The CIB1 transcription factor regulates light- and heat-inducible cell elongation via a two-step HLH/bHLH system.

Light and high temperature promote plant cell elongation. PHYTOCHROME INTERACTING FACTOR4 (PIF4, a typical basic helix-loop-helix [bHLH] transcriptional activator) and the non-DNA binding atypical HLH inhibitors PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and LONG HYPOCOTYL IN FAR-RED 1 (HFR1) competitively regulate cell elongation in response to light conditions and high temperature. However, the underlying mechanisms have not been fully clarified. Here, we show that in Arabidopsis thaliana, the bHLH transcription factor CRYPTOCHROME-INTERACTING BASIC HELIX-LOOP-HELIX 1 (CIB1) positively regulates cell elongation under the control of PIF4, PAR1, and HFR1. Furthermore, PIF4 directly regulates CIB1 expression by interacting with its promoter, and PAR1 and HFR1 interfere with PIF4 binding to the CIB1 promoter. CIB1 activates genes that function in cell elongation, and PAR1 interferes with the DNA binding activity of CIB1, thus suppressing cell elongation. Hence, two antagonistic HLH/bHLH systems, the PIF4-PAR1/HFR1 and CIB1-PAR1 systems, regulate cell elongation in response to light and high temperature. We thus demonstrate the important role of non-DNA binding small HLH proteins in the transcriptional regulation of cell elongation in plants.

Structural basis for Na(+) transport mechanism by a light-driven Na(+) pump.

Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

Red-Tuning of the Channelrhodopsin Spectrum Using Long Conjugated Retinal Analogues.

As optogenetic studies become more popular, the demand for red-shifted channelrhodopsin is increasing, because blue-green light is highly scattered or absorbed by animal tissues. In this study, we developed a red-shifted channelrhodopsin by elongating the conjugated double-bond system of the native chromophore, all -trans-retinal (ATR1). Analogues of ATR1 and ATR2 (3,4-didehydro-retinal) in which an extra C═C bond is inserted at different positions (C6-C7, C10-C11, and C14-C15) were synthesized and introduced into a widely used channelrhodopsin variant, C1C2 (a chimeric protein of channelrhodopsin-1 and channelrhodopsin-2 from Chlamydomonas reinhardtii). C1C2 bearing these retinal analogues as chromophores showed broadened absorption spectra toward the long-wavelength side and photocycle intermediates similar to the conducting state of channelrhodopsin. However, the position of methyl groups on the retinal polyene chain influenced the yield of the pigment, absorption maximum, and photocycle pattern to a variable degree. The lack of a methyl group at position C9 of the analogues considerably decreased the yield of the pigment, whereas a methyl group at position C15 exhibited a large red-shift in the absorption spectra of the C1C2 analogue. Expansion of the chromophore binding pocket by mutation of aromatic residue Phe265 to Ala improved the yield of the pigment bearing elongated ATR1 analogues without a great alteration of the photocycle kinetics of C1C2. Our results show that elongation of the conjugated double-bond system of retinal is a promising strategy for improving the ability of channelrhodopsin to absorb long-wavelength light passing through the biological optical window.

Alternative Formation of Red-Shifted Channelrhodopsins: Noncovalent Incorporation with Retinal-Based Enamine-Type Schiff Bases and Mutated Channelopsin.

Red-shifted channelrhodopsins (ChRs) are attractive for optogenetic tools. We developed a new type of red-shifted ChRs that utilized noncovalent incorporation of retinal and 3,4-dehydroretinal-based enamine-type Schiff bases and mutated channelopsin, C1C2-K296G. These ChRs exhibited absorption maxima that were shifted 10-30 nm toward longer wavelengths than that of C1C2-ChR regenerated with all-trans-retinal.

Chimeras of channelrhodopsin-1 and -2 from Chlamydomonas reinhardtii exhibit distinctive light-induced structural changes from channelrhodopsin-2.

