Channelrhodopsins are microbial-type rhodopsins that function as light-gated cation channels. Understanding how the detailed architecture of the protein governs its dynamics and specificity for ions is important, because it has the potential to assist in designing site-directed channelrhodopsin mutants for specific neurobiology applications. Here we use bioinformatics methods to derive accurate alignments of channelrhodopsin sequences, assess the sequence conservation patterns and find conserved motifs in channelrhodopsins, and use homology modeling to construct three-dimensional structural models of channelrhodopsins. The analyses reveal that helices C and D of channelrhodopsins contain Cys, Ser, and Thr groups that can engage in both intra- and inter-helical hydrogen bonds. We propose that these polar groups participate in inter-helical hydrogen-bonding clusters important for the protein conformational dynamics and for the local water interactions. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.

Channelrhodopsins serve as photoreceptors that control the motility behavior of green flagellate algae and act as light-gated ion channels when heterologously expressed in animal cells. Here, we report direct measurements of proton transfer from the retinylidene Schiff base in several channelrhodopsin variants expressed in HEK293 cells. A fast outward-directed current precedes the passive channel current that has the opposite direction at physiological holding potentials. This rapid charge movement occurs on the timescale of the M intermediate formation in microbial rhodopsins, including that for channelrhodopsin from Chlamydomonas augustae and its mutants, reported in this study. Mutant analysis showed that the glutamate residue corresponding to Asp(85) in bacteriorhodopsin acts as the primary acceptor of the Schiff-base proton in low-efficiency channelrhodopsins. Another photoactive-site residue corresponding to Asp(212) in bacteriorhodopsin serves as an alternative proton acceptor and plays a more important role in channel opening than the primary acceptor. In more efficient channelrhodopsins from Chlamydomonas reinhardtii, Mesostigma viride, and Platymonas (Tetraselmis) subcordiformis, the fast current was apparently absent. The inverse correlation of the outward proton transfer and channel activity is consistent with channel function evolving in channelrhodopsins at the expense of their capacity for active proton transport.

Potassium-selective channelrhodopsins (KCRs) are light-gated K+ channels recently found in the stramenopile protist Hyphochytrium catenoides. When expressed in neurons, KCRs enable high-precision optical inhibition of spiking (optogenetic silencing). KCRs are capable of discriminating K+ from Na+ without the conventional K+ selectivity filter found in classical K+ channels. The genome of H. catenoides also encodes a third paralog that is more permeable for Na+ than for K+. To identify structural motifs responsible for the unusual K+ selectivity of KCRs, we systematically analyzed a series of chimeras and mutants of this protein. We found that mutations of three critical residues in the paralog convert its Na+-selective channel into a K+-selective one. Our characterization of homologous proteins from other protists (Colponema vietnamica, Cafeteria burkhardae, and Chromera velia) and metagenomic samples confirmed the importance of these residues for K+ selectivity. We also show that Trp102 and Asp116, conserved in all three H. catenoides paralogs, are necessary, although not sufficient, for K+ selectivity. Our results provide the foundation for further engineering of KCRs for optogenetic needs. IMPORTANCE Recently discovered microbial light-gated ion channels (channelrhodopsins) with a higher permeability for K+ than for Na+ (potassium-selective channelrhodopsins [kalium channelrhodopsins, or KCRs]) demonstrate an alternative K+ selectivity mechanism, unrelated to well-characterized "selectivity filters" of voltage- and ligand-gated K+ channels. KCRs can be used for optogenetic inhibition of neuronal firing and potentially for the development of gene therapies to treat neurological and cardiovascular disorders. In this study, we identified structural motifs that determine the K+ selectivity of KCRs that provide the foundation for their further improvement as optogenetic tools.

Optical control of the heart muscle is a promising strategy for cardiology because it is more specific than traditional electrical stimulation, and allows a higher temporal resolution than pharmacological interventions. Anion channelrhodopsins (ACRs) from cryptophyte algae expressed in cultured neonatal rat ventricular cardiomyocytes produced inhibitory currents at less than one-thousandth of the light intensity required by previously available optogenetic tools, such as the proton pump archaerhodopsin-3 (Arch). Because of their greater photocurrents, ACRs permitted complete inhibition of cardiomyocyte electrical activity under conditions in which Arch was inefficient. Most importantly, ACR expression allowed precisely controlled shortening of the action potential duration by switching on the light during its repolarization phase, which was not possible with previously used optogenetic tools. Optical shortening of cardiac action potentials may benefit pathophysiology research and the development of optogenetic treatments for cardiac disorders such as the long QT syndrome.

