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Channelrhodopsins (ChRs) are light-gated cation channels derived from algae that have shown experimental utility in optogenetics; for example, neurons expressing ChRs can be optically controlled with high temporal precision within systems as complex as freely moving mammals. Although ChRs have been broadly applied to neuroscience research, little is known about the molecular mechanisms by which these unusual and powerful proteins operate. Here we present the crystal structure of a ChR (a C1C2 chimaera between ChR1 and ChR2 from Chlamydomonas reinhardtii ) at 2.3 Å resolution. The structure reveals the essential molecular architecture of ChRs, including the retinal-binding pocket and cation conduction pathway. This integration of structural and electrophysiological analyses provides insight into the molecular basis for the remarkable function of ChRs, and paves the way for the precise and principled design of ChR variants with novel properties. Channelrhodopsins are light-gated cation channels used in optogenetics; here, the high-resolution crystal structure of a channelrhodopsin from Chlamydomonas reinhardtii is determined. Structure of key optogenetics reagent The channelrhodopsins are light-gated ion channels, found in algae, that have rapidly become familiar in the neuroscience lab as optogenetics reagents: the activities of neurons expressing channelrhodopsins can be optically controlled within systems as complicated as living mammals. The X-ray crystal structure of a chimaera of two channelrhodopsins has now been determined at 2.3 Å resolution. The structure reveals the molecular architecture of this ion channel, including the retinal-binding pocket and cation conduction pathway. This work paves the way for the design of new channelrhodopsin variants with enhanced properties.

In the present study we analyze the role of polarization in the biological activity of Electromagnetic Fields (EMFs)/Electromagnetic Radiation (EMR). All types of man-made EMFs/EMR - in contrast to natural EMFs/EMR - are polarized. Polarized EMFs/EMR can have increased biological activity, due to: 1) Ability to produce constructive interference effects and amplify their intensities at many locations. 2) Ability to force all charged/polar molecules and especially free ions within and around all living cells to oscillate on parallel planes and in phase with the applied polarized field. Such ionic forced-oscillations exert additive electrostatic forces on the sensors of cell membrane electro-sensitive ion channels, resulting in their irregular gating and consequent disruption of the cell’s electrochemical balance. These features render man-made EMFs/EMR more bioactive than natural non-ionizing EMFs/EMR. This explains the increasing number of biological effects discovered during the past few decades to be induced by man-made EMFs, in contrast to natural EMFs in the terrestrial environment which have always been present throughout evolution, although human exposure to the latter ones is normally of significantly higher intensities/energy and longer durations. Thus, polarization seems to be a trigger that significantly increases the probability for the initiation of biological/health effects.

Recently, optical stimulation 1 , 2 , 3 has begun to unravel the neuronal processing that controls certain animal behaviours 4 , 5 . However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Here, we show an approach based on radio-frequency magnetic-field heating of nanoparticles to remotely activate temperature-sensitive cation channels in cells. Superparamagnetic ferrite nanoparticles were targeted to specific proteins on the plasma membrane of cells expressing TRPV1, and heated by a radio-frequency magnetic field. Using fluorophores as molecular thermometers, we show that the induced temperature increase is highly localized. Thermal activation of the channels triggers action potentials in cultured neurons without observable toxic effects. This approach can be adapted to stimulate other cell types and, moreover, may be used to remotely manipulate other cellular machinery for novel therapeutics. Radiofrequency magnetic-field heating of nanoparticles can activate temperature-sensitive ion channels, offering an alternative to optical methods for stimulating cells remotely.

