In vivo spatiotemporal control of voltage-gated ion channels by using photoactivatable peptidic toxins.

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 (NaV) 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 NaV channel function under all conditions tested. By creating multiple photoactivatable toxins, we demonstrate the broad applicability of this toxin-photoactivation technology.

Photopharmacology of Ion Channels through the Light of the Computational Microscope.

The optical control and investigation of neuronal activity can be achieved and carried out with photoswitchable ligands. Such compounds are designed in a modular fashion, combining a known ligand of the target protein and a photochromic group, as well as an additional electrophilic group for tethered ligands. Such a design strategy can be optimized by including structural data. In addition to experimental structures, computational methods (such as homology modeling, molecular docking, molecular dynamics and enhanced sampling techniques) can provide structural insights to guide photoswitch design and to understand the observed light-regulated effects. This review discusses the application of such structure-based computational methods to photoswitchable ligands targeting voltage- and ligand-gated ion channels. Structural mapping may help identify residues near the ligand binding pocket amenable for mutagenesis and covalent attachment. Modeling of the target protein in a complex with the photoswitchable ligand can shed light on the different activities of the two photoswitch isomers and the effect of site-directed mutations on photoswitch binding, as well as ion channel subtype selectivity. The examples presented here show how the integration of computational modeling with experimental data can greatly facilitate photoswitchable ligand design and optimization. Recent advances in structural biology, both experimental and computational, are expected to further strengthen this rational photopharmacology approach.

Effects of electromagnetic fields on neuronal ion channels: a systematic review.

Many aspects of chemistry and biology are mediated by electromagnetic field (EMF) interactions. The central nervous system (CNS) is particularly sensitive to EMF stimuli. Studies have explored the direct effect of different EMFs on the electrical properties of neurons in the last two decades, particularly focusing on the role of voltage-gated ion channels (VGCs). This work aims to systematically review published evidence in the last two decades detailing the effects of EMFs on neuronal ion channels as per the PRISM guidelines. Following a predetermined exclusion and inclusion criteria, 22 papers were included after searches on three online databases. Changes in calcium homeostasis, attributable to the voltage-gated calcium channels, were found to be the most commonly reported result of EMF exposure. EMF effects on the neuronal landscape appear to be diverse and greatly dependent on parameters, such as the field's frequency, exposure time, and intrinsic properties of the irradiated tissue, such as the expression of VGCs. Here, we systematically clarify how neuronal ion channels are particularly affected and differentially modulated by EMFs at multiple levels, such as gating dynamics, ion conductance, concentration in the membrane, and gene and protein expression. Ion channels represent a major transducer for EMF-related effects on the CNS.

Specific residues in the cytoplasmic domain modulate photocurrent kinetics of channelrhodopsin from Klebsormidium nitens.

Channelrhodopsins (ChRs) are light-gated ion channels extensively applied as optogenetics tools for manipulating neuronal activity. All currently known ChRs comprise a large cytoplasmic domain, whose function is elusive. Here, we report the cation channel properties of KnChR, one of the photoreceptors from a filamentous terrestrial alga Klebsormidium nitens, and demonstrate that the cytoplasmic domain of KnChR modulates the ion channel properties. KnChR is constituted of a 7-transmembrane domain forming a channel pore, followed by a C-terminus moiety encoding a peptidoglycan binding domain (FimV). Notably, the channel closure rate was affected by the C-terminus moiety. Truncation of the moiety to various lengths prolonged the channel open lifetime by more than 10-fold. Two Arginine residues (R287 and R291) are crucial for altering the photocurrent kinetics. We propose that electrostatic interaction between the rhodopsin domain and the C-terminus domain accelerates the channel kinetics. Additionally, maximal sensitivity was exhibited at 430 and 460 nm, the former making KnChR one of the most blue-shifted ChRs characterized thus far, serving as a novel prototype for studying the molecular mechanism of color tuning of the ChRs. Furthermore, KnChR would expand the optogenetics tool kit, especially for dual light applications when short-wavelength excitation is required.

