Probing plant signal processing optogenetically by two channelrhodopsins.

Early plant responses to different stress situations often encompass cytosolic Ca2+ increases, plasma membrane depolarization and the generation of reactive oxygen species1-3. However, the mechanisms by which these signalling elements are translated into defined physiological outcomes are poorly understood. Here, to study the basis for encoding of specificity in plant signal processing, we used light-gated ion channels (channelrhodopsins). We developed a genetically engineered channelrhodopsin variant called XXM 2.0 with high Ca2+ conductance that enabled triggering cytosolic Ca2+ elevations in planta. Plant responses to light-induced Ca2+ influx through XXM 2.0 were studied side by side with effects caused by an anion efflux through the light-gated anion channelrhodopsin ACR1 2.04. Although both tools triggered membrane depolarizations, their activation led to distinct plant stress responses: XXM 2.0-induced Ca2+ signals stimulated production of reactive oxygen species and defence mechanisms; ACR1 2.0-mediated anion efflux triggered drought stress responses. Our findings imply that discrete Ca2+ signals and anion efflux serve as triggers for specific metabolic and transcriptional reprogramming enabling plants to adapt to particular stress situations. Our optogenetics approach unveiled that within plant leaves, distinct physiological responses are triggered by specific ion fluxes, which are accompanied by similar electrical signals.

High correlated color temperature artificial lighting impairs retinal pigment epithelium integrity and chloride ion transport: A potential mechanism for choroidal thinning.

Among the environmental factors contributing to myopia, the role of correlated color temperature (CCT) of ambient light emerges as a key element warranting in-depth investigation. The choroid, a highly vascularized and dynamic structure, often undergoes thinning during the progression of myopia, though the precise mechanism remains elusive. The retinal pigment epithelium (RPE), the outermost layer of the retina, plays a pivotal role in regulating the transport of ion and fluid between the subretinal space and the choroid. A hypothesis suggests that variations in choroidal thickness (ChT) may be modulated by transepithelial fluid movement across the RPE. Our experimental results demonstrate that high CCT illumination significantly compromised the integrity of tight junctions in the RPE and disrupted chloride ion transport. This functional impairment of the RPE may lead to a reduction in fluid transfer across the RPE, consequently resulting in choroidal thinning and potentially accelerating axial elongation. Our findings provide support for the crucial role of the RPE in regulating ChT. Furthermore, we emphasize the potential hazards posed by high CCT artificial illumination on the RPE, the choroid, and refractive development, underscoring the importance of developing eye-friendly artificial light sources to aid in the prevention and control of myopia.

Discovery of a new light-driven Li+/Na+-pumping rhodopsin with DTG motif.

Microbial pumping rhodopsin is a seven-transmembrane retinal binding protein, which is light-driven ion pump with a functional key motif. Ion-pumping with the key motif and charged amino acids in the rhodopsin is biochemically important. The rhodopsins with DTG motif have been discovered in various eubacteria, and they function as H+ pump. Especially, the DTG motif rhodopsins transported H+ despite the replacement of a proton donor by Gly. We investigated Methylobacterium populi rhodopsin (MpR) in one of the DTG motif rhodopsin clades. To determine which ions the MpR transport, we tested with various monovalent ion solutions and determined that MpR transports Li+/Na+. By replacing the three negatively charged residues residues which are located in helix B, Glu32, Glu33, and Asp35, we concluded that the residues play a critical role in the transport of Li+/Na+. The MpR E33Q transported H+ in place of Li+/Na+, suggesting that Glu33 is a Li+/Na+ binding site on the cytoplasmic side. Gly93 in MpR was replaced by Asp to convert from the Li+/Na+ pump to the H+ pump, resulting in MpR G93D transporting H+. Dissociation constant (Kd) values of Na+ for MpR WT and E33Q were determined to be 4.0 and 72.5 mM, respectively. These results indicated the mechanism by which MpR E33Q transports H+. Up to now, various ion-pumping rhodopsins have been discovered, and Li+/Na+-pumping rhodopsins were only found in the NDQ motif in NaR. Here, we report a new light-driven Na+ pump MpR and have determined the important residues required for Li+/Na+-pumping different from previously known NaR.

