Channel Gating in Kalium Channelrhodopsin Slow Mutants.

Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is the first discovered natural light-gated ion channel that shows higher selectivity to K+ than to Na+ and therefore is used to silence neurons with light (optogenetics). Replacement of the conserved cysteine residue in the transmembrane helix 3 (Cys110) with alanine or threonine results in a >1,000-fold decrease in the channel closing rate. The phenotype of the corresponding mutants in channelrhodopsin 2 is attributed to breaking of a specific interhelical hydrogen bond (the "DC gate"). Unlike CrChR2 and other ChRs with long distance "DC gates", the HcKCR1 structure does not reveal any hydrogen bonding partners to Cys110, indicating that the mutant phenotype is likely caused by disruption of direct interaction between this residue and the chromophore. In HcKCR1_C110A, fast photochemical conversions corresponding to channel gating were followed by dramatically slower absorption changes. Full recovery of the unphotolyzed state in HcKCR1_C110A was extremely slow with two time constants 5.2 and 70 min. Analysis of the light-minus-dark difference spectra during these slow processes revealed accumulation of at least four spectrally distinct blue light-absorbing photocycle intermediates, L, M1 and M2, and a UV light-absorbing form, typical of bacteriorhodopsin-like channelrhodopsins from cryptophytes. Our results contribute to better understanding of the mechanistic links between the chromophore photochemistry and channel conductance, and provide the basis for using HcKCR1_C110A as an optogenetic tool.

Channelrhodopsins: From Phototaxis to Optogenetics.

Channelrhodopsins stand out among other retinal proteins because of their capacity to generate passive ionic currents following photoactivation. Owing to that, channelrhodopsins are widely used in neuroscience and cardiology as instruments for optogenetic manipulation of the activity of excitable cells. Photocurrents generated by channelrhodopsins were first discovered in the cells of green algae in the 1970s. In this review we describe this discovery and discuss the current state of research in the field.

Structures of channelrhodopsin paralogs in peptidiscs explain their contrasting K+ and Na+ selectivities.

Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is a light-gated channel used for optogenetic silencing of mammalian neurons. It selects K+ over Na+ in the absence of the canonical tetrameric K+ selectivity filter found universally in voltage- and ligand-gated channels. The genome of H. catenoides also encodes a highly homologous cation channelrhodopsin (HcCCR), a Na+ channel with >100-fold larger Na+ to K+ permeability ratio. Here, we use cryo-electron microscopy to determine atomic structures of these two channels embedded in peptidiscs to elucidate structural foundations of their dramatically different cation selectivity. Together with structure-guided mutagenesis, we show that K+ versus Na+ selectivity is determined at two distinct sites on the putative ion conduction pathway: in a patch of critical residues in the intracellular segment (Leu69/Phe69, Ile73/Ser73 and Asp116) and within a cluster of aromatic residues in the extracellular segment (primarily, Trp102 and Tyr222). The two filters are on the opposite sides of the photoactive site involved in channel gating.

Sequential absorption of two photons creates a bistable form of RubyACR responsible for its strong desensitization.

Channelrhodopsins with red-shifted absorption, rare in nature, are highly desired for optogenetics because light of longer wavelengths more deeply penetrates biological tissue. RubyACRs (Anion ChannelRhodopsins), a group of four closely related anion-conducting channelrhodopsins from thraustochytrid protists, are the most red-shifted channelrhodopsins known with absorption maxima up to 610 nm. Their photocurrents are large, as is typical of blue- and green-absorbing ACRs, but they rapidly decrease during continuous illumination (desensitization) and extremely slowly recover in the dark. Here, we show that long-lasting desensitization of RubyACRs results from photochemistry not observed in any previously studied channelrhodopsins. Absorption of a second photon by a photocycle intermediate with maximal absorption at 640 nm (P640) renders RubyACR bistable (i.e., very slowly interconvertible between two spectrally distinct forms). The photocycle of this bistable form involves long-lived nonconducting states (Llong and Mlong), formation of which is the reason for long-lasting desensitization of RubyACR photocurrents. Both Llong and Mlong are photoactive and convert to the initial unphotolyzed state upon blue or ultraviolet (UV) illumination, respectively. We show that desensitization of RubyACRs can be reduced or even eliminated by using ns laser flashes, trains of short light pulses instead of continuous illumination to avoid formation of Llong and Mlong, or by application of pulses of blue light between pulses of red light to photoconvert Llong to the initial unphotolyzed state.

Potassium-selective channelrhodopsins.

Since their discovery 21 years ago, channelrhodopsins have come of age and have become indispensable tools for optogenetic control of excitable cells such as neurons and myocytes. Potential therapeutic utility of channelrhodopsins has been proven by partial vision restoration in a human patient. Previously known channelrhodopsins are either proton channels, non-selective cation channels almost equally permeable to Na+ and K+ besides protons, or anion channels. Two years ago, we discovered a group of channelrhodopsins that exhibit over an order of magnitude higher selectivity for K+ than for Na+. These proteins, known as "kalium channelrhodopsins" or KCRs, lack the canonical tetrameric selectivity filter found in voltage- and ligand-gated K+ channels, and use a unique selectivity mechanism intrinsic to their individual protomers. Mutant analysis has revealed that the key residues responsible for K+ selectivity in KCRs are located at both ends of the putative cation conduction pathway, and their role has been confirmed by high-resolution KCR structures. Expression of KCRs in mouse neurons and human cardiomyocytes enabled optical inhibition of these cells' electrical activity. In this minireview we briefly discuss major results of KCR research obtained during the last two years and suggest some directions of future research.