Charge Transport by Light-Activated Rhodopsins Determined by Electrophysiological Recordings.

Electrophysiological experiments are required to determine the ion transport properties of light-activated currents from microbial rhodopsin expressing cells. The recordings set the quantitative basis for correlation with spectroscopic data and for understanding of channel gating, ion transport vectoriality, or ion selectivity. This chapter focuses on voltage-clamp recordings of channelrhodopsin-2-expressing cells, and it will describe different illumination protocols that reveal the kinetic properties of gating. While the opening and closing reaction is determined from a single turnover upon a short laser flash, desensitization of the light-gated currents is studied under continuous illumination. Recovery from the desensitized state is probed after prolonged illumination with a subsequent light activation upon different dark intervals. Compiling the experimental data will define a minimum number of states in kinetic schemes used to describe the light-gated currents in channelrhodopsins, and emphasis will be given on how to correlate the results with the different time-resolved spectroscopic experiments.

Schizorhodopsins: A family of rhodopsins from Asgard archaea that function as light-driven inward H+ pumps.

Schizorhodopsins (SzRs), a rhodopsin family first identified in Asgard archaea, the archaeal group closest to eukaryotes, are present at a phylogenetically intermediate position between typical microbial rhodopsins and heliorhodopsins. However, the biological function and molecular properties of SzRs have not been reported. Here, SzRs from Asgardarchaeota and from a yet unknown microorganism are expressed in Escherichia coli and mammalian cells, and ion transport assays and patch clamp analyses are used to demonstrate SzR as a novel type of light-driven inward H+ pump. The mutation of a cytoplasmic glutamate inhibited inward H+ transport, suggesting that it functions as a cytoplasmic H+ acceptor. The function, trimeric structure, and H+ transport mechanism of SzR are similar to that of xenorhodopsin (XeR), a light-driven inward H+ pumping microbial rhodopsins, implying that they evolved convergently. The inward H+ pump function of SzR provides new insight into the photobiological life cycle of the Asgardarchaeota.

Controlling Engineered P2X Receptors with Light.

This chapter details methods to express and modify ATP-gated P2X receptor channels so that they can be controlled using light. Following expression in cells, a photoswitchable tool compound can be used to covalently modify mutant P2X receptors, as previously demonstrated for homomeric P2X2 and P2X3 receptors, and heteromeric P2X2/3 receptors. Engineered P2X receptors can be rapidly and reversibly opened and closed by different wavelengths of light. Light-activated P2X receptors can be mutated further to impart ATP-insensitivity if required. This method offers control of specific P2X receptor channels with high spatiotemporal precision to study their roles in physiology and pathophysiology.

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.

X-ray induces mechanical and heat allodynia in mouse via TRPA1 and TRPV1 activation.

Radiotherapy-related pain is a common adverse reaction with a high incidence among cancer patients undergoing radiotherapy and remarkably reduces the quality of life. However, the mechanisms of ionizing radiation-induced pain are largely unknown. In this study, mice were treated with 20 Gy X-ray to establish ionizing radiation-induced pain model. X-ray evoked a prolonged mechanical, heat, and cold allodynia in mice. Transient receptor potential vanilloid 1 and transient receptor potential ankyrin 1 were significantly upregulated in lumbar dorsal root ganglion. The mechanical and heat allodynia could be transiently reverted by intrathecal injection of transient receptor potential vanilloid 1 antagonist capsazepine and transient receptor potential ankyrin 1 antagonist HC-030031. Additionally, the phosphorylated extracellular regulated protein kinases (ERK) and Jun NH2-terminal Kinase (JNK) in pain neural pathway were induced by X-ray treatment. Our findings indicated that activation of transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 is essential for the development of X-ray-induced allodynia. Furthermore, our findings suggest that targeting on transient receptor potential vanilloid 1 and transient receptor potential ankyrin 1 may be promising prevention strategies for X-ray-induced allodynia in clinical practice.

Domain insertion permissibility-guided engineering of allostery in ion channels.