Whole body potassium as a biomarker for potassium uptake using a mouse model.

Potassium is known for its effect on modifiable chronic diseases like hypertension, cardiac disease, diabetes (type-2), and bone health. In this study, a new method, neutron generator based neutron activation analysis (NAA), was utilized to measure potassium (K) in mouse carcasses. A DD110 neutron generator based NAA assembly was used for irradiation.Thirty-two postmortem mice (n= 16 males and 16 females, average weight [Formula: see text] and [Formula: see text] g) were employed for this study. Soft-tissue equivalent mouse phantoms were prepared for the calibration. All mice were irradiated for 10 minutes, and the gamma spectrum with 42K was collected using a high efficiency, high purity germanium (HPGe) detector. A lead shielding assembly was designed and developed around the HPGe detector to obtain an improved detection limit. Each mouse sample was irradiated and measured twice to reduce uncertainty. The average potassium concentration was found to be significantly higher in males [Formula: see text] compared to females [Formula: see text]. We also observed a significant correlation between potassium concentration and the weight of the mice. The detection limit for potassium quantification with the NAA system was 46 ppm. The radiation dose to the mouse was approximately 56 [Formula: see text] mSv for 10-min irradiation. In conclusion, this method is suitable for estimating individual potassium concentration in small animals. The direct evaluation of total body potassium in small animals provides a new way to estimate potassium uptake in animal models. This method can be adapted later to quantify potassium in the human hand and small animals in vivo. When used in vivo, it is also expected to be a valuable tool for longitudinal assessment, kinetics, and health outcomes.

History and Perspectives of Ion-Transporting Rhodopsins.

The first light-sensing proteins used in optogenetics were rhodopsins. The word "rhodopsin" originates from the Greek words "rhodo" and "opsis," indicating rose and sight, respectively. Although the classical meaning of rhodopsin is the red-colored pigment in our eyes, the modern meaning of rhodopsin encompasses photoactive proteins containing a retinal chromophore in animals and microbes. Animal and microbial rhodopsins possess 11-cis and all-trans retinal, respectively, to capture light in seven transmembrane α-helices, and photoisomerizations into all-trans and 13-cis forms, respectively, initiate each function. We are able to find ion-transporting proteins in microbial rhodopsins, such as light-gated channels and light-driven pumps, which are the main tools in optogenetics. In this chapter, historical aspects and molecular properties of rhodopsins are introduced. In the first part, "what is rhodopsin?", general introduction of rhodopsin is presented. Then, molecular mechanism of bacteriorodopsin, a light-driven proton pump and the best-studied microbial rhodopsin, is described. In the section of channelrhodopsin, the light-gated ion channel, molecular properties, and several variants are introduced. As the history has proven, understanding the molecular mechanism of microbial rhodopsins is a prerequisite for useful functional design of optogenetics tools in future.

Diversity, Mechanism, and Optogenetic Application of Light-Driven Ion Pump Rhodopsins.

Ion-transporting microbial rhodopsins are widely used as major molecular tools in optogenetics. They are categorized into light-gated ion channels and light-driven ion pumps. While the former passively transport various types of cations and anions in a light-dependent manner, light-driven ion pumps actively transport specific ions, such as H+, Na+, Cl-, against electrophysiological potential by using light energy. Since the ion transport by these pumps induces hyperpolarization of membrane potential and inhibit neural firing, light-driven ion-pumping rhodopsins are mostly applied as inhibitory optogenetics tools. Recent progress in genome and metagenome sequencing identified more than several thousands of ion-pumping rhodopsins from a wide variety of microbes, and functional characterization studies has been revealing many new types of light-driven ion pumps one after another. Since light-gated channels were reviewed in other chapters in this book, here the rapid progress in functional characterization, molecular mechanism study, and optogenetic application of ion-pumping rhodopsins were reviewed.