A stomatal ion channel The stomata on the undersides of leaves control the exchange of carbon dioxide and water between plants and the atmosphere. Stomatal pore aperture is regulated by transport of ions and metabolites across guard-cell membranes. Perhaps surprisingly, until now no plant plasma membrane anion channel subunits have been cloned — and the homologues of animal anion channels have been shown not to encode functional ion channels in plants. Now two groups working independently have identified a protein that is an essential component for S-type anion channel function and is required for stomatal closure in response to a variety of physiological and stress stimuli. Termed SLAC1, it is a distant homologue of fungal and bacterial dicarboxylate/malic acid transport proteins. One of two related studies that describe the identification of a protein which is an essential component for S-type anion channel function and is required for stomatal closure in response to a variety of physiological and stress stimuli including carbon dioxide and ozone. Stomatal pores, formed by two surrounding guard cells in the epidermis of plant leaves, allow influx of atmospheric carbon dioxide in exchange for transpirational water loss. Stomata also restrict the entry of ozone — an important air pollutant that has an increasingly negative impact on crop yields, and thus global carbon fixation 1 and climate change 2 . The aperture of stomatal pores is regulated by the transport of osmotically active ions and metabolites across guard cell membranes 3 , 4 . Despite the vital role of guard cells in controlling plant water loss 3 , 4 , ozone sensitivity 1 , 2 and CO 2 supply 2 , 5 , 6 , 7 , the genes encoding some of the main regulators of stomatal movements remain unknown. It has been proposed that guard cell anion channels function as important regulators of stomatal closure and are essential in mediating stomatal responses to physiological and stress stimuli 3 , 4 , 8 . However, the genes encoding membrane proteins that mediate guard cell anion efflux have not yet been identified. Here we report the mapping and characterization of an ozone-sensitive Arabidopsis thaliana mutant, slac1 . We show that SLAC1 ( SLOW ANION CHANNEL-ASSOCIATED 1 ) is preferentially expressed in guard cells and encodes a distant homologue of fungal and bacterial dicarboxylate/malic acid transport proteins. The plasma membrane protein SLAC1 is essential for stomatal closure in response to CO 2 , abscisic acid, ozone, light/dark transitions, humidity change, calcium ions, hydrogen peroxide and nitric oxide. Mutations in SLAC1 impair slow (S-type) anion channel currents that are activated by cytosolic Ca 2+ and abscisic acid, but do not affect rapid (R-type) anion channel currents or Ca 2+ channel function. A low homology of SLAC1 to bacterial and fungal organic acid transport proteins, and the permeability of S-type anion channels to malate 9 suggest a vital role for SLAC1 in the function of S-type anion channels.

We restored visual function to animal models of human blindness using a chemical compound that photosensitizes a mammalian ion channel. Virus-mediated expression of this light sensor in surviving retinal cells of blind mice restored light responses in vitro, reanimated innate light avoidance, and enabled learned visually guided behavior. The treatment also restored light responses to the retina of blind dogs. Patients that might benefit from this treatment would need to have intact ganglion cell and nerve fiber layers. In general, these are patients diagnosed with retinitis pigmentosa and some forms of Leber congenital amaurosis. Patients diagnosed with other types of blindness, for example, age-related macular degeneration or diabetic retinopathy, would not be candidates for this treatment. Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration ( rd1 ) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0 460 ). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0 460 was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0 460 was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0 460 in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0 460 in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0 460 was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation.

