Light-gated Anion Channels Research Articles

Optogenetic Determination of Dynamic and Cell-Type-Specific Inhibitory Reversal Potentials.

The reversal potential refers to the membrane potential at which the net current flow through a channel reverses direction. The reversal potential is determined by transmembrane ion gradients and, in turn, determines how the channel's activity will affect the membrane potential. Traditional investigation into the reversal potential of inhibitory ligand-gated ion channels (EInh) has relied upon the activation of endogenous receptors, such as the GABA-A receptor (GABAAR). There are, however, challenges associated with activating endogenous receptors, including agonist delivery, isolating channel responses, and the effects of receptor saturation and desensitization. Here, we demonstrate the utility of using a light-gated anion channel, stGtACR2, to probe EInh in the rodent brain. Using mice of both sexes, we demonstrate that the properties of this optically activated channel make it a suitable proxy for studying GABAAR receptor-mediated inhibition. We validate this agonist-independent optogenetic strategy in vitro and in vivo and further show how it can accurately capture differences in EInh dynamics following manipulations of endogenous ion fluxes. This allows us to explore distinct resting EInh differences across genetically defined neuronal subpopulations. Using this approach to challenge ion homeostasis mechanisms in neurons, we uncover cell-specific EInh dynamics that are supported by the differential expression of endogenous ion handling mechanisms. Our findings therefore establish an effective optical strategy for revealing novel aspects of inhibitory reversal potentials and thereby expand the repertoire of optogenetics.

Isospectral intermediates in the photochemical reaction cycle of anion channelrhodopsin GtACR1

The most effective tested optogenetic tools available for neuronal silencing are the light-gated anion channel proteins found in the cryptophyte alga Guillardia theta (GtACRs). Molecular mechanisms of GtACRs, including the photointermediates responsible for the open channel state, are of great interest for understanding their exceptional conductance. In this study, the photoreactions of GtACR1 and its D234N, A75E, and S97E mutants were investigated using multichannel time-resolved absorption spectroscopy. For each of the proteins, the analysis showed two early microsecond transitions between K-like and L-like forms and two late millisecond recovery steps. Spectral forms associated with potential molecular intermediates of the proteins were derived and their evolutions in time were analyzed. The results indicate the presence of isospectral intermediates in the photocycles and expand the range of potential intermediates responsible for the open channel state.

Combining different ion-selective channelrhodopsins to control water flux by light

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.

Cre-dependent ACR2-expressing reporter mouse strain for efficient long-lasting inhibition of neuronal activity

Optogenetics is a powerful tool for manipulating neuronal activity by light illumination with high temporal and spatial resolution. Anion-channelrhodopsins (ACRs) are light-gated anion channels that allow researchers to efficiently inhibit neuronal activity. A blue light-sensitive ACR2 has recently been used in several in vivo studies; however, the reporter mouse strain expressing ACR2 has not yet been reported. Here, we generated a new reporter mouse strain, LSL-ACR2, in which ACR2 is expressed under the control of Cre recombinase. We crossed this strain with a noradrenergic neuron-specific driver mouse (NAT-Cre) to generate NAT-ACR2 mice. We confirmed Cre-dependent expression and function of ACR2 in the targeted neurons by immunohistochemistry and electrophysiological recordings in vitro, and confirmed physiological function using an in vivo behavioral experiment. Our results show that the LSL-ACR2 mouse strain can be applied for optogenetic inhibition of targeted neurons, particularly for long-lasting continuous inhibition, upon crossing with Cre-driver mouse strains. The LSL-ACR2 strain can be used to prepare transgenic mice with homogenous expression of ACR2 in targeted neurons with a high penetration ratio, good reproducibility, and no tissue invasion.

Optogenetic control of the guard cell membrane potential and stomatal movement by the light-gated anion channel GtACR1.

Guard cells control the aperture of plant stomata, which are crucial for global fluxes of CO2 and water. In turn, guard cell anion channels are seen as key players for stomatal closure, but is activation of these channels sufficient to limit plant water loss? To answer this open question, we used an optogenetic approach based on the light-gated anion channelrhodopsin 1 (GtACR1). In tobacco guard cells that express GtACR1, blue- and green-light pulses elicit Cl- and NO3 - currents of -1 to -2 nA. The anion currents depolarize the plasma membrane by 60 to 80 mV, which causes opening of voltage-gated K+ channels and the extrusion of K+ As a result, continuous stimulation with green light leads to loss of guard cell turgor and closure of stomata at conditions that provoke stomatal opening in wild type. GtACR1 optogenetics thus provides unequivocal evidence that opening of anion channels is sufficient to close stomata.

