Blue-shifted ancyromonad channelrhodopsins for multiplex optogenetics

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 that must be found in nature, as they are difficult to engineer without disrupting channel function. Scanning numerous sequence databases, we identified three robust 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 close Nutomonas longa homolog (peak absorption at ∼440 nm) generates inward cation currents in mammalian cells under physiological conditions, significantly exceeding those by previously known tools. Measurements of transient absorption changes and pH titration of purified Ancyromonas sigmoides ACR ( Ans ACR) combined with mutant analysis revealed the roles of the residues in the photoactive site. Both ancyromonad ACRs allowed optogenetic silencing of mouse cortical neurons in brain slices. Ans ACR expression in the cholinergic neurons enabled photoinhibition of pharyngeal muscle contraction in live worms. Ans ACR could be activated by near-infrared two-photon illumination, which is required to control specific neurons in thick tissue. Our results improved the mechanistic understanding of light-gated channel function and expanded the optogenetic toolkit.