WAChRs are excitatory opsins sensitive to indoor lighting

  1. Amanda J. Tose
  2. Alberto A. Nava
  3. Sara N. McGrath
  4. Alan R. Mardinly
  5. Alexander Naka

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Abstract

Hundreds of novel opsins have been characterized since the advent of optogenetics, but low experimental throughput has limited the scale of opsin engineering campaigns. We modified an automated patch-clamp system with a multispectral light source and a custom light path to enable high-throughput electrophysiological measurements of opsin functional properties. Using this approach, we screened over 1,750 opsins from a range of families. We discovered that the F240A mutation of the light-gated potassium channel WiChR abolished potassium selectivity, turning it into a sensitive excitatory channel that we dubbed “WAChR”. We systematically mutated WAChR and identified variants that expand the frontier of speed-sensitivity tradeoffs. Multiple WAChR variants produced large inward currents in response to indoor ambient office light, and responded to irradiances as low as 15 nW/mm 2 , something that we did not observe with other ultra-sensitive opsins. In vivo recording from the mouse cortex confirmed that WAChRs exhibit enhanced sensitivity in neurons. These ambient-light sensitive channels should be broadly useful for neuroscience research and vision restoration therapies.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17889080.

    Summary 

    This study reports the discovery and characterization of a class of highly light-sensitive, excitatory opsins termed "WAChRs" that were engineered from the potassium-selective channelrhodopsin WiChR. WAChRs were found to respond to irradiances as low as 15 nW/mm2, which are achievable even with indoor ambient office light and at least an order of magnitude more sensitive than previously reported opsins. The authors adapted a commercially available automated patch-clamp system to enable high-throughput measurements of light-evoked currents in mammalian cells across 7 different wavelengths. They used this platform to characterize the optogenetic performance of a small set (~20) of known opsins, which uncovered that a F240A mutation that converts WiChR from K+-selective to more broadly cation permeable results in a highly light-sensitive, excitatory opsin dubbed "WAChR". They then used this dataset in conjunction with prior work in the literature to train protein language models to suggest mutations in WAChR to improve functional properties such as activation threshold, amplitude, and speed. The authors characterized these variants using their high-throughput optogenetic automated patch clamp, and further characterized a subset using manual patch clamp to demonstrate the enhanced sensitivity of WAChRs relative to existing opsins. Finally, the authors expressed WAChRs in vivo in the mouse brain and demonstrated that WAChR evokes a larger increase in firing rate in response to light compared to a state-of-the-art opsin, ChRmine. Overall, this study presents a valuable new tool that represents an improvement over existing opsins. However, the study could be strengthened by characterizing existing opsins with the same trafficking tags and promoters as WAChRs to address potential mechanisms by which WAChRs outperform existing tools. Nevertheless, the comprehensive benchmarking of a large set of opsins represents an important resource for the field, and the novel opsins presented in this work should help unlock new avenues for both basic research and translational applications.

    Major points

     1.    Figure 5: The authors note that ex3mV1Co and CoChR-3M were not fused with any signal peptides, whereas other opsins were fused with the standard GFSE cassette. While the authors justify this difference by noting that prior work did not use these peptides, it would be helpful if the authors could also characterize these two opsins with the same GFSE fusion to determine the extent to which trafficking of the opsins accounts for differences in the threshold and other properties. This would be particularly useful for ex3mV1Co where the threshold, sensitivity, and amplitude are closer to those of the WAChRs (Fig. 5B).

    2.    Figure 7: For in vivo experiments, the authors drive expression of WAChR using the EF1a promoter but drive expression of ChRmine using the CAG promoter. In the Methods, the authors recognize that this is less than ideal, but the in vivo results would be more compelling and thorough if the authors also assessed the performance of AAV-EF1a-ChRmine. Moreover, to determine the extent to which the observed increase in firing for WAChR relative to ChRmine may be attributed to differences in expression, the authors should consider performing a quantitative assessment of relative expression levels of each opsin for both AAV-CAG-ChRmine and AAV-EF1a-WAChR-m, either through fluorescence or Western blotting.

    Minor points

    1.    Figure S1: The text indicates that the cleavable LucyRho (LR) tag was prepended in some cases to enhance surface expression. Could the authors clarify the experiments/constructs in which the LR tag was used?

    2.    Figure 2: The sample size for WiChR F240A is listed as n = 2, whereas the sample size for ChRmine is n = 39. The interpretability of these data would be improved if the authors could comment on why the sample size for WiChR F240A is so low, as well as any other variants for which the sample size is low (assuming this is not a typo). For instance, does this reflect variability in expression/toxicity, or is this a result of some aspect of the manual patch clamp platform?

    3.    Figure 5/S6: In Fig. S6, it appears from the representative images of opsin expression that AAV-CoChR-3M may be expressed at a lower level compared to the other opsins. It would be helpful if the authors could provide quantification of expression level (either based on fluorescence or Western blot) for the opsins in Figs. 5 and S6 to better assess any impacts of expression level on the patch clamp results.

    4.    Figure S6C: There may be a typo on the label for the P2A construct in the figure, which does not currently match what is written in the figure caption (GFS vs GSE). Could the authors double check the figure and correct the label if necessary?

    5.    Figure 7A: Most of the rest of Figure 7 presents data directly comparing ChRmine with WAChR-m, so the authors may consider moving Fig. 7A to the Supplementary Information to enhance consistency.