Optical silencing of activity provides a way to test the necessity of neurons in behaviour. Two light-gated anion channels, GtACR1 and GtACR2, have recently been shown to potently inhibit activity in cultured mammalian neurons and in Drosophila. Here, we test the usefulness of these channels in larval zebrafish, using spontaneous coiling behaviour as the assay.

Channelrhodopsins guide algal phototaxis and are widely used as optogenetic probes for control of membrane potential with light. "Bacteriorhodopsin-like" cation channelrhodopsins (BCCRs) from cryptophytes differ in primary structure from other CCRs, lacking usual residues important for their cation conductance. Instead, the sequences of BCCR match more closely those of rhodopsin proton pumps, containing residues responsible for critical proton transfer reactions. We report 19 new BCCRs which, together with the earlier 6 known members of this family, form three branches (subfamilies) of a phylogenetic tree. Here, we show that the conductance mechanisms in two subfamilies differ with respect to involvement of the homolog of the proton donor in rhodopsin pumps. Two BCCRs from the genus Rhodomonas generate photocurrents that rapidly desensitize under continuous illumination. Using a combination of patch clamp electrophysiology, absorption, Raman spectroscopy, and flash photolysis, we found that the desensitization is due to rapid accumulation of a long-lived nonconducting intermediate of the photocycle with unusually blue-shifted absorption with a maximum at 330 nm. These observations reveal diversity within the BCCR family and contribute to deeper understanding of their independently evolved cation channel function.IMPORTANCE Cation channelrhodopsins, light-gated channels from flagellate green algae, are extensively used as optogenetic photoactivators of neurons in research and recently have progressed to clinical trials for vision restoration. However, the molecular mechanisms of their photoactivation remain poorly understood. We recently identified cryptophyte cation channelrhodopsins, structurally different from those of green algae, which have separately evolved to converge on light-gated cation conductance. This study reveals diversity within this new protein family and describes a subclade with unusually rapid desensitization that results in short transient photocurrents in continuous light. Such transient currents have not been observed in the green algae channelrhodopsins and are potentially useful in optogenetic protocols. Kinetic UV-visible (UV-vis) spectroscopy and photoelectrophysiology reveal that the desensitization is caused by rapid accumulation of a nonconductive photointermediate in the photochemical reaction cycle. The absorption maximum of the intermediate is 330 nm, the shortest wavelength reported in any rhodopsin, indicating a novel chromophore structure.

Many channelrhodopsins are permeable to protons. We found that in neurons, activation of a high-current channelrhodopsin, CheRiff, led to significant acidification, with faster acidification in the dendrites than in the soma. Experiments with patterned optogenetic stimulation in monolayers of HEK cells established that the acidification was due to proton transport through the opsin, rather than through other voltage-dependent channels. We identified and characterized two opsins which showed large photocurrents, but small proton permeability, PsCatCh2.0 and ChR2-3M. PsCatCh2.0 showed excellent response kinetics and was also spectrally compatible with simultaneous voltage imaging with QuasAr6a. Stimulation-evoked acidification is a possible source of disruptions to cell health in scientific and prospective therapeutic applications of optogenetics. Channelrhodopsins with low proton permeability are a promising strategy for avoiding these problems.

Channelrhodopsins are light-gated ion channels used to control excitability of designated cells in large networks with high spatiotemporal resolution. While ChRs selective for H+, Na+, K+ and anions have been discovered or engineered, Ca2+-selective ChRs have not been reported to date. Here, we analyse ChRs and mutant derivatives with regard to their Ca2+ permeability and improve their Ca2+ affinity by targeted mutagenesis at the central selectivity filter. The engineered channels, termed CapChR1 and CapChR2 for calcium-permeable channelrhodopsins, exhibit reduced sodium and proton conductance in connection with strongly improved Ca2+ permeation at negative voltage and low extracellular Ca2+ concentrations. In cultured cells and neurons, CapChR2 reliably increases intracellular Ca2+ concentrations. Moreover, CapChR2 can robustly trigger Ca2+ signalling in hippocampal neurons. When expressed together with genetically encoded Ca2+ indicators in Drosophila melanogaster mushroom body output neurons, CapChRs mediate light-evoked Ca2+ entry in brain explants.

KCR channelrhodopsins (K+-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K+ selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K+ selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K+ selectivity; rather than forming the symmetrical filter of canonical K+ channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K+ selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K+ selectivity also provides a framework for next-generation optogenetics.