Cytosolic Ca2+ in guard cells plays an important role in stomatal movement responses to environmental stimuli. These cytosolic Ca2+ increases result from Ca2+ influx through Ca2+-permeable channels in the plasma membrane and Ca2+ release from intracellular organelles in guard cells. However, the genes encoding defined plasma membrane Ca2+-permeable channel activity remain unknown in guard cells and, with some exceptions, largely unknown in higher plant cells. Here, we report the identification of two Arabidopsis (Arabidopsis thaliana) cation channel genes, CNGC5 and CNGC6, that are highly expressed in guard cells. Cytosolic application of cyclic GMP (cGMP) and extracellularly applied membrane-permeable 8-Bromoguanosine 3′,5′-cyclic monophosphate-cGMP both activated hyperpolarization-induced inward-conducting currents in wild-type guard cells using Mg2+ as the main charge carrier. The cGMP-activated currents were strongly blocked by lanthanum and gadolinium and also conducted Ba2+, Ca2+, and Na+ ions. cngc5 cngc6 double mutant guard cells exhibited dramatically impaired cGMP-activated currents. In contrast, mutations in CNGC1, CNGC2, and CNGC20 did not disrupt these cGMP-activated currents. The yellow fluorescent protein-CNGC5 and yellow fluorescent protein-CNGC6 proteins localize in the cell periphery. Cyclic AMP activated modest inward currents in both wild-type and cngc5cngc6 mutant guard cells. Moreover, cngc5 cngc6 double mutant guard cells exhibited functional abscisic acid (ABA)-activated hyperpolarization-dependent Ca2+-permeable cation channel currents, intact ABA-induced stomatal closing responses, and whole-plant stomatal conductance responses to darkness and changes in CO2 concentration. Furthermore, cGMP-activated currents remained intact in the growth controlled by abscisic acid2 and abscisic acid insensitive1 mutants. This research demonstrates that the CNGC5 and CNGC6 genes encode unique cGMP-activated nonselective Ca2+-permeable cation channels in the plasma membrane of Arabidopsis guard cells.

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near −65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor–based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure–function relationships of the light-gated pore.

Ultrasound (US) neuromodulation, especially sonogenetics, has been demonstrated with potential applications in noninvasive and targeted treatment of various neurological disorders. Despite the growing interest, the mechanism for US neuromodulation remains elusive, and the optimal condition for eliciting a neural response with minimal adverse effect has not been identified. Here, we investigate the Piezo1 activation and intracellular calcium response elicited by acoustical streaming induced shear stress under various US exposure conditions. We find that Piezo1 activation and resultant intracellular calcium response depend critically on shear stress amplitude and pulse length of the stimulation. Under the same insonification acoustic energy, we further identify an optical pulse length that leads to maximum cell deformation, Piezo1 activation, and calcium response with minimal injury, confirmed by numerical modeling of Piezo1 channel gating dynamics. Our results provide insight into the mechanism of ultrasonic activation of Piezo1 and highlight the importance of optimizing US exposure conditions in sonogenetics applications.

The naturally occurring channelrhodopsin variant anion channelrhodopsin-1 (ACR1), discovered in the cryptophyte algae Guillardia theta , exhibits large light-gated anion conductance and high anion selectivity when expressed in heterologous settings, properties that support its use as an optogenetic tool to inhibit neuronal firing with light. However, molecular insight into ACR1 is lacking owing to the absence of structural information underlying light-gated anion conductance. Here we present the crystal structure of G. theta ACR1 at 2.9 Å resolution. The structure reveals unusual architectural features that span the extracellular domain, retinal-binding pocket, Schiff-base region, and anion-conduction pathway. Together with electrophysiological and spectroscopic analyses, these findings reveal the fundamental molecular basis of naturally occurring light-gated anion conductance, and provide a framework for designing the next generation of optogenetic tools. The crystal structure of anion channelrhodopsin-1 (ACR1) from the algae Guillardia theta provides insights into the basis of anion conductance.

Photoactivatable drugs targeting ligand-gated ion channels open up new opportunities for light-guided therapeutic interventions. Photoactivable toxins targeting ion channels have the potential to control excitable cell activities with low invasiveness and high spatiotemporal precision. As proof-of-concept, we develop HwTxIV-Nvoc, a UV light-cleavable and photoactivatable peptide that targets voltage-gated sodium (Na V ) channels and validate its activity in vitro in HEK293 cells, ex vivo in brain slices and in vivo on mice neuromuscular junctions. We find that HwTxIV-Nvoc enables precise spatiotemporal control of neuronal Na V channel function under all conditions tested. By creating multiple photoactivatable toxins, we demonstrate the broad applicability of this toxin-photoactivation technology. Photoactivable toxins targeting ion channels have great potential to control cell activity. Here the authors report HwTxIV-Nvoc, a UV light-cleavable and photoactivatable peptide that targets voltage-gated sodium channels; they validate this in cells, brain slices and in vivo on mice neuromuscular junctions.