Blue-shifted ancyromonad channelrhodopsins for multiplex optogenetics

Abstract Light-gated ion channels from protists (channelrhodopsins or ChRs) are optogenetic tools widely used for controlling neurons and cardiomyocytes. Multiplex optogenetic applications require spectrally separated molecules, which are difficult to engineer without disrupting channel function. Scanning numerous sequence databases, we identified three naturally blue-shifted ChRs from ancyromonads. They form a separate branch on the phylogenetic tree and contain residue motifs characteristic of anion ChRs (ACRs). However, only two conduct chloride, whereas the closely related Nutomonas longa homolog generates inward cation currents in mammalian cells under physiological conditions, significantly exceeding those by previously known tools with similar spectral maxima (peak absorption at ∼440 nm). Measurements of transient absorption changes and pH titration of purified proteins combined with mutant analysis revealed the roles of the residues in the photoactive site. Ancyromonad ChRs could be activated by near-infrared two-photon illumination, a technique that enables the deeper-tissue optogenetic activation of specific neurons in three dimensions. Both ancyromonad ACRs allowed optogenetic silencing of mouse cortical neurons in brain slices. Ancyromonas sigmoides ACR (AnsACR) expression in cholinergic neurons enabled photoinhibition of pharyngeal muscle contraction in live worms. Overall, our results deepen the mechanistic understanding of light-gated channel function and expand the optogenetic toolkit with potent, blue-shifted ChRs. Impact statement Ancyromonad channelrhodopsins advance our understanding of ionic selectivity and wavelength regulation in light-gated ion channels, and expand the toolkit for multiplexing with red-shifted fluorescent sensors. Competing Interest Statement The authors have declared no competing interest. Footnotes New data (including 31 new figure panels) and their discussion were added in the revision.