Cation and anion channelrhodopsins (CCRs and ACRs, respectively) primarily from two algal species, Chlamydomonas reinhardtii and Guillardia theta, have become widely used as optogenetic tools to control cell membrane potential with light. We mined algal and other protist polynucleotide sequencing projects and metagenomic samples to identify 75 channelrhodopsin homologs from four channelrhodopsin families, including one revealed in dinoflagellates in this study. We carried out electrophysiological analysis of 33 natural channelrhodopsin variants from different phylogenetic lineages and 10 metagenomic homologs in search of sequence determinants of ion selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins. Our results show that association of a reduced number of glutamates near the conductance path with anion selectivity depends on a wider protein context, because prasinophyte homologs with a glutamate pattern identical to that in cryptophyte ACRs are cation selective. Desensitization is also broadly context dependent, as in one branch of stramenopile ACRs and their metagenomic homologs, its extent roughly correlates with phylogenetic relationship of their sequences. Regarding spectral tuning, we identified two prasinophyte CCRs with red-shifted spectra to 585 nm. They exhibit a third residue pattern in their retinal-binding pockets distinctly different from those of the only two types of red-shifted channelrhodopsins known (i.e., the CCR Chrimson and RubyACRs). In cryptophyte ACRs we identified three specific residue positions in the retinal-binding pocket that define the wavelength of their spectral maxima. Lastly, we found that dinoflagellate rhodopsins with a TCP motif in the third transmembrane helix and a metagenomic homolog exhibit channel activity. IMPORTANCE Channelrhodopsins are widely used in neuroscience and cardiology as research tools and are considered prospective therapeutics, but their natural diversity and mechanisms remain poorly characterized. Genomic and metagenomic sequencing projects are producing an ever-increasing wealth of data, whereas biophysical characterization of the encoded proteins lags behind. In this study, we used manual and automated patch clamp recording of representative members of four channelrhodopsin families, including a family in dinoflagellates that we report in this study. Our results contribute to a better understanding of molecular determinants of ionic selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins.

Optogenetic silencing allows time-resolved functional interrogation of defined neuronal populations. However, the limitations of inhibitory optogenetic tools impose stringent constraints on experimental paradigms. The high light power requirement of light-driven ion pumps and their effects on intracellular ion homeostasis pose unique challenges, particularly in experiments that demand inhibition of a widespread neuronal population in vivo. Guillardia theta anion-conducting channelrhodopsins (GtACRs) are promising in this regard, due to their high single-channel conductance and favorable photon-ion stoichiometry. However, GtACRs show poor membrane targeting in mammalian cells, and the activity of such channels can cause transient excitation in the axon due to an excitatory chloride reversal potential in this compartment. Here, we address these problems by enhancing membrane targeting and subcellular compartmentalization of GtACRs. The resulting soma-targeted GtACRs show improved photocurrents, reduced axonal excitation and high light sensitivity, allowing highly efficient inhibition of neuronal activity in the mammalian brain.

Water transport through water channels, aquaporins (AQPs), is vital for many physiological processes including epithelial fluid secretion, cell migration and adipocyte metabolism. Water flux through AQPs is driven by the osmotic gradient that results from concentration differences of solutes including ions. Here, we developed a novel optogenetic toolkit that combines the light-gated anion channel GtACR1 either with the light-gated K+ channel HcKCR1 or the new Na+ channelrhodopsin HcNCR1 with high Na+ permeability, to manipulate water transport in Xenopus oocytes non-invasively. Water efflux through AQP was achieved by light-activating K+ and Cl- efflux through HcKCR1 and GtACR1. Contrarily, when GtACR1 was co-expressed with HcNCR1, inward movement of Na+ and Cl- was light-triggered, and the resulting osmotic gradient led to water influx through AQP1. In sum, we demonstrate a novel optogenetic strategy to manipulate water movement into or out of Xenopus oocytes non-invasively. This approach provides a new avenue to interfere with water homeostasis as a means to study related biological phenomena across cell types and organisms.