Allostery is a fundamental principle of protein regulation that remains hard to engineer, particularly in membrane proteins such as ion channels. Here we use human Inward Rectifier K+ Channel Kir2.1 to map site-specific permissibility to the insertion of domains with different biophysical properties. We find that permissibility is best explained by dynamic protein properties, such as conformational flexibility. Several regions in Kir2.1 that are equivalent to those regulated in homologs, such as G-protein-gated inward rectifier K+ channels (GIRK), have differential permissibility; that is, for these sites permissibility depends on the structural properties of the inserted domain. Our data and the well-established link between protein dynamics and allostery led us to propose that differential permissibility is a metric of latent allosteric capacity in Kir2.1. In support of this notion, inserting light-switchable domains into sites with predicted latent allosteric capacity renders Kir2.1 activity sensitive to light.

Light-Induced Opening of the TRP Channel in Isolated Membrane Patches Excised from Photosensitive Microvilli from Drosophila Photoreceptors.

Drosophila phototransduction occurs in light-sensitive microvilli arranged in a longitudinal structure of the photoreceptor, termed the rhabdomere. Rhodopsin (Rh), isomerized by light, couples to G-protein, which activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG), inositol trisphosphate and H+. This pathway opens the light-dependent channels, transient receptor potential (TRP) and transient receptor potential like (TRPL). PLC and TRP are held together in a protein assembly by the scaffold protein INAD. We report that the channels can be photoactivated in on-cell rhabdomeric patches and in excised patches by DAG. In excised patches, addition of PLC-activator, m-3M3FBS, or G-protein-activator, GTP-γ-S, opened TRP. These reagents were ineffective in PLC-mutant norpA and in the presence of PLC inhibitor U17322. However, DAG activated TRP even when PLC was pharmacologically or mutationally suppressed. These observations indicate that PLC, G-protein, and TRP were retained functional in these patches. DAG also activated TRP in the protein kinase C (PKC) mutant, inaC, excluding the possibility that PKC could mediate DAG-dependent TRP activation. Labeling diacylglycerol kinase (DGK) by fusion of fluorescent mCherry (mCherry-DGK) indicates that DGK, which returns DAG to dark levels, is highly expressed in the microvilli. In excised patches, TRP channels could be light-activated in the presence of GTP, which is required for G-protein activation. The evidence indicates that the proteins necessary for phototransduction are retained functionally after excision and that DAG is necessary and sufficient for TRP opening. This work opens up unique possibilities for studying, in sub-microscopic native membrane patches, the ubiquitous phosphoinositide signaling pathway and its regulatory mechanisms in unprecedented detail.

Optogenetic manipulation of intracellular calcium by BACCS promotes differentiation of MC3T3-E1 cells.

Bone remodeling is maintained through the balance between bone formation by osteoblasts and bone resorption by osteoclasts. Previous studies suggested that intracellular Ca2+ signaling plays an important role in the differentiation of osteoblasts; however, the molecular mechanism of Ca2+ signaling in the differentiation of osteoblasts remains unclear. To elucidate the effect of Ca2+ signaling in osteoblasts, we employed an optogenetic tool, blue light-activated Ca2+ channel switch (BACCS). BACCS was used to spatiotemporally control intracellular Ca2+ with blue light stimulation. MC3T3-E1 cells, which have been used as a model of differentiation from preosteoblast to osteoblast, were promoted to differentiate by BACCS expression and rhythmical blue light stimulation. The results indicated that intracellular Ca2+ change from the outside of the cells can regulate signaling for differentiation of MC3T3-E1 cells. Our findings provide evidence that Ca2+ could cause osteoblast differentiation.

An Atomistic Model of a Precursor State of Light-Induced Channel Opening of Channelrhodopsin.