Channelrhodopsin-2 (ChR2) from the green alga Chlamydomonas reinhardtii functions as a light-gated cation channel that has been developed as an optogenetic tool to stimulate specific nerve cells in animals and control their behavior by illumination. The molecular mechanism of ChR2 has been extensively studied by a variety of spectroscopic methods, including light-induced difference Fourier transform infrared (FTIR) spectroscopy, which is sensitive to structural changes in the protein upon light activation. An atomic structure of channelrhodopsin was recently determined by x-ray crystallography using a chimera of channelrhodopsin-1 (ChR1) and ChR2. Electrophysiological studies have shown that ChR1/ChR2 chimeras are less desensitized upon continuous illumination than native ChR2, implying that there are some structural differences between ChR2 and chimeras. In this study, we applied light-induced difference FTIR spectroscopy to ChR2 and ChR1/ChR2 chimeras to determine the molecular basis underlying these functional differences. Upon continuous illumination, ChR1/ChR2 chimeras exhibited structural changes distinct from those in ChR2. In particular, the protonation state of a glutamate residue, Glu-129 (Glu-90 in ChR2 numbering), in the ChR chimeras is not changed as dramatically as in ChR2. Moreover, using mutants stabilizing particular photointermediates as well as time-resolved measurements, we identified some differences between the major photointermediates of ChR2 and ChR1/ChR2 chimeras. Taken together, our data indicate that the gating and desensitizing processes in ChR1/ChR2 chimeras are different from those in ChR2 and that these differences should be considered in the rational design of new optogenetic tools based on channelrhodopsins.

The regulatory mechanism of ion permeation through a channelrhodopsin derived from Mesostigma viride (MvChR1).

The five glutamate (E) residues of transmembrane (TM)-2 of channelrhodopsin (CrChR)-2 are conserved among several members of the ChR family. A point mutation of one of them, E97, to a nonpolar alanine (E97A) reduced the photocurrent amplitude without influencing other photocurrent properties. The charge at this position is also the determinant of the Gd(3+)-dependent block of the channel. It has thus been suggested that E97 interacts with hydrated cations to facilitate their permeation and that these residues are the primary binding sites of Gd(3+). However, the counterpart of this position is alanine for MvChR1 from Mesostigma viride. Here we investigated the ion permeation and the Gd(3+)-dependent channel block of MvChR1. We found that the high-affinity binding site of Gd(3+) was absent in MvChR1, but was dependent on the negativity at this position. However, the ion permeation through the channel was markedly interfered with a negative charge at this position. Based on these findings, it is proposed that the ions can pass through the pore with minimal interaction with this position.

Regulation of later neurogenic stages of adult-derived neural stem/progenitor cells by L-type Ca2+ channels.

In the adult hippocampus, new neurons are continuously generated and incorporated into the local circuitry in a manner dependent on the network activity. Depolarization evoked by neurotransmitters has been assumed to activate L-type Ca2+ channels (LTCC) which regulate the intracellular Ca2+ -dependent signaling cascades. The process of neurogenesis contains several stages such as proliferation, fate determination, selective death/survival and maturation. Here, we investigated which stage of neurogenesis is under the regulation of LTCC using a clonal line of neural stem/progenitor cells, PZ5, which was derived from adult rat hippocampus. Although undifferentiated PZ5 cells were type 1-like cells expressing both nestin and glial fibrillary acidic protein, they generated neuronal, astrocytic and oligodendrocytic populations in differentiation medium containing retinoic acid. Proliferation of undifferentiated PZ5 cells was dependent on neither the LTCC antagonist, nimodipine (Nimo) nor the LTCC agonists, Bay K 8644 (BayK) or FPL 64176 (FPL), whereas the fraction of neuronal population that expressed both ßIII-tubulin and MAP2 was reduced by Nimo but increased by BayK or FPL. At an earlier period of differentiation (e.g., day 4), the fraction of PZ5 cells expressing HuC/D, pan-neuronal marker, was not affected either by the LTCC activation or inhibition. At a later period of differentiation (e.g., day 9), the fraction of dying neurons was decreased by LTCC activation and increased by LTCC inhibition. It is suggested that the LTCC activation facilitates the survival and maturation of immature neurons, and that its inhibition facilitates the neuronal death.

Expression of a truncated form of the endoplasmic reticulum chaperone protein, σ1 receptor, promotes mitochondrial energy depletion and apoptosis.