Manipulation of neuronal activity through genetically targeted actuator molecules is a powerful approach for studying information flow in the brain. In these approaches the genetically targeted component, a receptor or a channel, is activated either by a small molecule (chemical genetics) or by light from a physical source (optogenetics). We developed a hybrid technology that allows control of the same neurons by both optogenetic and chemical genetic means. The approach is based on engineered chimeric fusions of a light-generating protein (luciferase) to a light-activated ion channel (channelrhodopsin). Ionic currents then can be activated by bioluminescence upon activation of luciferase by its substrate, coelenterazine (CTZ), as well as by external light. In cell lines, expression of the fusion of Gaussia luciferase to Channelrhodopsin-2 yielded photocurrents in response to CTZ. Larger photocurrents were produced by fusing the luciferase to Volvox Channelrhodopsin-1. This version allowed chemical modulation of neuronal activity when expressed in cultured neurons: CTZ treatment shifted neuronal responses to injected currents and sensitized neurons to fire action potentials in response to subthreshold synaptic inputs. These luminescent channelrhodopsins--or luminopsins--preserve the advantages of light-activated ion channels, while extending their capabilities. Our proof-of-principle results suggest that this novel class of tools can be improved and extended in numerous ways.

Channelrhodopsin-1 and 2 (ChR1 and ChR2) form cation channels that are gated by light through an unknown mechanism. We tested the DC-gate hypothesis that C167 and D195 are involved in the stabilization of the cation-permeable state of ChRWR/C1C2 which consists of TM1-5 of ChR1 and TM6-7 of ChR2 and ChRFR which consists of TM1-2 of ChR1 and TM3-7 of ChR2. The cation permeable state of each ChRWR and ChRFR was markedly prolonged in the order of several tens of seconds when either C167 or D195 position was mutated to alanine (A). Therefore, the DC-gate function was conserved among these chimeric ChRs. We next investigated the kinetic properties of the ON/OFF response of these bi-stable ChR mutants as they are important in designing the photostimulation protocols for the optogenetic manipulation of neuronal activities. The turning-on rate constant of each photocurrent followed a linear relationship to 0-0.12 mW mm(-2) of blue LED light or to 0-0.33 mW mm(-2) of cyan LED light. Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance. As the magnitude of the photocurrent was mostly determined by the turning-on rate constant and the irradiation time, the minimal irradiance that effectively evoked an action potential (threshold irradiance) was decreased with time only if the neuron, which expresses bi-stable ChRs, has a certain large membrane time constant (eg. τm > 20 ms). On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time. Based on these quantitative data, we would propose that these bi-stable ChRs would be most suitable for enhancing the intrinsic activity of excitatory pyramidal neurons at a minimal magnitude of irradiance.

Optogenetic inhibition of specific neuronal types in the brain enables analysis of neural circuitry and is promising for the treatment of a number of neurological disorders. Anion channelrhodopsins (ACRs) from the cryptophyte alga Guillardia theta generate larger photocurrents than other available inhibitory optogenetic tools, but more rapid channels are needed for temporally precise inhibition, such as single-spike suppression, of high-frequency firing neurons. Faster ACRs have been reported, but their potential advantages for time-resolved inhibitory optogenetics have not so far been verified in neurons. We report RapACR, nicknamed so for "rapid," an ACR from Rhodomonas salina, that exhibits channel half-closing times below 10 ms and achieves equivalent inhibition at 50-fold lower light intensity in lentivirally transduced cultured mouse hippocampal neurons as the second-generation engineered Cl--conducting channelrhodopsin iC++. The upper limit of the time resolution of neuronal silencing with RapACR determined by measuring the dependence of spiking recovery after photoinhibition on the light intensity was calculated to be 100 Hz, whereas that with the faster of the two G. theta ACRs was 13 Hz. Further acceleration of RapACR channel kinetics was achieved by site-directed mutagenesis of a single residue in transmembrane helix 3 (Thr111 to Cys). We also show that mutation of another ACR (Cys to Ala at the same position) with a greatly extended lifetime of the channel open state acts as a bistable photochromic tool in mammalian neurons. These molecules extend the time domain of optogenetic neuronal silencing while retaining the high light sensitivity of Guillardia ACRs.

Channelrhodopsins (ChRs) are light-gated ion channels widely used as optogenetic tools for manipulating neuronal activity. The currently characterized ChR families include green algal and cryptophyte cation-conducting ChRs (CCRs) and cryptophyte, haptophyte, and stramenopile anion-conducting ChRs (ACRs). Here, we report the discovery of a new family of phylogenetically distinct ChRs encoded by marine giant viruses and acquired from their unicellular green algal hosts. These previously unknown viral and green algal ChRs act as ACRs when expressed in cultured neuroblastoma-derived cells and are likely involved in behavioral responses to light.

