Direct detection of the chloride release and uptake reactions of Natronomonas pharaonis halorhodopsin.

Membrane transport proteins undergo multistep conformational changes to fulfill the transport of substrates across biological membranes. Substrate release and uptake are the most important events of these multistep reactions that accompany significant conformational changes. Thus, their relevant structural intermediates should be identified to better understand the molecular mechanism. However, their identifications have not been achieved for most transporters due to the difficulty of detecting the intermediates. Herein, we report the success of these identifications for a light-driven chloride transporter halorhodopsin (HR). We compared the time course of two flash-induced signals during a single transport cycle. One is a potential change of Cl--selective membrane, which enabled us to detect tiny Cl--concentration changes due to the Cl- release and the subsequent Cl--uptake reactions by HR. The other is the absorbance change of HR reflecting the sequential formations and decays of structural intermediates. Their comparison revealed not only the intermediates associated with the key reactions but also the presence of two additional Cl--binding sites on the Cl--transport pathways. The subsequent mutation studies identified one of the sites locating the protein surface on the releasing side. Thus, this determination also clarified the Cl--transport pathway from the initial binding site until the release to the medium.

In Vivo Neocortical [K]o Modulation by Targeted Stimulation of Astrocytes.

A normally functioning nervous system requires normal extracellular potassium ion concentration ([K]o). Throughout the nervous system, several processes, including those of an astrocytic nature, are involved in [K]o regulation. In this study we investigated the effect of astrocytic photostimulation on [K]o. We hypothesized that in vivo photostimulation of eNpHR-expressing astrocytes leads to a decreased [K]o. Using optogenetic and electrophysiological techniques we showed that stimulation of eNpHR-expressing astrocytes resulted in a significantly decreased resting [K]o and evoked K responses. The amplitude of the concomitant spreading depolarization-like events also decreased. Our results imply that astrocytic membrane potential modification could be a potential tool for adjusting the [K]o.

Microbial Halorhodopsins: Light-Driven Chloride Pumps.

Early research on the four microbial rhodopsins discovered in the archaeal Halobacterium salinarum revealed a structural template that served as a scaffold for two different functions: light-driven ion transport and phototaxis. Bacteriorhodopsin and halorhodopsin are proton and chloride pumps, respectively, while sensory rhodopsin I and II are responsible for phototactic behavior of the archaea. Halorhodopsins have been identified in various other species. Besides this group of archaeal halorhodopsins distinct chloride transporting rhodopsins groups have recently been identified in other organism like Flavobacteria or Cyanobacteria. Halorhodopsin from Natronomonas pharaonis is the best-studied homologue because of its facile expression and purification and its advantageous properties, which was the reason to introduce this protein as neural silencer into the new field of optogenetics. Two other major families of genetically encoded silencing proteins, proton pumps and anion channels, extended the repertoire of optogenetic tools. Here, we describe the functional and structural characteristics of halorhodopsins. We will discuss the data in light of common principles underlying the mechanism of ion pumps and sensors and will review biophysical and biochemical aspects of neuronal silencers.

Cortical cholinergic signaling controls the detection of cues.

The cortical cholinergic input system has been described as a neuromodulator system that influences broadly defined behavioral and brain states. The discovery of phasic, trial-based increases in extracellular choline (transients), resulting from the hydrolysis of newly released acetylcholine (ACh), in the cortex of animals reporting the presence of cues suggests that ACh may have a more specialized role in cognitive processes. Here we expressed channelrhodopsin or halorhodopsin in basal forebrain cholinergic neurons of mice with optic fibers directed into this region and prefrontal cortex. Cholinergic transients, evoked in accordance with photostimulation parameters determined in vivo, were generated in mice performing a task necessitating the reporting of cue and noncue events. Generating cholinergic transients in conjunction with cues enhanced cue detection rates. Moreover, generating transients in noncued trials, where cholinergic transients normally are not observed, increased the number of invalid claims for cues. Enhancing hits and generating false alarms both scaled with stimulation intensity. Suppression of endogenous cholinergic activity during cued trials reduced hit rates. Cholinergic transients may be essential for synchronizing cortical neuronal output driven by salient cues and executing cue-guided responses.

Visceral hypersensitivity induced by optogenetic activation of the amygdala in conscious rats.

In vivo optogenetics identifies brain circuits controlling behaviors in conscious animals by using light to alter neuronal function and offers a novel tool to study the brain-gut axis. Using adenoviral-mediated expression, we aimed to investigate whether photoactivation with channelrhodopsin (ChR2) or photoinhibition with halorhodopsin (HR3.0) of fibers originating from the central nucleus of the amygdala (CeA) at the bed nucleus of the stria terminalis (BNST) had any effect on colonic sensitivity. We also investigated whether there was any deleterious effect of the adenovirus on the neuronal population or the neuronal phenotype within the CeA-BNST circuitry activated during the optogenetic stimulation. In male rats, the CeA was infected with vectors expressing ChR2 or HR3.0 and fiber optic cannulae were implanted on the BNST. After 8-10 wk, the response to graded, isobaric colonic distension was measured with and without laser stimulation of CeA fibers at the BNST. Immunohistochemistry and histology were used to evaluate vector expression, neuronal integrity, and neurochemical phenotype. Photoactivation of CeA fibers at the BNST with ChR2 induced colonic hypersensitivity, whereas photoinhibition of CeA fibers at the BNST with HR3.0 had no effect on colonic sensitivity. Control groups treated with virus expressing reporter proteins showed no abnormalities in neuronal morphology, neuronal number, or neurochemical phenotype following laser stimulation. Our experimental findings reveal that optogenetic activation of discrete brain nuclei can be used to advance our understanding of complex visceral nociceptive circuitry in a freely moving rat model. NEW & NOTEWORTHY Our findings reveal that optogenetic technology can be employed as a tool to advance understanding of the brain-gut axis. Using adenoviral-mediated expression of opsins, which were activated by laser light and targeted by fiber optic cannulae, we examined central nociceptive circuits mediating visceral pain in a freely moving rat. Photoactivation of amygdala fibers in the stria terminalis with channelrhodopsin induced colonic hypersensitivity, whereas inhibition of the same fibers with halorhodopsin did not alter colonic sensitivity.