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.

Green-Sensitive, Long-Lived, Step-Functional Anion Channelrhodopsin-2 Variant as a High-Potential Neural Silencing Tool

Anion channelrhodopsin-2 (GtACR2) was identified from the alga Guillardia theta as a light-gated anion channel, providing a powerful neural silencing tool for optogenetics. To expand its molecular properties, we produced here GtACR2 variants by strategic mutations on the four residues around the retinal chromophore (i.e., R129, G152, P204, and C233). After the screening with the Escherichia coli expression system, we estimated spectral sensitivities and the anion channeling function by using the HEK293 expression system. Among the mutants, triple (R129M/G152S/C233A) and quadruple (R129M/G152S/P204T/C233A) mutants showed the significantly red-shifted absorption maxima (λmax = 498 and 514 nm, respectively) and the long-lived channel-conducting states (the half-life times were 3.4 and 5.4 s, respectively). In addition, both mutants can be activated and inactivated by different wavelengths, representing their step-functional ability. We nicknamed the quadruple mutant "GLaS-ACR2" from its green-sensitive, long-lived, step-functional properties. The unique characteristics of GLaS-ACR2 suggest its high potential as a neural silencing tool.

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.

Quantitation of the neural silencing activity of anion channelrhodopsins in Caenorhabditis elegans and their applicability for long-term illumination

Ion pumps and channels are responsible for a wide variety of biological functions. Ion pumps transport only one ion during each stimulus-dependent reaction cycle, whereas ion channels conduct a large number of ions during each cycle. Ion pumping rhodopsins such as archaerhodopsin-3 (Arch) are often utilized as light-dependent neural silencers in animals, but they require a high-density light illumination of around 1 mW/mm2. Recently, anion channelrhodopsins -1 and -2 (GtACR1 and GtACR2) were discovered as light-gated anion channels from the cryptophyte algae Guillardia theta. GtACRs are therefore expected to silence neural activity much more efficiently than Arch. In this study, we successfully expressed GtACRs in neurons of the nematode Caenorhabditis elegans (C. elegans) and quantitatively evaluated how potently GtACRs can silence neurons in freely moving C. elegans. The results showed that the light intensity required for GtACRs to cause locomotion paralysis was around 1 µW/mm2, which is three orders of magnitude smaller than the light intensity required for Arch. As attractive features, GtACRs are less harmfulness to worms and allow stable neural silencing effects under long-term illumination. Our findings thus demonstrate that GtACRs possess a hypersensitive neural silencing activity in C. elegans and are promising tools for long-term neural silencing.

Structural mechanisms of selectivity and gating in anion channelrhodopsins.

Both designed and natural anion-conducting channelrhodopsins (dACRs and nACRs, respectively) have been widely applied in optogenetics (enabling selective inhibition of target-cell activity during animal behaviour studies), but each class exhibits performance limitations, underscoring trade-offs in channel structure-function relationships. Therefore, molecular and structural insights into dACRs and nACRs will be critical not only for understanding the fundamental mechanisms of these light-gated anionchannels, but also to create next-generation optogenetic tools. Here we report crystal structures of the dACR iC++, along with spectroscopic, electrophysiological and computational analyses that provide unexpected insights into pH dependence, substrate recognition, channel gating and ion selectivity of both dACRs and nACRs. These results enabled us to create an anion-conducting channelrhodopsin integrating the key features of large photocurrent and fast kinetics alongside exclusive anion selectivity.

Optical inhibition of larval zebrafish behaviour with anion channelrhodopsins

BackgroundOptical 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.ResultsWhen the GtACRs were expressed in spinal neurons of embryonic zebrafish and actuated with blue or green light, spontaneous movement was inhibited. In GtACR1-expressing fish, only 3 μW/mm2 of light was sufficient to have an effect; GtACR2, which is poorly trafficked, required slightly stronger illumination. No inhibition was seen in non-expressing siblings. After light offset, the movement of GtACR-expressing fish increased, which suggested that termination of light-induced neural inhibition may lead to activation. Consistent with this, two-photon imaging of spinal neurons showed that blue light inhibited spontaneous activity in spinal neurons of GtACR1-expressing fish, and that the level of intracellular calcium increased following light offset.ConclusionsThese results show that GtACR1 and GtACR2 can be used to optically inhibit neurons in larval zebrafish with high efficiency. The activity elicited at light offset needs to be taken into consideration in experimental design, although this property can provide insight into the effects of transiently stimulating a circuit.