Structural Foundations of Potassium Selectivity in Channelrhodopsins.

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.

Biophysical characterization of light-gated ion channels using planar automated patch clamp.

Channelrhodopsins (ChRs) are proteins that guide phototaxis in protists and exhibit light-gated channel conductance when their genes are heterologously expressed in mammalian cells. ChRs are widely used as molecular tools to control neurons and cardiomyocytes with light (optogenetics). Cation- and anion-selective ChRs (CCRs and ACRs, respectively) enable stimulation and inhibition of neuronal activity by depolarization and hyperpolarization of the membrane, respectively. More than 400 natural ChR variants have been identified so far, and high-throughput polynucleotide sequencing projects add many more each year. However, electrophysiological characterization of new ChRs lags behind because it is mostly done by time-consuming manual patch clamp (MPC). Here we report using a high-throughput automated patch clamp (APC) platform, SyncroPatch 384i from Nanion Technologies, for ChR research. We find that this instrument can be used for determination of the light intensity dependence and current-voltage relationships in ChRs and discuss its advantages and limitations.

Kalium channelrhodopsins are natural light-gated potassium channels that mediate optogenetic inhibition.

Channelrhodopsins are used widely for optical control of neurons, in which they generate photoinduced proton, sodium or chloride influx. Potassium (K+) is central to neuron electrophysiology, yet no natural K+-selective light-gated channel has been identified. Here, we report kalium channelrhodopsins (KCRs) from Hyphochytrium catenoides. Previously known gated potassium channels are mainly ligand- or voltage-gated and share a conserved K+-selectivity filter. KCRs differ in that they are light-gated and have independently evolved an alternative K+ selectivity mechanism. The KCRs are potent, highly selective of K+ over Na+, and open in less than 1 ms following photoactivation. The permeability ratio PK/PNa of 23 makes H. catenoides KCR1 (HcKCR1) a powerful hyperpolarizing tool to suppress excitable cell firing upon illumination, demonstrated here in mouse cortical neurons. HcKCR1 enables optogenetic control of K+ gradients, which is promising for the study and potential treatment of potassium channelopathies such as epilepsy, Parkinson's disease and long-QT syndrome and other cardiac arrhythmias.

Cation and Anion Channelrhodopsins: Sequence Motifs and Taxonomic Distribution.

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.

The crystal structure of bromide-bound GtACR1 reveals a pre-activated state in the transmembrane anion tunnel.

The crystal structure of the light-gated anion channel GtACR1 reported in our previous Research Article (Li et al., 2019) revealed a continuous tunnel traversing the protein from extracellular to intracellular pores. We proposed the tunnel as the conductance channel closed by three constrictions: C1 in the extracellular half, mid-membrane C2 containing the photoactive site, and C3 on the cytoplasmic side. Reported here, the crystal structure of bromide-bound GtACR1 reveals structural changes that relax the C1 and C3 constrictions, including a novel salt-bridge switch mechanism involving C1 and the photoactive site. These findings indicate that substrate binding induces a transition from an inactivated state to a pre-activated state in the dark that facilitates channel opening by reducing free energy in the tunnel constrictions. The results provide direct evidence that the tunnel is the closed form of the channel of GtACR1 and shed light on the light-gated channel activation mechanism.

Probing Channelrhodopsin Electrical Activity in Algal Cell Populations.

Photoelectric recording from populations of phototactic flagellate algae provides a means to study channelrhodopsin functions in vivo. Technical simplicity, versatility, high sensitivity, and reproducibility are the advantages of this assay over recording from individual algal cells by the suction pipette technique. Here we describe the principles and procedures of this assay.

Emerging Diversity of Channelrhodopsins and Their Structure-Function Relationships.

Cation and anion channelrhodopsins (CCRs and ACRs, respectively) from phototactic algae have become widely used as genetically encoded molecular tools to control cell membrane potential with light. Recent advances in polynucleotide sequencing, especially in environmental samples, have led to identification of hundreds of channelrhodopsin homologs in many phylogenetic lineages, including non-photosynthetic protists. Only a few CCRs and ACRs have been characterized in detail, but there are indications that ion channel function has evolved within the rhodopsin superfamily by convergent routes. The diversity of channelrhodopsins provides an exceptional platform for the study of structure-function evolution in membrane proteins. Here we review the current state of channelrhodopsin research and outline perspectives for its further development.

RubyACRs, nonalgal anion channelrhodopsins with highly red-shifted absorption.

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.

Conductance Mechanisms of Rapidly Desensitizing Cation Channelrhodopsins from Cryptophyte Algae.

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.

Opposite Charge Movements Within the Photoactive Site Modulate Two-Step Channel Closing in GtACR1.

