient absorption changes monitored at the wavelength of the M intermediate absorption in the AnsACR_G86E mutant (red) compared to the WT (black). (I) Laser-flash-induced photocurrents of the AnsACR_G86E mutant recorded at the external pH 7.4 (black) and 5.4 (red). The arrow shows the increase in the channel current upon acidification.
The online version of this article includes the following source data for Figure 4:
Source data 1. Source data for the numerical values shown in (A-D, F, and G).
Color tuning of ancyromonad ChRs.
(A) Amino acid residues of the retinal-binding pocket tested by mutagenesis in this study. (B) The corresponding residues in the GtACR1 structure (6edq). (C-K) The photocurrent action spectra of the indicated mutants compared to the respective WTs. The data points are the mean ± SEM values (the n values are provided in the Source Data 1).
The online version of this article includes the following source data for Figure 5:
Source data 1. Source data for the numbers of cells sampled and numerical values shown in (C-K).
2P excitation of ancyromonad ACRs.
(A) The 2P photocurrent action spectra. The data points are the mean ± SEM values (n = 5 cells for each variant). (B-F) The mean normalized photocurrent traces recorded upon 2P excitation from the indicated ChR variants (GtACR1 and GtACR2 are included for comparison) at +20 mV in the Cl--based external solution (n = 6 cells for each variant). The illumination (the duration of which is shown as the bars on top) was 15 mW at the λmax for each variant. (G, H) The amplitude of photocurrent measured at the peak time (G) and at the end of 5-s illumination (H). (I) Desensitization at the end of illumination. In G-I, the symbols are the data from individual cells, the lines are mean ± SEM values (n = 6 cells for each variant). For more detail see Methods.
The online version of this article includes the following source data for Figure 6:
Source data 1. Source data for the numerical values shown in (A and G-I).
Photoactivation of AnsACR and FtACR inhibits the action potentials of mouse cortical neurons.
(A, B) Representative membrane voltage traces of neurons expressing AnsACR (A) and FtACR (B) in response to −0.1 nA (left), 0 nA (middle), and 0.5 nA (right) injections without (top) and with (bottom) 470 nm light pulses (power density of 38.7 mW mm-2). (C, D) The frequencies of action potentials evoked by different current injections with (blue) and without (black) photoactivation of AnsACR (C) and FtACR (D). For all panels, data points from male mice are indicated by squares and female mice by circles. One male and one female mouse were used for each of the AnsACR and FtACR experiments. Data are mean ± SEM.
**, p ≤ 0.01 for for comparison between dark and light stimulation at 0.2-0.5 nA current injection by the multiple Wilcoxon matched-pairs signed rank test with Benjamini, Krieger, and Yekutieli’s corrections.
The online version of this article includes the following source data for Figure 7:
Source data 1. Source data for the numerical values shown in (C, D).
Photoinhibition of pharyngeal pumping in live C. elegans expressing AnsACR in the cholinergic neurons.
(A) A scheme of the genetic construct for AnsACR expression in the cholinergic neurons. HA, homology arms. (B) A zoomed-in section of an EPG recording. AP, action potential; TB, terminal bulb. The double-headed arrow shows the interval between two successive R1 spikes used to calculate pharyngeal pumping frequency. (C) Electropharyngeogram recordings from an AnsACR-expressing worm illuminated with 470 nm light at the indicated irradiances. The blue bar shows the duration of illumination. (D) The frequency of the pharyngeal pumping calculated from recordings as shown in A. The symbols are the mean values, and the error bars are the SEM values (n = 11 worms for the WT and 13 worms per condition for the transgenic worms). The numbers are the irradiance values in mW mm-2; ret is retinal. (E) The dependence of the pharyngeal pumping frequency on the irradiance calculated from the 30-60 s segment of the data shown in B. The symbols are the data from individual worms; the lines are the mean and SEM values. The empty and filled symbols for the transgenic worms show the data from two independently created transgenic lines.
The online version of this article includes the following source data for Figure 8:
Source data 1. Source data for the numerical values shown in (D, E).
The protein alignment of the 7TM domains of ChR variants identified and characterized in this study and the previously known GtACR1.
The amino acid residues are colored according to their chemical properties. The residue ruler is according to the GtACR1 sequence. The lines show the transmembrane helices TM1-TM7 of GtACR1. The arrows point to the positions corresponding to Asp68 (black), Ser97 (red), and Asp234 (blue) of GtACR1.
Action spectra and photocurrents of ACRs from Ancoracysta twista and Odontella aurita.
In the left panels, the action spectra. The data points are the mean ± SEM values; n = 6 cells for each variant. In the middle and right panels, the photocurrents recorded by manual patch clamping in the Cl- bath (middle) and Asp- bath (right) at the holding voltages increased in 20-mV increments from −60 mV. The dark cyan bars show the duration of illumination (500, 520 and 510 nm, respectively, for AtACR, OaACR1, and OaACR2).
Comparison of the absorption and action spectra of individual ancyromonad ChRs.
(A-C) Overlays of the absorption and action spectra from the main text Figures 1B and C.
Desensitization at the end of 1-s illumination.
(A-E) Normalized photocurrent traces recorded from the indicated ChRs at −60 mV. The colored rectangles show the duration of illumination. The numbers are photon flux densities multiplied by 1016. (F) The dependence of desensitization, calculated as the peak minus end current divided by the peak current and multiplied by 100%, on the photon flux density. The data points are mean ± SEM (n = 8 cells for each variant).
The online version of this article includes the following source data for Figure 1 – figure supplement 4:
Source data 1. Source data for the numerical values shown in (F).
