WAChRs are excitatory opsins sensitive to indoor lighting
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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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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 …
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!
- 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).
- Zhang, Z. et al. Protein language models learn evolutionary statistics of interacting sequence motifs. Proc. Natl. Acad. Sci. U. S. A. 121, e2406285121 (2024).
- Morizumi, T. et al. Structures of channelrhodopsin paralogs in peptidiscs explain their contrasting K+ and Na+ selectivities. Nat. Commun. 14, 4365 (2023).
- 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).
- Govorunova, E. G., Sineshchekov, O. A. & Spudich, J. L. Potassium-selective channelrhodopsins. Biophys. Physicobiol. 20, e201011 (2023).
- Tajima, S. et al. Structural basis for ion selectivity in potassium-selective channelrhodopsins. Cell 186, 4325–4344.e26 (2023).
- Kishi, K. E. et al. Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine. Cell 185, 672–689.e23 (2022).
- Morizumi, T. et al. Structural insights into light-gating of potassium-selective channelrhodopsin. Nat. Commun. 16, 1283 (2025).
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This is fascinating. I have 2 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. Would also be very curious to know what organisms can tolerate a trait like this.
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.
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