Light-gated rhodopsin cation channels from chlorophyte algae have transformed neuroscience research through their use as membrane-depolarizing optogenetic tools for targeted photoactivation of neuron firing. Photosuppression of neuronal action potentials has been limited by the lack of equally efficient tools for membrane hyperpolarization. We describe anion channel rhodopsins (ACRs), a family of light-gated anion channels from cryptophyte algae that provide highly sensitive and efficient membrane hyperpolarization and neuronal silencing through light-gated chloride conduction. ACRs strictly conducted anions, completely excluding protons and larger cations, and hyperpolarized the membrane of cultured animal cells with much faster kinetics at less than one-thousandth of the light intensity required by the most efficient currently available optogenetic proteins. Natural ACRs provide optogenetic inhibition tools with unprecedented light sensitivity and temporal precision.

We demonstrate that two rhodopsins, identified from cDNA sequences, function as low-and high-light-intensity phototaxis receptors in the eukaryotic alga Chlamydomonas reinhardtii. Each of the receptors consists of an Ϸ300-residue seven-transmembrane helix domain with a retinal-binding pocket homologous to that of archaeal rhodopsins, followed by Ϸ400 residues of additional membraneassociated portion. The function of the two rhodopsins, Chlamydomonas sensory rhodopsins A and B (CSRA and CSRB), as phototaxis receptors is demonstrated by in vivo analysis of photoreceptor electrical currents and motility responses in transformants with RNA interference (RNAi) directed against each of the rhodopsin genes. The kinetics, fluence dependencies, and action spectra of the photoreceptor currents differ greatly in transformants in accord with the relative amounts of photoreceptor pigments expressed. The data show that CSRA has an absorption maximum near 510 nm and mediates a fast photoreceptor current that saturates at high light intensity. In contrast, CSRB absorbs maximally at 470 nm and generates a slow photoreceptor current saturating at low light intensity. The relative wavelength dependence of CSRA and CSRB activity in producing phototaxis responses matches precisely the wavelength dependence of the CSRA-and CSRB-generated currents, demonstrating that each receptor mediates phototaxis. The saturation of the two photoreceptor currents at different light fluence levels extends the range of light intensity to which the organism can respond. Further, at intensities where both operate, their light signals are integrated at the level of membrane depolarization caused by the two photoreceptor currents.retinal protein ͉ photoreceptor ͉ receptor currents ͉ signal transduction U nicellular flagellate algae optimize their light environment by motility responses. Phototaxis (or oriented movement) guides them toward or away from a light source, whereas photophobic responses prevent their crossing a light͞dark border (1). In Chlamydomonas reinhardtii these photomotility responses are mediated by retinal-containing receptor(s), as shown by retinal reconstitution studies in blind mutants (2-5). Moreover, it has been established that the native chromophore of the photoreceptor protein(s) is 6-s-trans all-trans-retinal, as in archaeal rhodopsins, and its alltrans͞13-cis isomerization is required for triggering behavioral responses (3,4,6).A complex photoreceptor apparatus is used to track the light source. The photoreceptor molecules appear to be localized in a small portion of the plasma membrane overlying the eyespot. Light absorption͞reflection by the eyespot modulates the photoreceptor illumination during helical swimming if the helix axis does not coincide with the light direction (7). This modulated illumination serves as a signal for the correction of the swimming path.A cascade of electrical phenomena plays a key role in the signal transduction. Photoexcitation of the receptor molecules results in the generation of photoreceptor...