The σ1 receptor (σ(1)R) regulates endoplasmic reticulum (ER)/mitochondrial interorganellar Ca(2+) mobilization through the inositol 1,4,5-trisphosphate receptor (IP(3)R). Here, we observed that expression of a novel splice variant of σ(1)R, termed short form σ(1)R (σ(1)SR), has a detrimental effect on mitochondrial energy production and cell survival. σ(1)SR mRNA lacks 47 ribonucleotides encoding exon 2, resulting in a frameshift and formation of a truncated receptor. σ(1)SR localizes primarily in the ER at perinuclear regions and forms a complex with σ(1)R but not with IP(3)R in the mitochondrion-associated ER membrane. Overexpression of both σ(1)R and the truncated isoform promotes mitochondrial elongation with increased ER mitochondrial contact surface. σ(1)R overexpression increases the efficiency of mitochondrial Ca(2+) uptake in response to IP(3)R-driven stimuli, whereas σ(1)SR overexpression reduces it. Most importantly, σ(1)R promotes ATP production via increased mitochondrial Ca(2+) uptake, promoting cell survival in the presence of ER stress. By contrast, σ(1)SR suppresses ATP production following ER stress, enhancing cell death. Taken together, the newly identified σ(1)SR isoform interferes with σ(1)R function relevant to mitochondrial energy production under ER stress conditions, promoting cellular apoptosis.

Optogenetic manipulation of neural and non-neural functions.

Optogenetic manipulation of the neuronal activity enables one to analyze the neuronal network both in vivo and in vitro with precise spatio-temporal resolution. Channelrhodopsins (ChRs) are light-sensitive cation channels that depolarize the cell membrane, whereas halorhodopsins and archaerhodopsins are light-sensitive Cl(-) and H(+) transporters, respectively, that hyperpolarize it when exogenously expressed. The cause-effect relationship between a neuron and its function in the brain is thus bi-directionally investigated with evidence of necessity and sufficiency. In this review we discuss the potential of optogenetics with a focus on three major requirements for its application: (i) selection of the light-sensitive proteins optimal for optogenetic investigation, (ii) targeted expression of these selected proteins in a specific group of neurons, and (iii) targeted irradiation with high spatiotemporal resolution. We also discuss recent progress in the application of optogenetics to studies of non-neural cells such as glial cells, cardiac and skeletal myocytes. In combination with stem cell technology, optogenetics may be key to successful research using embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) derived from human patients through optical regulation of differentiation-maturation, through optical manipulation of tissue transplants and, furthermore, through facilitating survival and integration of transplants.

Driving Neurogenesis in Neural Stem Cells with High Sensitivity Optogenetics.

Optogenetic stimulation of neural stem cells (NSCs) enables their activity-dependent photo-modulation. This provides a spatio-temporal tool for studying activity-dependent neurogenesis and for regulating the differentiation of the transplanted NSCs. Currently, this is mainly driven by viral transfection of channelrhodopsin-2 (ChR2) gene, which requires high irradiance and complex in vivo/vitro stimulation systems. Additionally, despite the extensive application of optogenetics in neuroscience, the transcriptome-level changes induced by optogenetic stimulation of NSCs have not been elucidated yet. Here, we made transformed NSCs (SFO-NSCs) stably expressing one of the step-function opsin (SFO)-variants of chimeric channelrhodopsins, ChRFR(C167A), which is more sensitive to blue light than native ChR2, via a non-viral transfection system using piggyBac transposon. We set up a simple low-irradiance optical stimulation (OS)-incubation system that induced c-fos mRNA expression, which is activity-dependent, in differentiating SFO-NSCs. More neuron-like SFO-NCSs, which had more elongated axons, were differentiated with daily OS than control cells without OS. This was accompanied by positive/negative changes in the transcriptome involved in axonal remodeling, synaptic plasticity, and microenvironment modulation with the up-regulation of several genes involved in the Ca2+-related functions. Our approach could be applied for stem cell transplantation studies in tissue with two strengths: lower carcinogenicity and less irradiance needed for tissue penetration.

Functional emergence of a column-like architecture in layer 5 of mouse somatosensory cortex in vivo.