Channelrhodopsins (ChRs) are microbial light-gated ion channels with a retinal chromophore and are widely utilized in optogenetics to precisely control neuronal activity with light. Despite increasing understanding of their structures and photoactivation kinetics, the atomistic mechanism of light gating and ion conduction remains elusive. Here, we present an atomic structural model of a chimeric ChR in a precursor state of the channel opening determined by an accurate hybrid molecular simulation technique and a statistical theory of internal water distribution. The photoactivated structure features extensive tilt of the chromophore accompanied by redistribution of water molecules in its binding pocket, which is absent in previously known photoactivated structures of analogous photoreceptors, and widely agrees with structural and spectroscopic experimental evidence of ChRs. The atomistic model manifests a photoactivated ion-conduction pathway that is markedly different from a previously proposed one and successfully explains experimentally observed mutagenic effects on key channel properties.

Crystal structure of the natural anion-conducting channelrhodopsin GtACR1.

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.

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 anion channels, 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.

A new copper ionophore DPMQ protects cells against ultraviolet B irradiation by inhibiting the TRPV1 channel.

Copper is more likely than iron to generate reactive oxygen species (ROS) in a redox reaction due to its higher electrochemical reactivity. This study examined the effect of a newly synthesized Cu2+ binding compound, (E)-2-(4-(dimethylamino)phenylimino)methyl)quinolin-8-ol (DPMQ), on ultraviolet B (UVB) irradiation-induced cytotoxicity in human dermal fibroblasts. DPMQ induced Cu2+ influx as effectively as disulfiram, a Cu2+ ionophore anticancer drug. However, disulfiram induced ROS generation, mitochondrial dysfunction, and apoptosis in fibroblasts in a Cu2+ -dependent manner, whereas DPMQ was not only nontoxic, but protected cells against UVB irradiation-induced apoptosis in a Cu2+ -independent manner. UVB irradiation induced a Ca2+ -dependent increase in ROS generation, a decrease in Nrf2 levels, and activation of the mitochondrial apoptotic pathway, and these effects were prevented by DPMQ, which also increased Nrf2 nuclear translocation in a Cu2+ -independent manner. UVB irradiation activated 12-lipoxygenase and 12-hydroxyeicosatetraenoic acid (12-HETE), a product of 12-lipoxygenase, activated the TRPV1 channel. DMPQ did not act as a Ca2+ chelator, but inhibited the cytosolic Ca2+ increase induced by 12-HETE or capsaicin, but not that induced by bradykinin or ATP. Blockade of Ca2+ influx by pharmacological inhibition or silencing of the TRPV1 channel or chelation of cytosolic Ca2+ inhibited the UVB irradiation-induced Nrf2 reduction, ROS generation, mitochondrial dysfunction, and apoptosis. Taken together, our results suggest that Ca2+ influx via the TRPV1 channel is responsible for UVB irradiation-induced cytotoxicity and that DPMQ protects cells against UVB irradiation by inhibiting the TRPV1 channel and stabilizing Nrf2, and could thus be a potentially useful compound for the treatment of free radical-induced diseases.

Kinetic profiles of photocurrents in cells expressing two types of channelrhodopsin genes.

Channelrhodopsin-2 (ChR2), a light-activated cation-selective ion channel, has been widely used as a tool in optogenetic research. ChR2 is specifically sensitive to wavelengths less than 550 nm. One of the methods to expand the sensitivity of a channelrhodopsin to a wider range of wavelengths is to express another channelrhodopsin in the cells by the transduction of an additional gene. Here, we report the characteristic features of cells expressing two types of channelrhodopsins, each having different wavelength sensitivities. In HEK293 cells stably expressing ChR2, photocurrents were elicited at stimuli of 400-550 nm, and the wavelength sensitivity range was expanded by the additional transduction of the modified Volvox channelrhodopsin-1 (mVChR1) gene, which has broad wavelength sensitivities, ranging from 400 to 600 nm. However, the photocurrent at 550 nm was lower than that of the mVChR1-expressing cell; moreover, the turning-on and turning-off constants were delayed, and the deactivation rates were decreased. Meanwhile, the response to lower light intensity was improved by the additional gene. Thus, the transduction of an additional gene is a useful method to improve the light and wavelength sensitivities, as well as photocurrent kinetic profiles, of channelrhodopsins.