Microbial sensory rhodopsins are a family of membrane-embedded photoreceptors in prokaryotic and eukaryotic organisms. Structures of archaeal rhodopsins, which function as light-driven ion pumps or photosensors, have been reported. We present the structure of a eubacterial rhodopsin, which differs from those of previously characterized archaeal rhodopsins in its chromophore and cytoplasmic-side portions. Anabaena sensory rhodopsin exhibits light-induced interconversion between stable 13-cis and all-trans states of the retinylidene protein. The ratio of its cis and trans chromophore forms depends on the wavelength of illumination, thus providing a mechanism for a single protein to signal the color of light, for example, to regulate color-sensitive processes such as chromatic adaptation in photosynthesis. Its cytoplasmic half channel, highly hydrophobic in the archaeal rhodopsins, contains numerous hydrophilic residues networked by water molecules, providing a connection from the photoactive site to the cytoplasmic surface believed to interact with the receptor's soluble 14-kilodalton transducer.Over the past 4 years, microbial genomics has revealed a large family of photoactive, seventransmembrane-helix retinylidene proteins called microbial rhodopsins in phylogenetically diverse species, including haloarchaea, proteobacteria, cyanobacteria, fungi, and algae (1-4). *To whom correspondence should be addressed. hudel@uci.edu (H.L.) or john.l.spudich@uth.tmc.edu (J.L.S. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThe first members of this family were discovered in halophilic archaea: the light-driven ion pumps bacteriorhodopsin and halorhodopsin and the phototaxis receptors sensory rhodopsins I and II. These four related haloarchaeal pigments are among the bestcharacterized membrane proteins in terms of structure and function, and nearly all of our knowledge of the properties of microbial rhodopsins, such as isomeric configuration and conformation of their chromophore, photochemical reactions, light-induced conformational changes in the protein, and function, derives from the study of these four, including atomic resolution structures that have been obtained for three of them (5-9). Studies of nonhaloarchaeal rhodopsins, of which >800 are known to exist (10, 11), are needed to examine the diversity of properties of this widespread family (12). Anabaena sensory rhodopsin, a recently discovered sensory representative outside of archaea (2), is well suited for exploration. It is the only bacterial sensory rhodopsin so far expressed in a photoactive form. Unlike the haloarchaeal sensory rhodopsins, which transmit signals to other integral membrane proteins, its function appears to involve modulation of a soluble cytoplasmic transducer, analogous to animal visual pigments (2).In this study, we report the structure of the retinal-complexed protein at 2.0 Å resolution, obtained by X-ray diffraction of crystals grown in a cubic lipid phase (table S1). The overall membrane-embedded seven-he...

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 the transformative technology of optogenetics. 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: (i) anion-conducting channelrhodopsins (ACRs) from cryptophyte algae, enabling efficient optogenetic neural suppression, (ii) cryptophyte cation-conducting channelrhodopsins (CCRs), structurally distinct from the green algae CCRs used extensively for neural activation, and (iii) enzymerhodopsins, with light-gated guanylylcyclase or kinase activity promising for optogenetic control of signal transduction.

A second group of proteorhodopsin-encoding genes (blue-absorbing proteorhodopsin, BPR) differing by 20 -30% in predicted primary structure from the first-discovered green-absorbing (GPR) group has been detected in picoplankton from Hawaiian deep sea water. Here we compare BPR and GPR absorption spectra, photochemical reactions, and proton transport activity. The photochemical reaction cycle of Hawaiian deep ocean BPR in cells is 10-fold slower than that of GPR with very low accumulation of a deprotonated Schiff base intermediate in cells and exhibits mechanistic differences, some of which are due to its glutamine residue rather than leucine at position 105. In contrast to GPR and other characterized microbial rhodopsins, spectral titrations of BPR indicate that a second titratable group, in addition to the retinylidene Schiff base counterion Asp-97, modulates the absorption spectrum near neutral pH. Mutant analysis confirms that Asp-97 and Glu-108 are proton acceptor and proton donor, respectively, in retinylidene Schiff base proton transfer reactions during the BPR photocycle as previously shown for GPR, but BPR contains an alternative acceptor evident in its D97N mutant, possibly the same as the second titratable group modulating the absorption spectrum. BPR, similar to GPR, carries out outward light-driven proton transport in Escherichia coli vesicles but with a reduced translocation rate attributable to its slower photocycle. In energized E. coli cells at physiological pH, the net effect of BPR photocycling is to generate proton currents dominated by a triggered proton influx, rather than efflux as observed with GPR-containing cells. Reversal of the proton current with the K ؉ -ionophore valinomycin supports that the influx is because of voltagegated channels in the E. coli cell membrane. These observations demonstrate diversity in photochemistry and mechanism among proteorhodopsins. Calculations of photon fluence rates at different ocean depths show that the difference in photocycle rates between GPR and BPR as well as their different absorption maxima may be explained as an adaptation to the different light intensities available in their respective marine environments. Finally, the results raise the possibility of regulatory (i.e. sensory) rather than energy harvesting functions of some members of the proteorhodopsin family.