To investigate how the functional architecture is organized in layer 5 (L5) of the somatosensory cortex of a mouse in vivo, the input-output relationship was investigated using an all-optical approach. The neural activity in L5 was optically recorded using a Ca2+ sensor, R-CaMP2, through a microprism inserted in the cortex under two-photon microscopy, while the L5 was regionally excited using optogenetics. The excitability was spread around the blue-light irradiated region, but the horizontal propagation was limited to within a certain distance (λ < 130 µm from the center of the illumination spot). When two regions were photostimulated with a short interval, the excitability of each cluster was reduced. Therefore, a column-like architecture had functionally emerged with reciprocal inhibition through a minimal number of synaptic relays. This could generate a synchronous output from a region of L5 with simultaneous enhancement of the signal-to-noise ratio by silencing of the neighboring regions.

Optogenetic study of the response interaction among multi-afferent inputs in the barrel cortex of rats.

We investigated the relationship between whisker mechanoreceptive inputs and the neural responses to optical stimulation in layer 2/upper 3 (L2/U3) of the barrel cortex using optogenetics since, ideally, we should investigate interactions among inputs with spatiotemporal acuity. Sixteen whisker points of a transgenic rat (W-TChR2V4), that expresses channelrhodopsin 2 (ChR2)-Venus conjugate (ChR2V) in the peripheral nerve endings surrounding the whisker follicles, were respectively connected one-by-one with 16 LED-coupled optical fibres, which illuminated the targets according to a certain pattern in order to evaluate interactions among the inputs in L2/U3. We found that the individual L2/U3 neurons frequently received excitatory inputs from multiple whiskers that were arrayed in a row. Although the interactions among major afferent inputs (MAIs) were negligible, negative interactions with the surrounding inputs suggest that the afferent inputs were integrated in the cortical networks to enhance the contrast of an array to its surroundings. With its simplicity, reproducibility and spatiotemporal acuity, the optogenetic approach would provide an alternative way to understand the principles of afferent integration in the cortex and should complement knowledge obtained by experiments using more natural stimulations.

Expanding the Toolbox of Upconversion Nanoparticles for In Vivo Optogenetics and Neuromodulation.

Optogenetics is an optical technique that exploits visible light for selective neuromodulation with spatio-temporal precision. Despite enormous effort, the effective stimulation of targeted neurons, which are located in deeper structures of the nervous system, by visible light, remains a technical challenge. Compared to visible light, near-infrared illumination offers a higher depth of tissue penetration owing to a lower degree of light attenuation. Herein, an overview of advances in developing new modalities for neural circuitry modulation utilizing upconversion-nanoparticle-mediated optogenetics is presented. These developments have led to minimally invasive optical stimulation and inhibition of neurons with substantially improved selectivity, sensitivity, and spatial resolution. The focus is to provide a comprehensive review of the mechanistic basis for evaluating upconversion parameters, which will be useful in designing, executing, and reporting optogenetic experiments.

Organelle Optogenetics: Direct Manipulation of Intracellular Ca2+ Dynamics by Light.

As one of the ubiquitous second messengers, the intracellular Ca2+, has been revealed to be a pivotal regulator of various cellular functions. Two major sources are involved in the initiation of Ca2+-dependent signals: influx from the extracellular space and release from the intracellular Ca2+ stores such as the endoplasmic/sarcoplasmic reticulum (ER/SR). To manipulate the Ca2+ release from the stores under high spatiotemporal precision, we established a new method termed "organelle optogenetics." That is, one of the light-sensitive cation channels (channelrhodopsin-green receiver, ChRGR), which is Ca2+-permeable, was specifically targeted to the ER/SR. The expression specificity as well as the functional operation of the ER/SR-targeted ChRGR (ChRGRER) was evaluated using mouse skeletal myoblasts (C2C12): (1) the ChRGRER co-localized with the ER-marker KDEL; (2) no membrane current was generated by light under whole-cell clamp of cells expressing ChRGRER; (3) an increase of fluorometric Ca2+ was evoked by the optical stimulation (OS) in the cells expressing ChRGRER in a manner independent on the extracellular Ca2+ concentration ([Ca2+]o); (4) the ΔF/F0 was sensitive to the inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and (5) the store-operated Ca2+ entry (SOCE) was induced by the OS in the ChRGRER-expressing cells. Our organelle optogenetics effectively manipulated the ER/SR to release Ca2+ from intracellular stores. The use of organelle optogenetics would reveal the neuroscientific significance of intracellular Ca2+ dynamics under spatiotemporal precision.

Targeted expression of step-function opsins in transgenic rats for optogenetic studies.

