Microbial rhodopsins are a family of photoactive retinylidene proteins widespread throughout the microbial world. They are notable for their diversity of function, using variations of a shared seven-transmembrane helix design and similar photochemical reactions to carry out distinctly different light-driven energy and sensory transduction processes. Their study has contributed to our understanding of how evolution modifies protein scaffolds to create new protein chemistry, and their use as tools to control membrane potential with light is fundamental to optogenetics for research and clinical applications. We review the currently known functions and present more in-depth assessment of three functionally and structurally distinct types discovered over the past two years: (a) anion channelrhodopsins (ACRs) from cryptophyte algae, which enable efficient optogenetic neural suppression; (b) cryptophyte cation channelrhodopsins (CCRs), structurally distinct from the green algae CCRs used extensively for neural activation and from cryptophyte ACRs; and (c) enzymerhodopsins, with light-gated guanylyl cyclase or kinase activity promising for optogenetic control of signal transduction.

Light control of motility behavior (phototaxis and photophobic responses) in green flagellate algae is mediated by sensory rhodopsins homologous to phototaxis receptors and light-driven ion transporters in prokaryotic organisms. In the phototaxis process, excitation of the algal sensory rhodopsins leads to generation of transmembrane photoreceptor currents. When expressed in animal cells, the algal phototaxis receptors function as light-gated cation channels, which has earned them the name “channelrhodopsins.” Channelrhodopsins have become useful molecular tools for light control of cellular activity. Only four channelrhodopsins, identified in Chlamydomonas reinhardtii and Volvox carteri , have been reported so far. By screening light-induced currents among algal species, we identified that the phylogenetically distant flagellate Mesostigma viride showed photoelectrical responses in vivo with properties suggesting a channelrhodopsin especially promising for optogenetic use. We cloned an M. viride channelrhodopsin, MChR1, and studied its channel activity upon heterologous expression. Action spectra in HEK293 cells match those of the photocurrents observed in M. viride cells. Comparison of the more divergent MChR1 sequence to the previously studied phylogenetically clustered homologs and study of several MChR1 mutants refine our understanding of the sequence determinants of channelrhodopsin function. We found that MChR1 has the most red-shifted and pH-independent spectral sensitivity so far reported, matches or surpasses known channelrhodopsins’ channel kinetics features, and undergoes minimal inactivation upon sustained illumination. This combination of properties makes MChR1 a promising candidate for optogenetic applications. IMPORTANCE Channelrhodopsins that function as phototaxis receptors in flagellate algae have recently come into the spotlight as genetically encoded single-molecule optical switches for turning on neuronal firing or other cellular processes, a technique called “optogenetics.” Only one of four currently known channelrhodopsins is widely used in optogenetics, although electrical currents recorded in diverse flagellates suggest the existence of a large variety of such proteins. We applied a strategy for the search for new channelrhodopsins with desirable characteristics by measuring rhodopsin-mediated photocurrents in microalgae, which helped us identify MChR1, a new member of the channelrhodopsin family. MChR1 exhibits several sought-after characteristics and thus expands the available optogenetic toolbox. The divergence of the MChR1 sequence from those of the four known channelrhodopsins contributes to our understanding of diversity in the primary structures of this subfamily of sensory rhodopsins.

Channelrhodopsins are light-gated ion channels widely used to control neuronal firing with light (optogenetics). We report two previously unknown families of anion channelrhodopsins (ACRs), one from the heterotrophic protists labyrinthulea and the other from haptophyte algae. Four closely related labyrinthulea ACRs, named RubyACRs here, exhibit a unique retinal-binding pocket that creates spectral sensitivities with maxima at 590 to 610 nm, the most red-shifted channelrhodopsins known, long-sought for optogenetics, and more broadly the most red-shifted microbial rhodopsins thus far reported. We identified three spectral tuning residues critical for the red-shifted absorption. Photocurrents recorded from the RubyACR from Aurantiochytrium limacinum (designated AlACR1) under single-turnover excitation exhibited biphasic decay, the rate of which was only weakly voltage dependent, in contrast to that in previously characterized cryptophyte ACRs, indicating differences in channel gating mechanisms between the two ACR families. Moreover, in A. limacinum we identified three ACRs with absorption maxima at 485, 545, and 590 nm, indicating color-sensitive photosensing with blue, green, and red spectral variation of ACRs within individual species of the labyrinthulea family. We also report functional energy transfer from a cytoplasmic fluorescent protein domain to the retinal chromophore bound within RubyACRs. Significance Our identification and characterization of two anion channelrhodopsins families, one from nonphotosynthetic microorganisms, shows that light-gated anion conductance is more widely spread among eukaryotic lineages than previously thought. The strongly red-shifted absorption spectra of the subset that we designate RubyACRs make them promising candidates for the long-sought inhibitory optogenetic tools producing large passive currents activated by red light, enabling deep tissue penetration. Previously only low-efficiency ion-pumping rhodopsins were available for neural inhibition at similar long wavelengths. The unusual residue composition of the retinal-binding pocket in RubyACRs expands our understanding of color tuning in rhodopsins. Finally, activation of chloride currents by energy transfer from a cytoplasmic fluorescent tag on RubyACRs introduces a potential dimension in molecular engineering of optogenetic tools.

Using structural modeling comparisons and mutagenesis, amino acid residue 105 was found to function as a spectral tuning switch in marine proteorhodopsins (PR). Changes at this position account for most of the spectral difference between blue-absorbing PRs (B-PRs), and green-absorbing PRs (G-PRs). Here we analyzed a Red Sea variant (RS29) from a new family of PRs that is composed of G-PR type variants that possess glutamine instead of leucine at position 105 like in B-PRs. The absorption spectrum as well as photocycle of RS29 variant were measured and compared to point-mutated ‘position 105’ PRs. Unexpectedly, the absorption maximum of RS29 was 515 nm, a smaller blue shift compared to the 498 nm maximum of G-PR_L105Q. We found that two additional residues at positions 65 and 70 each contribute a small red shift to the absorption spectrum of G-PR and therefore appear to account for the intermediate absorption maximum of RS29 by their opposing influences on the spectrum. Our results show that in addition to the retinal pocket position 105 determinant, other residues predicted to be outside the retinal pocket fine-tune the absorption spectra of marine PRs. The RS29 photochemical reaction cycle was found to be 2 orders of magnitude slower than that of G-PR with a t_1/2 of >600 ms. This result raises the possibility of regulatory (i.e. sensory) rather than energy harvesting functions for some members of the PR family.