An inhibitory role of Arg-84 in anion channelrhodopsin-2 expressed in Escherichia coli

Anion channelrhodopsin-2 (ACR2) was recently identified from the cryptophyte algae Guillardia theta and has become a focus of interest in part because of its novel light-gated anion channel activity and its extremely high neural silencing activity. In this study, we tried to express ACR2 in Escherichia coli cells as a recombinant protein. The E. coli cells expressing ACR2 showed an increase in pH upon blue-light illumination in the presence of monovalent anions and the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating an inward anion channel activity. Then, taking advantage of the E. coli expression system, we performed alanine-scanning mutagenesis on conserved basic amino acid residues. One of them, R84A, showed strong signals compared with the wild-type, indicating an inhibitory role of R84 on Cl− transportation. The signal was strongly enhanced in R84E, whereas R84K was less effective than the wild-type (i.e., R84). These results suggest that the positive charge at position 84 is critical for the inhibition. Thus we succeeded in functional expression of ACR2 in E. coli and found the inhibitory role of R84 during the anion transportation.

Natural Anion Channel Rhodopsins: A New Family of Tools for Optogenetic Neural Inhibition

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 efficient tools for membrane hyperpolarization. Anion Channel Rhodopsins (ACRs), natural light-gated anion channels recently discovered in cryptophyte algae [1], show distant homology to cation-conducting channelrhodopsins (CCRs), but strictly conduct anions. GtACR1 and GtACR2 from Guillardia theta hyperpolarize animal cell plasma membranes with much faster kinetics at less than one-thousandth of the light intensity required by the most efficient currently available optogenetic proteins. Their anion specificity, large currents, and fast kinetics enable ACRs to hyperpolarize and silence neurons through light-gated chloride conduction with unprecedented light sensitivity and temporal precision. The GtACRs are also the most conductive rhodopsin channels known, with 25-fold greater unitary conductance than the most commonly studied CCR, channelrhodopsin-2 [1]. Our initial findings on the gating mechanism of ACRs reveal that the structural basis for their anion conductance is fundamentally different from that of engineered chloride-conducting CCR mutants, not attributable to simply a modification of the CCR selectivity filter. During our analysis we identified an inverted mutant constitutively open in the dark and closed by light, the first such mutant of any light-gated channel, valuable for probing the structure of the channel open state. Also we identified a “step function” GtACR1 mutant useful in optogenetics [2].1. E.G. Govorunova, O.A. Sineshchekov, R. Janz, X. Liu, and J.L. Spudich (2015) Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics. Science 349:647-650.2. O.A. Sineshchekov, E.G. Govorunova, H. Li, and J.L. Spudich (2015) Gating mechanisms of a natural anion channelrhodopsin. Proc. Natl. Acad. Sci. USA, submitted.

NEUROSCIENCE. Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics.

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.

Structure-guided transformation of channelrhodopsin into a light-activated chloride channel.

Using light to silence electrical activity in targeted cells is a major goal of optogenetics. Available optogenetic proteins that directly move ions to achieve silencing are inefficient, pumping only a single ion per photon across the cell membrane rather than allowing many ions per photon to flow through a channel pore. Building on high-resolution crystal-structure analysis, pore vestibule modeling, and structure-guided protein engineering, we designed and characterized a class of channelrhodopsins (originally cation-conducting) converted into chloride-conducting anion channels. These tools enable fast optical inhibition of action potentials and can be engineered to display step-function kinetics for stable inhibition, outlasting light pulses and for orders-of-magnitude-greater light sensitivity of inhibited cells. The resulting family of proteins defines an approach to more physiological, efficient, and sensitive optogenetic inhibition.

Conversion of Channelrhodopsin into a Light-Gated Chloride Channel

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.

Discovery of Brainwide Neural-Behavioral Maps via Multiscale Unsupervised Structure Learning

A single nervous system can generate many distinct motor patterns. Identifying which neurons and circuits control which behaviors has been a laborious piecemeal process, usually for one observer-defined behavior at a time. We present a fundamentally different approach to neuron-behavior mapping. We optogenetically activated 1054 identified neuron lines in Drosophila larvae and tracked the behavioral responses from 37,780 animals. Application of multiscale unsupervised structure learning methods to the behavioral data enabled us to identify 29 discrete, statistically distinguishable, observer-unbiased behavioral phenotypes. Mapping the neural lines to the behavior(s) they evoke provides a behavioral reference atlas for neuron subsets covering a large fraction of larval neurons. This atlas is a starting point for connectivity- and activity-mapping studies to further investigate the mechanisms by which neurons mediate diverse behaviors.