Rats are excellent animal models for experimental neuroscience. However, the application of optogenetics in rats has been hindered because of the limited number of established transgenic rat strains. To accomplish cell-type specific targeting of an optimized optogenetic molecular tool, we generated ROSA26/CAG-floxed STOP-ChRFR(C167A)-Venus BAC rats that conditionally express the step-function mutant channelrhodopsin ChRFR(C167A) under the control of extrinsic Cre recombinase. In primary cultured cortical neurons derived from this reporter rat, only Cre-positive cells expressing ChRFR(C167A) became bi-stable, that is, their excitability was enhanced by blue light and returned to the baseline by yellow~red light. In bigenic pups carrying the Phox2B-Cre driver, ChRFR(C167A) was specifically expressed in the rostral parafacial respiratory group (pFRG) in the medulla, where endogenous Phox2b immunoreactivity was detected. These neurons were sensitive to blue light with an increase in the firing frequency. Thus, this transgenic rat actuator/reporter system should facilitate optogenetic studies involving the effective in vivo manipulation of the activities of specific cell fractions using light of minimal intensity.

Restoration of visual response in aged dystrophic RCS rats using AAV-mediated channelopsin-2 gene transfer.

PURPOSE: To investigate whether the channelopsin-2 (Chop2) gene would restore visual responses in 10-month-old dystrophic Royal College of Surgeons (aged RCS; rdy/rdy) rats, the authors transferred the Chop2 gene into the retinal cells of aged RCS rats using the adenoassociated virus (AAV) vector. METHODS: The N-terminal fragment (residues 1-315) of Chop2 was fused to a fluorescent protein, Venus, in frame at the end of the Chop2 coding fragment. The viral vector construct (AAV-Chop2V) for the expression of the Chop2V in the retina was made by subcloning into an adenoassociated virus vector, including the CAG promoter. To evaluate the expression profile of Chop2V in the retina, the rats were killed and the eyes were removed and fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline. Retinal wholemount specimens and cryosections were made. Under anesthetized conditions, electrodes for the recording of visually evoked potentials (VEPs) were implanted onto the visual cortex in aged-RCS (rdy/rdy) rats. AAV-Chop2V vectors were then injected into the vitreous cavity of the left eyes. As a control, AAV-Venus vectors were applied to the right eyes. VEPs were evoked by the flash of a blue, white, or red light-emitting diode (LED) and were recorded from the visual cortex of the rats at various time points after the AAV vector injection. RESULTS: Chop2V fluorescence was predominantly observed in retinal ganglion cells (RGCs). Some fluorescence was observed in the inner nuclear layer and the inner plexiform layer neurites. A tendency of recovery was observed in the VEPs of aged RCS (rdy/rdy) rats after the AAV-Chop2V injection but not after the AAV-Venus injection. The visual response of AAV-Chop2V-injected aged RCS (rdy/rdy) rats was less sensitive to the blue LED flash than that of nondystrophic RCS (+/+) rats. The AAV-Chop2V-injected aged RCS (rdy/rdy) rats were insensitive to the red LED flash, which evoked a robust VEP in the RCS (+/+) rats. CONCLUSIONS: The visual response of aged RCS (rdy/rdy) rats was partially restored by transduction of the Chop2 gene through AAV into the inner retinal neurons, mainly RGCs. These results suggest that the transduction of Chop2 would provide a new strategy to treat some retinitis pigmentosa (RP) symptoms independent of their etiology.

Synaptic vesicle dynamics in the mossy fiber-CA3 presynaptic terminals of mouse hippocampus.

The mossy fiber (MF)-CA3 synapse in the hippocampus is unique in the CNS because of its wide dynamic range of transmitter release during short- and long-term plasticity. The presynaptic mechanisms underlying the fidelity of transmission were investigated for the MF-CA3 synapses. The relative size of readily releasable pool (RRP) of vesicles was estimated by counting the number of docked vesicles at an active zone (AZ) on the transmission electron microscopy (TEM) image. The size of the releasable pool and the exo-endocytosis kinetics were directly measured from individual large MF boutons in hippocampal slices of transgenic mice that selectively express synaptopHluorin (SpH), a pH-sensitive GFP fused to the lumenal aspect of one of the vesicular membrane proteins, VAMP-2, in these boutons. Here we found (1) there are distinct two vesicle pools, the resting pool which is resistant to exocytosis, and the releasable pool, (2) the initially docked vesicles are easily depleted and the RRP is maintained by refilling from the reserve subpopulation of releasable pool ("reserve" releasable pool), and (3) the contribution of rapid reuse of recycled vesicles is relatively small. Therefore, the fidelity of transmission is suggested to be ensured by the rapid refilling rate of RRP.