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.

A Visible-Light-Regulated Chloride Transport Channel Inspired by Rhodopsin.

Inspired by the light-regulating capabilities of naturally occurring rhodopsin, we have constructed a visible-light-regulated Cl- -transport membrane channel based on a supramolecular host-guest interaction. A natural retinal chromophore, capable of a visible-light response, is used as the guest and grafted into the artificial channel. Upon introduction of an ethyl-urea-derived pillar[6]arene (Urea-P6) host, threading or de-threading of the retinal and selective bonding of Cl- can be utilized to regulate ion transport. Based on the visible-light responsiveness of the host-guest interaction, Cl- transport can be regulated by visible light between ON and OFF states. Visible-light-regulated Cl- transport as a chemical model permits to understand comparable biological ion-selective transport behaviors. Furthermore, this result also supplies a smart visible-light-responsive Cl- transporter, which may have applications in natural photoelectric conversion and photo-controlled delivery systems.

Accumulation of ambient phosphate into the periplasm of marine bacteria is proton motive force dependent.

Bacteria acquire phosphate (Pi) by maintaining a periplasmic concentration below environmental levels. We recently described an extracellular Pi buffer which appears to counteract the gradient required for Pi diffusion. Here, we demonstrate that various treatments to outer membrane (OM) constituents do not affect the buffered Pi because bacteria accumulate Pi in the periplasm, from which it can be removed hypo-osmotically. The periplasmic Pi can be gradually imported into the cytoplasm by ATP-powered transport, however, the proton motive force (PMF) is not required to keep Pi in the periplasm. In contrast, the accumulation of Pi into the periplasm across the OM is PMF-dependent and can be enhanced by light energy. Because the conventional mechanism of Pi-specific transport cannot explain Pi accumulation in the periplasm we propose that periplasmic Pi anions pair with chemiosmotic cations of the PMF and millions of accumulated Pi pairs could influence the periplasmic osmolarity of marine bacteria.

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus.

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse. To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.

Thermal constraints on in vivo optogenetic manipulations.

A key assumption of optogenetics is that light only affects opsin-expressing neurons. However, illumination invariably heats tissue, and many physiological processes are temperature-sensitive. Commonly used illumination protocols increased the temperature by 0.2-2 °C and suppressed spiking in multiple brain regions. In the striatum, light delivery activated an inwardly rectifying potassium conductance and biased rotational behavior. Thus, careful consideration of light-delivery parameters is required, as even modest intracranial heating can confound interpretation of optogenetic experiments.

Photosynthesis in Arabidopsis Is Unaffected by the Function of the Vacuolar K+ Channel TPK3.

Photosynthesis is limited by the slow relaxation of nonphotochemical quenching, which primarily dissipates excess absorbed light energy as heat. Because the heat dissipation process is proportional to light-driven thylakoid lumen acidification, manipulating thylakoid ion and proton flux via transport proteins could improve photosynthesis. However, an important aspect of the current understanding of the thylakoid ion transportome is inaccurate. Using fluorescent protein fusions, we show that the Arabidopsis (Arabidopsis thaliana) two-pore K+ channel TPK3, which had been reported to mediate thylakoid K+ flux, localizes to the tonoplast, not the thylakoid. The localization of TPK3 outside of the thylakoids is further supported by the absence of TPK3 in isolated thylakoids as well as the inability of isolated chloroplasts to import TPK3 protein. In line with the subcellular localization of TPK3 in the vacuole, we observed that photosynthesis in the Arabidopsis null mutant tpk3-1, which carries a transfer DNA insertion in the first exon, remains unaffected. To gain a comprehensive understanding of how thylakoid ion flux impacts photosynthetic efficiency under dynamic growth light regimes, we performed long-term photosynthesis imaging of established and newly isolated transthylakoid K+- and Cl--flux mutants. Our results underpin the importance of the thylakoid ion transport proteins potassium cation efflux antiporter KEA3 and voltage-dependent chloride channel VCCN1 and suggest that the activity of yet unknown K+ channel(s), but not TPK3, is critical for optimal photosynthesis in dynamic light environments.

