The capture and utilization of light is an exquisitely evolved process. The single-component microbial opsins, although more limited than multicomponent cascades in processing, display unparalleled compactness and speed. Recent advances in understanding microbial opsins have been driven by molecular engineering for optogenetics and by comparative genomics. Here we provide a Primer on these light-activated ion channels and pumps, describe a group of opsins bridging prior categories, and explore the convergence of molecular engineering and genomic discovery for the utilization and understanding of these remarkable molecular machines.

Many organisms capture or sense sunlight using rhodopsin pigments, which are integral membrane proteins that bind retinal chromophores. Rhodopsins comprise two distinct protein families , type-1 (microbial rhodopsins) and type-2 (animal rhodopsins). The two families share similar topologies and contain seven transmembrane helices that form a pocket in which retinal is linked covalently as a protonated Schiff base to a lysine at the seventh transmembrane helix. Type-1 and type-2 rhodopsins show little or no sequence similarity to each other, as a consequence of extensive divergence from a common ancestor or convergent evolution of similar structures . Here we report a previously unknown and diverse family of rhodopsins-which we term the heliorhodopsins-that we identified using functional metagenomics and that are distantly related to type-1 rhodopsins. Heliorhodopsins are embedded in the membrane with their N termini facing the cell cytoplasm, an orientation that is opposite to that of type-1 or type-2 rhodopsins. Heliorhodopsins show photocycles that are longer than one second, which is suggestive of light-sensory activity. Heliorhodopsin photocycles accompany retinal isomerization and proton transfer, as in type-1 and type-2 rhodopsins, but protons are never released from the protein, even transiently. Heliorhodopsins are abundant and distributed globally; we detected them in Archaea, Bacteria, Eukarya and their viruses. Our findings reveal a previously unknown family of light-sensing rhodopsins that are widespread in the microbial world.

The introduction of two microbial opsin-based tools, channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), to neuroscience has generated interest in fast, multimodal, cell type-specific neural circuit control. Here we describe a cation-conducting channelrhodopsin (VChR1) from Volvox carteri that can drive spiking at 589 nm, with excitation maximum red-shifted ~70 nm compared with ChR2. These results demonstrate fast photostimulation with yellow light, thereby defining a functionally distinct third category of microbial rhodopsin proteins.Microbial proteins that can be rapidly activated by light have been adapted for research in neuroscience, including ChR2 and NpHR, which permit millisecond-precision optical control of genetically defined cell types in intact neural tissue 1-6 . Because ChR2 is a blue light-gated cation channel and NpHR is a yellow light-driven chloride pump, the combination of these two proteins allows independent neural excitation and inhibition in the same preparation. However, there has been enormous interest in developing a hypothetical third major optogenetic tool, namely a second cation channel with an action spectrum that is substantially red-shifted relative to ChR2, to allow tests of the interaction of cell types in circuit computation or behavior.Although efforts to develop a distinct light-activated excitatory protein have been focused on molecular engineering of ChR2, another approach would be to identify previously unknown microbial channelrhodopsins using genomic tools. One ChR2-related sequence from the spheroidal alga Volvox carteri (Fig. 1a) has been described, but the absorption spectrum of the protein and the photocycle dynamics are virtually identical to those of ChR2 (refs. 7 ,8 ). Therefore, we searched the genome database from the US Department of Energy Joint Genome Institute, discovered a second Volvox ChR (VChR1) that is more related to ChR1 (ref. 9) from Chlamydomonas reinhardtii, explored its properties in heterologous expression systems and functionally tested the codon-optimized opsin gene in mammalian neurons.We expressed VChR1 in Xenopus oocytes and HEK293 cells and observed evoked photocurrents similar to those of ChR1 from Chlamydomonas 9,10 . The photocurrents were graded with light intensity and showed inactivation from a fast peak toward a reduced stationary plateau (Fig. 1b) (Fig. 1b) 9 , and currents showed an inwardly rectifying current-voltage relationship (Fig. 1c).Certain primary structural differences between VChR1 and the Chlamydomonas ChRs suggested that the properties of VChR1 would be distinct from those of the other ChRs (Fig. 1d). On the basis of electrostatic potential and quantum mechanical-molecular mechanical calculations for bacteriorhodopsin and relatives, the counterion complex of the all-trans retinal Schiff base (RSB; Fig. 1d) should be critical for color tuning 11,12 , but these residues are conserved in both ChR1 and VChR1 (blue sequence, Fig. 1d). On the other hand, calculations and mutational experiments 11,12 predict that fo...

The field of optogenetics uses channelrhodopsins (ChRs) for light-induced neuronal activation. However, optimized tools for cellular inhibition at moderate light levels are lacking. We found that replacement of E90 in the central gate of ChR with positively charged residues produces chloride-conducting ChRs (ChloCs) with only negligible cation conductance. Molecular dynamics modeling unveiled that a high-affinity Cl(-)-binding site had been generated near the gate. Stabilizing the open state dramatically increased the operational light sensitivity of expressing cells (slow ChloC). In CA1 pyramidal cells, ChloCs completely inhibited action potentials triggered by depolarizing current injections or synaptic stimulation. Thus, by inverting the charge of the selectivity filter, we have created a class of directly light-gated anion channels that can be used to block neuronal output in a fully reversible fashion.