Guillardia theta anion channelrhodopsin 1 is a light-gated anion channel widely used as an optogenetic inhibitory tool. Our recently published crystal structure of its dark (closed) state revealed that the photoactive retinylidene chromophore is located midmembrane in a full-length intramolecular tunnel through the protein, the radius of which is less than that of a chloride ion. Here we show that acidic (glutamate) substitutions for residues within the inner half-tunnel enhance the fast channel closing and, for residues within the outer half-tunnel, enhance the slow channel closing. The magnitude of these effects was proportional to the distance of the mutated residue from the photoactive site. These data indicate that the local electrical field across the photoactive site controls fast and slow channel closing, involving outward and inward charge displacements. In the purified mutant proteins, we observed corresponding opposite changes in kinetics of the M photocycle intermediate. A correlation between fast closing and M rise and slow closing and M decay observed in the mutants suggests that the Schiff base proton is one of the displaced charges. Opposite signs of the effects indicate that deprotonation and reprotonation of the Schiff base take place on the same (outer) side of the membrane and explains opposite rectification of fast and slow channel closing. Оur comprehensive protein-wide acidic residue substitution screen shows that only mutations of the residues located in the intramolecular tunnel confer strong rectification, which confirms the prediction that the tunnel expands upon photoexcitation to form the anion pathway.

Crystal structure of a natural light-gated anion channelrhodopsin.

The anion channelrhodopsin GtACR1 from the alga Guillardia theta is a potent neuron-inhibiting optogenetics tool. Presented here, its X-ray structure at 2.9 Å reveals a tunnel traversing the protein from its extracellular surface to a large cytoplasmic cavity. The tunnel is lined primarily by small polar and aliphatic residues essential for anion conductance. A disulfide-immobilized extracellular cap facilitates channel closing and the ion path is blocked mid-membrane by its photoactive retinylidene chromophore and further by a cytoplasmic side constriction. The structure also reveals a novel photoactive site configuration that maintains the retinylidene Schiff base protonated when the channel is open. These findings suggest a new channelrhodopsin mechanism, in which the Schiff base not only controls gating, but also serves as a direct mediator for anion flux.

Extending the Time Domain of Neuronal Silencing with Cryptophyte Anion Channelrhodopsins.

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.

Bacteriorhodopsin-like channelrhodopsins: Alternative mechanism for control of cation conductance.

The recently discovered cation-conducting channelrhodopsins in cryptophyte algae are far more homologous to haloarchaeal rhodopsins, in particular the proton pump bacteriorhodopsin (BR), than to earlier known channelrhodopsins. They uniquely retain the two carboxylate residues that define the vectorial proton path in BR in which Asp-85 and Asp-96 serve as acceptor and donor, respectively, of the photoactive site Schiff base (SB) proton. Here we analyze laser flash-induced photocurrents and photochemical conversions in Guillardia theta cation channelrhodopsin 2 (GtCCR2) and its mutants. Our results reveal a model in which the GtCCR2 retinylidene SB chromophore rapidly deprotonates to the Asp-85 homolog, as in BR. Opening of the cytoplasmic channel to cations in GtCCR2 requires the Asp-96 homolog to be unprotonated, as has been proposed for the BR cytoplasmic channel for protons. However, reprotonation of the GtCCR2 SB occurs not from the Asp-96 homolog, but by proton return from the earlier protonated acceptor, preventing vectorial proton translocation across the membrane. In GtCCR2, deprotonation of the Asp-96 homolog is required for cation channel opening and occurs >10-fold faster than reprotonation of the SB, which temporally correlates with channel closing. Hence in GtCCR2, cation channel gating is tightly coupled to intramolecular proton transfers involving the same residues that define the vectorial proton path in BR.

The Expanding Family of Natural Anion Channelrhodopsins Reveals Large Variations in Kinetics, Conductance, and Spectral Sensitivity.

Natural anion channelrhodopsins (ACRs) discovered in the cryptophyte alga Guillardia theta generate large hyperpolarizing currents at membrane potentials above the Nernst equilibrium potential for Cl- and thus can be used as efficient inhibitory tools for optogenetics. We have identified and characterized new ACR homologs in different cryptophyte species, showing that all of them are anion-selective, and thus expanded this protein family to 20 functionally confirmed members. Sequence comparison of natural ACRs and engineered Cl--conducting mutants of cation channelrhodopsins (CCRs) showed radical differences in their anion selectivity filters. In particular, the Glu90 residue in channelrhodopsin 2, which needed to be mutated to a neutral or alkaline residue to confer anion selectivity to CCRs, is nevertheless conserved in all of the ACRs identified. The new ACRs showed a large variation of the amplitude, kinetics, and spectral sensitivity of their photocurrents. A notable variant, designated "ZipACR", is particularly promising for inhibitory optogenetics because of its combination of larger current amplitudes than those of previously reported ACRs and an unprecedentedly fast conductance cycle (current half-decay time 2-4 ms depending on voltage). ZipACR expressed in cultured mouse hippocampal neurons enabled precise photoinhibition of individual spikes in trains of up to 50 Hz frequency.

Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications.

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