Rhodopsin-Like Ionic Gate Fabricated with Graphene Oxide and Isomeric DNA Switch for Efficient Photocontrol of Ion Transport.

Rhodopsin, composed of opsin and isomeric retinal, acts as the primary photoreceptor by converting light into electric signals. Inspired by rhodopsin, we have fabricated a light-regulated ionic gate on the basis of the design of a graphene oxide (GO)-biomimetic DNA-nanochannel architecture. In this design, photoswitchable azobenzene (Azo)-DNA is introduced to the surface of porous anodic alumina (PAA) membrane. With modulation of the interaction between the GO blocker and Azo-DNA via flexibly regulating trans and cis states of Azo under the irradiation of visible and ultraviolet light, alternatively, the ionic gate is switched between ON and OFF states. This newly constructed ionic gate can possess high efficiency for the control of ion transport because of the high blocking property of GO and the rather tiny path within the barrier layer which are both first employed to fabricate ionic gate. We anticipate that this rhodopsin-like ionic gate may provide a new model and method for the investigation of ion channel, ion function, and ion quantity. In addition, because of the advantages of simple fabrication, good biocompatibility, and universality, this bioinspired system may have potential applications as optical sensors, in photoelectric transformation, and in controllable drug delivery.

Electrophysiological evidence for light-activated cation transport in calcifying corals.

Light has been demonstrated to enhance calcification rates in hermatypic coral species. To date, it remains unresolved whether calcifying epithelia change their ion transport activity during illumination, and whether such a process is mediated by the endosymbiotic algae or can be controlled by the coral host itself. Using a modified Ussing chamber in combination with H+ sensitive microelectrode measurements, the present work demonstrates that light triggers the generation of a skeleton positive potential of up to 0.9 mV in the hermatypic coral Stylophora pistillata. This potential is generated by a net flux of cations towards the skeleton and reaches its maximum at blue (450 nm) light. The effects of pharmacological inhibitors targeting photosynthesis 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and anion transport 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) were investigated by pH microelectrode measurements in coral tissues demonstrating a rapid decrease in tissue pH under illumination. However, these inhibitors showed no effect on the electrophysiological light response of the coral host. By contrast, metabolic inhibition by cyanide and deoxyglucose reversibly inhibited the light-induced cation flux towards the skeleton. These results suggest that ion transport across coral epithelia is directly triggered by blue light, independent of photosynthetic activity of algal endosymbionts. Measurements of this very specific and quantifiable physiological response can provide parameters to identify photoreception mechanisms and will help to broaden our understanding of the mechanistic link between light stimulation and epithelial ion transport, potentially relevant for calcification in hermatypic corals.

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.

Light-Driven Chloride Transport Kinetics of Halorhodopsin.

Despite growing interest in light-driven ion pumps for use in optogenetics, current estimates of their transport rates span two orders of magnitude due to challenges in measuring slow transport processes and determining protein concentration and/or orientation in membranes in vitro. In this study, we report, to our knowledge, the first direct quantitative measurement of light-driven Cl- transport rates of the anion pump halorohodopsin from Natronomonas pharaonis (NpHR). We used light-interfaced voltage clamp measurements on NpHR-expressing oocytes to obtain a transport rate of 219 (± 98) Cl-/protein/s for a photon flux of 630 photons/protein/s. The measurement is consistent with the literature-reported quantum efficiency of ∼30% for NpHR, i.e., 0.3 isomerizations per photon absorbed. To reconcile our measurements with an earlier-reported 20 ms rate-limiting step, or 35 turnovers/protein/s, we conducted, to our knowledge, novel consecutive single-turnover flash experiments that demonstrate that under continuous illumination, NpHR bypasses this step in the photocycle.

Low-temperature FTIR spectroscopy provides evidence for protein-bound water molecules in eubacterial light-driven ion pumps.