Desensitization at the end of 5-s illumination.
(A-E) Normalized photocurrent traces recorded from the indicated ChRs at +20 mV. The colored rectangles show the duration of illumination. (F) Desensitization at the end of illumination. The symbols are data from individual cells, the lines are mean ± SEM, n = 6 cells for each variant.
The online version of this article includes the following source data for Figure 1 – figure supplement 4:
Source data 1. Source data for the numerical values shown in (F).
Photocurrent amplitudes of anycromonad ChRs and comparison of NlCCR with CrChR2.
(A, B) Unbiased estimation of peak photocurrent amplitudes. The photocurrents were recorded at 20 mV for AnsACR and FtACR, and at −60 mV for NlCCR and PsChR2. *, p = 0.0047; **, p = 9.9E10-6 by the two-tailed, two-sample Kolmogorov-Smirnov test. (C, D) The Vr shifts measured upon replacing Na+ with NMDG+ in the external solution (C), and upon its acidification from pH 7.4 to 5.4 (D). In all panels, the symbols are the data from individual cells; the lines are the mean and SEM values; the numbers in brackets are the numbers of cells sampled.
The online version of this article includes the following source data for Figure 2 – figure supplement 1:
Source data 1. Source data for the numbers of cells sampled and numerical values shown in (A-D).
Photocurrent traces recorded from ancyromonad ChRs by manual patch clamping.
The photocurrents were recorded by manual patch clamping in the Cl- bath (left), Asp- bath (middle), and NMDG+ bath (right) at the holding voltages increased in 20-mV increments from −60 mV. The blue bars show the duration of 470 nm illumination.
Current-voltage relationships of photocurrent kinetic components in the three ancyromonad ChRs and laser-flash-evoked NlCCR photocurrents.
(A, B, and D). The voltage dependence of the amplitudes of the channel closing and opening components estimated by multi-componential approximation of the laser-flash-evoked photocurrents recorded from the indicated ancyromonad ChRs. (C) Laser-flash evoked photocurrent traces of NlCCR recorded at the holding voltages increased in 30-mV steps from −60 mV in the Cl--based bath (black) and at −60 mV in the Asp--based bath (red). The thin lines are experimental recordings, and the thick dashed lines are multiexponential approximations. The numbers are the τ values of the individual kinetic components.
The online version of this article includes the following source data for Figure 3 – figure supplement 1:
Source data 1. Source data for the numerical values shown in (A, B, and D).
Dependence of absorption on the Cl- concentration.
The lines are the absorption spectra of detergent-purified AnsACR at the indicated Cl- concentrations.
Continued analysis of the AnsACR_G86E mutant.
(A) The voltage dependence of the fast current at pH 7.4 (black) and 5.4 (red). (B) The voltage dependence of the channel current at pH 5.4 in the Cl--based bath (black) and the Asp--based bath (blue). (C) The difference spectra obtained upon alkalization on the purified mutant (red) and WT (black). (D) pH titration of the absorbance difference at 297 nm in the purified mutant (red) and WT (black).
The online version of this article includes the following source data for Figure 4 – figure supplement 2:
Source data 1. Source data for the numerical values shown in (A, B, and D).
Photocurrents in the AnsACR_Q48E mutant.
(A) Laser-flash evoked photocurrent traces of the mutant recorded at the holding voltages increased in 30-mV steps from −60 mV (red). A trace from the WT at −60 mV (black) is shown for comparison. The thin lines are experimental recordings, and the thick dashed lines are multiexponential approximations. The numbers are the τ values of the individual kinetic components. (B) The voltage dependence of the amplitudes of the kinetic components obtained by multiexponential approximation of the laser-flash-evoked photocurrents.
Mutations blue-shifting other microbial rhodopsins spectra do not affect ancyromonad ChRs.
The photocurrent action spectra of the indicated mutants compared to the respective WTs. The data points are the mean ± SEM values.
The online version of this article includes the following source data for Figure 5 – figure supplement 1:
Source data 1. Source data for the numbers of cells sampled and numerical values shown in (A-D).
Power dependence upon 2P illumination.
(A-C) The dependence of the photocurrent rise on the quadratic light power. The data points are the mean ± SEM (n = 5 cells for each variant). A 2nd-order polynomial function was fit to the data. The dashed lines show a linear approximation of the initial portion of the curve.
Characterization of AnsACR and FtACR expression in cortical neurons and axonal excitatory effect.
(A) Representative fluorescence images of 300 µm-thick brain slices expressing tdTomato and EYFP fused to the C termini of AnsACR (left) and FtACR (right) in cortical layer 2/3 pyramidal neurons. The axons of ACR+ layer 2/3 pyramidal neurons ramify in layer 5. L, layer. (B) Resting membrane potentials (left), input resistances (middle), and capacitances (right) of AnsACR+ and FtACR+ neurons. (C) Representative traces of light-evoked excitatory post-synaptic currents (EPSCs) recorded from ACR- pyramidal neurons in layer 2/3 in response to 10-ms 470 nm light pulses (power density of 38.7 mW mm-2). (D) Summary data of experiments in (C). The peak currents of light-evoked EPSCs were measured from the averaged current traces of three trials. In all panels, the data points from male mice are indicated by squares and female mice by circles. One male and one female mouse were used for each of the AnsACR and FtACR experiments. The data points are the mean ± SEM values.
The online version of this article includes the following source data for Figure 6 – figure supplement 1:
Source data 1. Source data for the numbers of cells sampled and numerical values shown in (B, D).