Myogenic Maturation by Optical-Training in Cultured Skeletal Muscle Cells.

Optogenetic techniques are powerful tools for manipulating biological processes in identified cells using light under high temporal and spatial resolutions. Here, we describe an optogenetic training strategy to promote morphological maturation and functional development of skeletal muscle cells in vitro. Optical stimulation with a rhythmical frequency facilitates specific structural alignment of sarcomeric proteins. Optical stimulation also depolarizes the membrane potential, and induces contractile responses in synchrony with the given pattern of light pulses. These results suggest that optogenetic techniques can be employed to manipulate activity-dependent processes during myogenic development and control contraction of photosensitive skeletal muscle cells with high temporal and special precision.

Kinetic characteristics of chimeric channelrhodopsins implicate the molecular identity involved in desensitization.

Channelrhodopsin (ChR)-1 and ChR2 were the first-identified members of ChRs which are a growing subfamily of microbial-type rhodopsins. Light absorption drives the generation of a photocurrent in cell membranes expressing ChR2. However, the photocurrent amplitude attenuates and becomes steady-state during prolonged irradiation. This process, called desensitization or inactivation, has been attributed to the accumulation of intermediates less conductive to cations. Here we provided evidence that the dark-adapted (DA) photocurrent before desensitization is kinetically different from the light-adapted (LA) one after desensitization, that is, the deceleration of both basal-to-conductive and conductive-to-basal transitions. When the kinetics were compared between the DA and LA photocurrents for the ChR1/2 chimeras, the transmembrane helices, TM1 and TM2, were the determinants of both basal-to-conductive and conductive-to-basal transitions, whereas TM4 may contribute to the basal-to-conductive transitions and TM5 may contribute to the conductive-to-basal transitions, respectively. The fact that the desensitization-dependent decrease of the basal-to-conductive and conductive-to-basal transitions was facilitated by the TM1 exchange from ChR2 to ChR1 and reversed by the further TM2 exchange suggests that the conformation change for the channel gating is predominantly regulated by the interaction between TM1 and TM2. Although the exchange of TM1 from ChR2 to ChR1 showed no obvious influence on the spectral sensitivity, this exchange significantly induced the desensitization-dependent blue shift. Therefore, the TM1 and 2 are the main structures involved in two features of the desensitization, the stabilization of protein conformation and the charge distribution around the retinal-Schiff base (RSB+).

Functional characterization of sodium-pumping rhodopsins with different pumping properties.

Sodium pumping rhodopsins (NaRs) are a unique member of the microbial-type I rhodopsin family which actively transport Na+ and H+ depending on ionic condition. In this study, we surveyed 12 different NaRs from various sources of eubacteria for their electrophysiological as well as spectroscopic properties. In mammalian cells several of these NaRs exhibited a Na+ based pump photocurrent and four interesting candidates were chosen for further characterization. Voltage dependent photocurrent amplitudes revealed a membrane potential-sensitive turnover rate, indicating the presence of an electrically-charged intermediate(s) in the photocycle reaction. The NaR from Salinarimonas rosea DSM21201 exhibited a red-shifted absorption spectrum, and slower kinetics compared to the first described sodium pump, KR2. Although the ratio of Na+ to H+ ion transport varied among the NaRs we tested, the NaRs from Flagellimonas sp_DIK and Nonlabens sp_YIK_SED-11 showed significantly higher Na+ selectivity when compared to KR2. All four further investigated NaRs showed a functional expression in dissociated hippocampal neuron culture and hyperpolarizing activity upon light-stimulation. Additionally, all four NaRs allowed optical inhibition of electrically-evoked neuronal spiking. Although efficiency of silencing was 3-5 times lower than silencing with the enhanced version of the proton pump AR3 from Halorubrum sodomense, our data outlines a new approach for hyperpolarization of excitable cells without affecting the intracellular and extracellular proton environment.