Light-driven H+, Na+ and Cl- pumps have been found in eubacteria, which convert light energy into a transmembrane electrochemical potential. A recent mutation study revealed asymmetric functional conversion between the two pumps, where successful functional conversions are achieved exclusively when mutagenesis reverses the evolutionary amino acid sequence changes. Although this fact suggests that the essential structural mechanism of an ancestral function is retained even after gaining a new function, questions regarding the essential structural mechanism remain unanswered. Light-induced difference FTIR spectroscopy was used to monitor the presence of strongly hydrogen-bonded water molecules for all eubacterial H+, Na+ and Cl- pumps, including a functionally converted mutant. This fact suggests that the strongly hydrogen-bonded water molecules are maintained for these new functions during evolution, which could be the reason for successful functional conversion from Na+ to H+, and from Cl- to H+ pumps. This also explains the successful conversion of the Cl- to the H+ pump only for eubacteria, but not for archaea. It is concluded that water-containing hydrogen-bonding networks constitute one of the essential structural mechanisms in eubacterial light-driven ion pumps.

Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin.

Microbial rhodopsins are well known as versatile and ubiquitous light-driven ion transporters and photosensors. While the proton transport mechanism has been studied in great detail, much less is known about various modes of anion transport. Until recently, only two main groups of light-driven anion pumps were known, archaeal halorhodopsins (HRs) and bacterial chloride pumps (known as ClRs or NTQs). Last year, another group of cyanobacterial anion pumps with a very distinct primary structure was reported. Here, we studied the chloride-transporting photocycle of a representative of this new group, Mastigocladopsis repens rhodopsin (MastR), using time-resolved spectroscopy in the infrared and visible ranges and site-directed mutagenesis. We found that, in accordance with its unique amino acid sequence containing many polar residues in the transmembrane region of the protein, its photocycle features a number of unusual molecular events not known for other anion-pumping rhodopsins. It appears that light-driven chloride ion transfers by MastR are coupled with translocation of protons and water molecules as well as perturbation of several polar sidechains. Of particular interest is transient deprotonation of Asp-85, homologous to the cytoplasmic proton donor of light-driven proton pumps (such as Asp-96 of bacteriorhodopsin), which may serve as a regulatory mechanism.

Spectroscopic characteristics of Rubricoccus marinus xenorhodopsin (RmXeR) and a putative model for its inward H+ transport mechanism.

A new group of microbial rhodopsins named xenorhodopsins (XeR), which are closely related to the cyanobacterial Anabaena sensory rhodopsin, show a light-driven "inward" proton transport activity, as reported for one representative of this group from Parvularcula oceani (PoXeR). In this study, we functionally and spectroscopically characterized a new member of the XeR clade from a marine bacterium Rubricoccus marinus SG-29T (RmXeR). Escherichia coli cells expressing recombinant RmXeR showed a light-induced alkalization of the cell suspension, which was strongly impaired by a protonophore, suggesting that RmXeR is a light-driven "inward" proton pump as is PoXeR. The spectroscopic properties of purified RmXeR were investigated and compared with those of PoXeR and a light-driven "outward" proton pump, bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum. Action spectroscopy revealed that RmXeR with all-trans retinal is responsible for the light-driven inward proton transport activity, but not with 13-cis retinal. From pH titration experiments and mutational analysis, we estimated the pKa values for the protonated Schiff base of the retinal chromophore and its counterion as 11.1 ± 0.07 and 2.1 ± 0.07, respectively. Of note, the direction of both the retinal composition change upon light-dark adaptation and the acid-induced spectral shift was opposite that of BR, which is presumably related to the opposite directions of ion transport (from outside to inside for RmXeR and from inside to outside for BR). Flash photolysis experiments revealed the appearances of three intermediates (L, M and O) during the photocycle. The proton uptake and release were coincident with the formation and decay of the M intermediate, respectively. Together with associated findings from other microbial rhodopsins, we propose a putative model for the inward proton transport mechanism of RmXeR.