Channelrhodopsin (ChR)-1 and ChR2 were the first-identified members of ChRs which are a growing subfamily of microbial-type rhodopsins. Light absorption drives the generation of a photocurrent in cell membranes expressing ChR2. However, the photocurrent amplitude attenuates and becomes steady-state during prolonged irradiation. This process, called desensitization or inactivation, has been attributed to the accumulation of intermediates less conductive to cations. Here we provided evidence that the dark-adapted (DA) photocurrent before desensitization is kinetically different from the light-adapted (LA) one after desensitization, that is, the deceleration of both basal-to-conductive and conductive-to-basal transitions. When the kinetics were compared between the DA and LA photocurrents for the ChR1/2 chimeras, the transmembrane helices, TM1 and TM2, were the determinants of both basal-to-conductive and conductive-to-basal transitions, whereas TM4 may contribute to the basal-to-conductive transitions and TM5 may contribute to the conductive-to-basal transitions, respectively. The fact that the desensitization-dependent decrease of the basal-to-conductive and conductive-to-basal transitions was facilitated by the TM1 exchange from ChR2 to ChR1 and reversed by the further TM2 exchange suggests that the conformation change for the channel gating is predominantly regulated by the interaction between TM1 and TM2. Although the exchange of TM1 from ChR2 to ChR1 showed no obvious influence on the spectral sensitivity, this exchange significantly induced the desensitization-dependent blue shift. Therefore, the TM1 and 2 are the main structures involved in two features of the desensitization, the stabilization of protein conformation and the charge distribution around the retinal-Schiff base (RSB+).

Channelrhodopsins (ChRs) are algal light-gated ion channels widely used as optogenetic tools for manipulating neuronal activity. ChRs desensitize under continuous bright-light illumination, resulting in a significant decline of photocurrents. Here we describe a metagenomically identified family of phylogenetically distinct anion-conducting ChRs (designated MerMAIDs). MerMAIDs almost completely desensitize during continuous illumination due to accumulation of a late non-conducting photointermediate that disrupts the ion permeation pathway. MerMAID desensitization can be fully explained by a single photocycle in which a long-lived desensitized state follows the short-lived conducting state. A conserved cysteine is the critical factor in desensitization, as its mutation results in recovery of large stationary photocurrents. The rapid desensitization of MerMAIDs enables their use as optogenetic silencers for transient suppression of individual action potentials without affecting subsequent spiking during continuous illumination. Our results could facilitate the development of optogenetic tools from metagenomic databases and enhance general understanding of ChR function.

The medial amygdala (MeA) receives pheromone information about conspecifics and has crucial functions in social behaviors. A previous study showed that activation of GABA neurons in the postero-dorsal MeA (MeApd) with channelrhodopsin-2H134R (ChR2) stimulates inter-male aggression. When performing these experiments using the faster channelrhodopsinH134R,E123T (ChETA), we find the opposite behavioral outcome. A systematic comparison between the two channelrhodopsin variants reveals that optogenetic activation of MeApd GABA neurons with ChETA suppresses aggression, whereas activation under ChR2 increases aggression. Although the mechanism for this paradoxical difference is not understood, we observe that activation of MeApd GABA neurons with ChR2 causes larger plateau depolarizations, smaller action potentials, and larger local inhibition than with ChETA. Thus, the channelrhodopsin variant used for in vivo optogenetic experiments can radically influence the behavioral outcome. Future work should continue to study the role of specific sub-populations of MeApd GABA neurons in aggression control.

Genetic engineering of natural light-gated ion channels has proven a powerful way to generate optogenetic tools for a wide variety of applications. In recent years, blue-light activated engineered anion-conducting channelrhodopsins (eACRs) have been developed, improved, and were successfully applied in vivo. We asked whether the approaches used to create eACRs can be transferred to other well-characterized cation-conducting channelrhodopsins (CCRs) to obtain eACRs with a broad spectrum of biophysical properties. We generated 22 variants using two conversion strategies applied to 11 CCRs and screened them for membrane expression, photocurrents and anion selectivity. We obtained two novel eACRs, Phobos and Aurora, with blue- and red-shifted action spectra and photocurrents similar to existing eACRs. Furthermore, step-function mutations greatly enhanced the cellular operational light sensitivity due to a slowed-down photocycle. These bi-stable eACRs can be reversibly toggled between open and closed states with brief light pulses of different wavelengths. All new eACRs reliably inhibited action potential firing in pyramidal CA1 neurons. In Drosophila larvae, eACRs conveyed robust and specific light-dependent inhibition of locomotion and nociception.