    6.    Figure 7C: The representative raster plots seem to suggest that there may be a higher level of baseline firing in AAV-CAG-ChRmine compared to AAV-EF1a-WAChR-m, which may not be captured by the Z-score metrics in Figs. 7D, S9, and S10. If this is a broad trend across all recorded units for ChRmine, it would be helpful for the authors to mention this in the text and provide some discussion for why this might be the case. If instead this is a feature of the specific unit selected, then it would help with clarity for the authors provide representative units for ChRmine and WAChR-m that are more closely matched in baseline firing. Additionally, displaying more of the pre-stimulation window in the data plotted in Fig. 7C would also allow for better visualization of baseline firing.

    Competing interests

    The authors declare that they have no competing interests.

    Competing interests

    The authors declare that they have no competing interests.

    Use of Artificial Intelligence (AI)

    The authors declare that they did not use generative AI to come up with new ideas for their review.

  2. Arcadia Science

    Thanks for the questions!

    Natural novelty -- do you see any evidence for natural variants for F240A? Would be super interesting if so, in case that could help identify other co-varying residues that could help with kinetics. Or even other substitutions at that site that give you more options for novelty engineering.

    Stepping back a bit: KCRs like WiChR were discovered quite recently and one of their defining features is the presence of a cluster of aromatic residues in the extracellular-facing vestibule of the ion pore domain. In WiChR these include F240 as well as F106, W120, F162, and F239. Along with D47 and N117, these residues have been shown to be critical for K+ selectivity, especially F240 and W120 (1).

    In other channelrhodopsins, these aromatic residues are typically hydrophilic. So F240 is a bit of an “unnatural” looking starting point, though it occupies a position that is not very conserved among CRs at large. By contrast, W120 is almost universally an arginine in other CRs.

    We’ve done a little bit of looking at coevolutionary patterns. Inspecting the categorical Jacobian (2) using ESM2 suggests that F240A and similar mutations are most strongly coupled to changes at D47 (also part of the K+ filter). But only a very small (single digit) number of KCRs have been discovered so far so there might not be a ton of signal to draw on here.

    There are however some detailed comparisons of another KCR, HcKCR1, with other non-KCRs that I think are instructive. One is with HcCCR (3–5) which is an Na+ selective channel that is very closely related to HcKCR1. The residue corresponding to F240 in WiChR is Y222 in HcKCR1 and T222 in HcCCR. The other is with ChRmine (6) where the residues corresponding to D47, W120, and F240 in WiChR (C29, W102, and Y222 in HcKCR1) are H33, R112, and E246. In ChRmine, these residues have been shown to affect kinetics (7).

    I think it’s a likely bet that there are improvements to WAChR that could be realized by some kind of coordinated remodeling of the hydrophobic residues in this region. We’ve explored here of course and we have found some intriguing things. Many edits do improve kinetics but often compromise sensitivity/current magnitude a lot; W120L is one that seems to speed things up with a moderate loss of sensitivity when added to WAChR. We haven’t tried to infill the entire aromatic cluster at once however, which would be interesting.

    Would also be very curious to know what organisms can tolerate a trait like this.

    Us too! As far as I know, there’s not much known about the ethology of K+ channelrhodopsins in general. They come from stramenopiles and channelrhodopsins in general are thought to be involved in phototaxis behaviors, but I don’t know if people have worked out what K+ selective channelrhodopsins specifically are doing.

    Do you have any insights into how that mutation, especially for a hydrophobic residue, might affect binding or folding/dynamics?

    I haven't taken a close look at where it sits on the structure, but that might provide some helpful insights into how to compensate for any hit you take on kinetics. Some really interesting molecular dynamics work has been done on HcKCR1 (6,8) which led to the design of variants with improved K+ selectivity and kinetics. We don’t really have many insights into WAChR at that level of detail, though I think it would be really interesting!

    1. Vierock, J. et al. WiChR, a highly potassium-selective channelrhodopsin for low-light one- and two-photon inhibition of excitable cells. Sci. Adv. 8, eadd7729 (2022).
    2. Zhang, Z. et al. Protein language models learn evolutionary statistics of interacting sequence motifs. Proc. Natl. Acad. Sci. U. S. A. 121, e2406285121 (2024).
    3. Morizumi, T. et al. Structures of channelrhodopsin paralogs in peptidiscs explain their contrasting K+ and Na+ selectivities. Nat. Commun. 14, 4365 (2023).
    4. Govorunova, E. G., Sineshchekov, O. A., Brown, L. S., Bondar, A.-N. & Spudich, J. L. Structural Foundations of Potassium Selectivity in Channelrhodopsins. MBio 13, e0303922 (2022).
    5. Govorunova, E. G., Sineshchekov, O. A. & Spudich, J. L. Potassium-selective channelrhodopsins. Biophys. Physicobiol. 20, e201011 (2023).
    6. Tajima, S. et al. Structural basis for ion selectivity in potassium-selective channelrhodopsins. Cell 186, 4325–4344.e26 (2023).
    7. Kishi, K. E. et al. Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine. Cell 185, 672–689.e23 (2022).
    8. Morizumi, T. et al. Structural insights into light-gating of potassium-selective channelrhodopsin. Nat. Commun. 16, 1283 (2025).
  3. Arcadia Science

    s.

    This is fascinating. I have 2 questions!

    1. Natural novelty -- do you see any evidence for natural variants for F240A? Would be super interesting if so, in case that could help identify other co-varying residues that could help with kinetics. Or even other substitutions at that site that give you more options for novelty engineering. Would also be very curious to know what organisms can tolerate a trait like this.

    2. Do you have any insights into how that mutation, especially for a hydrophobic residue, might affect binding or folding/dynamics? I haven't taken a close look at where it sits on the structure, but that might provide some helpful insights into how to compensate for any hit you take on kinetics.

  4. Version published to 10.1101/2025.09.12.675947 on bioRxiv