Dual optical control and mechanistic insights into photoswitchable group II and III metabotropic glutamate receptors.

G protein-coupled receptor (GPCR) signaling occurs in complex spatiotemporal patterns that are difficult to probe using standard pharmacological and genetic approaches. A powerful approach for dissecting GPCRs is to use light-controlled pharmacological agents that are tethered covalently and specifically to genetically engineered receptors. However, deficits in our understanding of the mechanism of such photoswitches have limited application of this approach and its extension to other GPCRs. In this study, we have harnessed the power of bioorthogonal tethering to SNAP and CLIP protein tags to create a family of light-gated metabotropic glutamate receptors (mGluRs). We define the mechanistic determinants of photoswitch efficacy, including labeling efficiency, dependence on photoswitch structure, length dependence of the linker between the protein tag and the glutamate ligand, effective local concentration of the glutamate moiety, and affinity of the receptor for the ligand. We improve the scheme for photoswitch synthesis as well as photoswitch efficiency, and generate seven light-gated group II/III mGluRs, including variants of mGluR2, 3, 6, 7, and 8. Members of this family of light-controlled receptors can be used singly or in specifically labeled, independently light-controlled pairs for multiplexed control of receptor populations.

Optogenetic termination of ventricular arrhythmias in the whole heart: towards biological cardiac rhythm management.

AIMS: Current treatments of ventricular arrhythmias rely on modulation of cardiac electrical function through drugs, ablation or electroshocks, which are all non-biological and rather unspecific, irreversible or traumatizing interventions. Optogenetics, however, is a novel, biological technique allowing electrical modulation in a specific, reversible and trauma-free manner using light-gated ion channels. The aim of our study was to investigate optogenetic termination of ventricular arrhythmias in the whole heart. METHODS AND RESULTS: Systemic delivery of cardiotropic adeno-associated virus vectors, encoding the light-gated depolarizing ion channel red-activatable channelrhodopsin (ReaChR), resulted in global cardiomyocyte-restricted transgene expression in adult Wistar rat hearts allowing ReaChR-mediated depolarization and pacing. Next, ventricular tachyarrhythmias (VTs) were induced in the optogenetically modified hearts by burst pacing in a Langendorff setup, followed by programmed, local epicardial illumination. A single 470-nm light pulse (1000 ms, 2.97 mW/mm2) terminated 97% of monomorphic and 57% of polymorphic VTs vs. 0% without illumination, as assessed by electrocardiogram recordings. Optical mapping showed significant prolongation of voltage signals just before arrhythmia termination. Pharmacological action potential duration (APD) shortening almost fully inhibited light-induced arrhythmia termination indicating an important role for APD in this process. CONCLUSION: Brief local epicardial illumination of the optogenetically modified adult rat heart allows contact- and shock-free termination of ventricular arrhythmias in an effective and repetitive manner after optogenetic modification. These findings could lay the basis for the development of fundamentally new and biological options for cardiac arrhythmia management.

Effects of 15 Hz square wave magnetic fields on the voltage-gated sodium and potassium channels in prefrontal cortex pyramidal neurons.

PURPOSE: Although magnetic fields have significant effects on neurons, little is known about the mechanisms behind their effects. The present study aimed to measure the effects of magnetic fields on ion channels in cortical pyramidal neurons. MATERIALS AND METHODS: Cortical pyramidal neurons of Kunming mice were isolated and then subjected to 15 Hz, 1 mT square wave (duty ratio 50%) magnetic fields stimulation. Sodium currents (INa), transient potassium currents (IA) and delayed rectifier potassium currents (IK) were recorded by whole-cell patch clamp method. RESULTS: We found that magnetic field exposure depressed channel current densities, and altered the activation kinetics of sodium and potassium channels. The inactivation properties of INa and IA were also altered. CONCLUSION: Magnetic field exposure alters ion channel function in neurons. It is likely that the structures of sodium and potassium channels were influenced by the applied field. Sialic acid, which is an important component of the channels, could be the molecule responsible for the reported results.