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.

Proteorhodopsins (PRs), members of the microbial rhodopsin superfamily of seven-transmembrane-helix proteins that use retinal chromophores, comprise the largest subfamily of rhodopsins, yet very little structural information is available. PRs are ubiquitous throughout the biosphere and their genes have been sequenced in numerous species of bacteria. They have been shown to exhibit ion-pumping activity like their archaeal homolog bacteriorhodopsin (BR). Here, the first crystal structure of a proteorhodopsin, that of a blue-light-absorbing proteorhodopsin (BPR) isolated from the Mediterranean Sea at a depth of 12 m (Med12BPR), is reported. Six molecules of Med12BPR form a doughnut-shaped C6 hexameric ring, unlike BR, which forms a trimer. Furthermore, the structures of two mutants of a related BPR isolated from the Pacific Ocean near Hawaii at a depth of 75 m (HOT75BPR), which show a C5 pentameric arrangement, are reported. In all three structures the retinal polyene chain is shifted towards helix C when compared with other microbial rhodopsins, and the putative proton-release group in BPR differs significantly from those of BR and xanthorhodopsin (XR). The most striking feature of proteorhodopsin is the position of the conserved active-site histidine (His75, also found in XR), which forms a hydrogen bond to the proton acceptor from the same molecule (Asp97) and also to Trp34 of a neighboring protomer. Trp34 may function by stabilizing His75 in a conformation that favors a deprotonated Asp97 in the dark state, and suggests cooperative behavior between protomers when the protein is in an oligomeric form. Mutation-induced alterations in proton transfers in the BPR photocycle in Escherichia coli cells provide evidence for a similar cross-protomer interaction of BPR in living cells and a functional role of the inter-protomer Trp34-His75 interaction in ion transport. Finally, Wat402, a key molecule responsible for proton translocation between the Schiff base and the proton acceptor in BR, appears to be absent in PR, suggesting that the ion-transfer mechanism may differ between PR and BR.

We characterize changes in isomeric states of the retinylidene chromophore during light-dark adaptation and photochemical reactions of Anabaena (Nostoc) sp. PCC7120 sensory rhodopsin (ASR). The results show that ASR represents a new type of microbial rhodopsin with a number of unusual characteristics. The three most striking are: (i) a primarily all-trans configuration of retinal in the dark-adapted state and (ii) a primarily 13-cis light-adapted state with a blue-shifted and lower extinction absorption spectrum, opposite of the case of bacteriorhodopsin; and (iii) efficient reversible light-induced interconversion between the 13-cis and all-trans unphotolyzed states of the pigment. The relative amount of ASR with cis and trans chromophore forms depends on the wavelength of illumination, providing a mechanism for single-pigment color sensing analogous to that of phytochrome pigments. In addition ASR exhibits unusually slow formation of L-like and M-like intermediates, with a dominant accumulation of M during the photocycle. Co-expression of ASR with its putative cytoplasmic transducer protein shifts the absorption maximum and strongly decreases the rate of dark adaptation of ASR, confirming interaction between the two proteins. Thus ASR, the first non-haloarchaeal sensory rhodopsin character-ized, demonstrates the diversity of photochemistry of microbial rhodopsins. Its photochromic properties and the position of its two ground state absorption maxima suggest it as a candidate for controlling differential photosynthetic light-harvesting pigment synthesis (chromatic adaptation) or other colorsensitive physiological responses in Anabaena cells.Over the past 5 years microbial genomics has revealed a large family of photoactive, seven-transmembrane helix, retinylidene proteins called microbial (or type 1) rhodopsins (1) in phylogenetically diverse species including haloarchaea (2), proteobacteria (3, 4), cyanobacteria (5), fungi, and algae (6 -8). The best characterized are the first discovered members of this family found in Halobacterium salinarum over 20 years ago: the light-driven ion pumps bacteriorhodopsin (BR) 1 and halorhodopsin and the phototaxis receptors sensory rhodopsins I and II (SRI and SRII). Nearly all of our knowledge of properties of type 1 rhodopsins (such as isomeric configuration and conformation of their chromophore, photochemical reactions, lightinduced conformational changes in the protein, and function) has been obtained from these four related pigments from H. salinarum. To examine the diversity of properties of the family, now known to be present in eubacteria and eukaryotic microorganisms as well as Archaea (1-8), studies of non-haloarchaeal pigments are needed. Anabaena sensory rhodopsin (ASR) is well suited for exploration (5). It is the first known prokaryotic sensory representative outside of haloarchaea, and its function appears to involve activation of a cytoplasmic transducer, unlike SRI and SRII, which transmit signals to other integral membrane proteins (methyl-accepting taxis t...

A recently discovered family of natural anion channelrhodopsins (ACRs) have the highest conductance among channelrhodopsins and exhibit exclusive anion selectivity, which make them efficient inhibitory tools for optogenetics. We report analysis of flashinduced absorption changes in purified wild-type and mutant ACRs, and of photocurrents they generate in HEK293 cells. Contrary to cation channelrhodopsins (CCRs), the ion conducting state of ACRs develops in an L-like intermediate that precedes the deprotonation of the retinylidene Schiff base (i.e., formation of an M intermediate). Channel closing involves two mechanisms leading to depletion of the conducting L-like state: (i) Fast closing is caused by a reversible L⇔M conversion. Glu-68 in Guillardia theta ACR1 plays an important role in this transition, likely serving as a counterion and proton acceptor at least at high and neutral pH. Incomplete suppression of M formation in the GtACR1_E68Q mutant indicates the existence of an alternative proton acceptor. (ii) Slow closing of the channel parallels irreversible depletion of the M-like and, hence, L-like state. Mutation of Cys-102 that strongly affected slow channel closing slowed the photocycle to the same extent. The L and M intermediates were in equilibrium in C102A as in the WT. In the position of Glu-123 in channelrhodopsin-2, ACRs contain a noncarboxylate residue, the mutation of which to Glu produced early Schiff base proton transfer and strongly inhibited channel activity. The data reveal fundamental differences between natural ACR and CCR conductance mechanisms and their underlying photochemistry, further confirming that these proteins form distinct families of rhodopsin channels.photochemical conversions | Schiff base | channel gating | channelrhodopsins | optogenetics T he genomes of cryptophyte algae harbor nucleotide sequences that encode anion-conducting channelrhodopsins (ACRs) (1, 2). Although these proteins show distant sequence homology to cation-conducting channelrhodopsins (CCRs) from green (chlorophyte) algae, they completely lack permeability for protons and metal cations and, thus, represent a distinct structural and functional class among microbial rhodopsins. Using ACRs permits optogenetic inhibition of neuronal firing at much lower light intensities than other currently used silencers (1).Analysis of photocurrents generated by ACR1 from Guillardia theta (GtACR1) in human embryonic kidney (HEK) cells under single-turnover conditions revealed that GtACR1 gating comprises two separate mechanisms with opposite dependencies on the membrane voltage and pH, and involving different amino acid residues (3). The first mechanism, characterized by fast closing of the channel, is regulated by Glu-68, a homolog of Glu-90 in channelrhodopsin-2 from the green alga Chlamydomonas reinhardtii (CrChR2). Replacement of Glu-90 with Arg in CrChR2 made the channel permeable for Cl − (4), whereas the same substitution of Glu-68 in GtACR1 did not influence its selectivity for anions, but inverted its gating, ren...