Asymmetric Functional Conversion of Eubacterial Light-driven Ion Pumps.

In addition to the well-known light-driven outward proton pumps, novel ion-pumping rhodopsins functioning as outward Na(+) and inward Cl(-) pumps have been recently found in eubacteria. They convert light energy into transmembrane electrochemical potential difference, similar to the prototypical archaeal H(+) pump bacteriorhodopsin (BR) and Cl(-) pump halorhodopsin (HR). The H(+), Na(+), and Cl(-) pumps possess the conserved respective DTE, NDQ, and NTQ motifs in the helix C, which likely serve as their functional determinants. To verify this hypothesis, we attempted functional interconversion between selected pumps from each category by mutagenesis. Introduction of the proton-pumping motif resulted in successful Na(+) → H(+) functional conversion. Introduction of the respective characteristic motifs with several additional mutations leads to successful Na(+) → Cl(-) and Cl(-) → H(+) functional conversions, whereas remaining conversions (H(+) → Na(+), H(+) → Cl(-), Cl(-) → Na(+)) were unsuccessful when mutagenesis of 4-6 residues was used. Phylogenetic analysis suggests that a H(+) pump is the common ancestor of all of these rhodopsins, from which Cl(-) pumps emerged followed by Na(+) pumps. We propose that successful functional conversions of these ion pumps are achieved exclusively when mutagenesis reverses the evolutionary amino acid sequence changes. Dependence of the observed functional conversions on the direction of evolution strongly suggests that the essential structural mechanism of an ancestral function is retained even after the gain of a new function during natural evolution, which can be evoked by a few mutations. By contrast, the gain of a new function needs accumulation of multiple mutations, which may not be easily reproduced by limited mutagenesis in vitro.

Structural Mechanism for Light-driven Transport by a New Type of Chloride Ion Pump, Nonlabens marinus Rhodopsin-3.

The light-driven inward chloride ion-pumping rhodopsin Nonlabens marinus rhodopsin-3 (NM-R3), from a marine flavobacterium, belongs to a phylogenetic lineage distinct from the halorhodopsins known as archaeal inward chloride ion-pumping rhodopsins. NM-R3 and halorhodopsin have distinct motif sequences that are important for chloride ion binding and transport. In this study, we present the crystal structure of a new type of light-driven chloride ion pump, NM-R3, at 1.58 Å resolution. The structure revealed the chloride ion translocation pathway and showed that a single chloride ion resides near the Schiff base. The overall structure, chloride ion-binding site, and translocation pathway of NM-R3 are different from those of halorhodopsin. Unexpectedly, this NM-R3 structure is similar to the crystal structure of the light-driven outward sodium ion pump, Krokinobacter eikastus rhodopsin 2. Structural and mutational analyses of NM-R3 revealed that most of the important amino acid residues for chloride ion pumping exist in the ion influx region, located on the extracellular side of NM-R3. In contrast, on the opposite side, the cytoplasmic regions of K. eikastus rhodopsin 2 were reportedly important for sodium ion pumping. These results provide new insight into ion selection mechanisms in ion pumping rhodopsins, in which the ion influx regions of both the inward and outward pumps are important for their ion selectivities.

Structure determination of α-helical membrane proteins by solution-state NMR: emphasis on retinal proteins.

The biochemical processes of living cells involve a numerous series of reactions that work with exceptional specificity and efficiency. The tight control of this intricate reaction network stems from the architecture of the proteins that drive the chemical reactions and mediate protein-protein interactions. Indeed, the structure of these proteins will determine both their function and interaction partners. A detailed understanding of the proximity and orientation of pivotal functional groups can reveal the molecular mechanistic basis for the activity of a protein. Together with X-ray crystallography and electron microscopy, NMR spectroscopy plays an important role in solving three-dimensional structures of proteins at atomic resolution. In the challenging field of membrane proteins, retinal-binding proteins are often employed as model systems and prototypes to develop biophysical techniques for the study of structural and functional mechanistic aspects. The recent determination of two 3D structures of seven-helical trans-membrane retinal proteins by solution-state NMR spectroscopy highlights the potential of solution NMR techniques in contributing to our understanding of membrane proteins. This review summarizes the multiple strategies available for expression of isotopically labeled membrane proteins. Different environments for mimicking lipid bilayers will be presented, along with the most important NMR methods and labeling schemes used to generate high-quality NMR spectra. The article concludes with an overview of types of conformational restraints used for generation of high-resolution structures of membrane proteins. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.

Electrostatic interactions and hydrogen bond dynamics in chloride pumping by halorhodopsin.

Translocation of negatively charged ions across cell membranes by ion pumps raises the question as to how protein interactions control the location and dynamics of the ion. Here we address this question by performing extensive molecular dynamics simulations of wild type and mutant halorhodopsin, a seven-helical transmembrane protein that translocates chloride ions upon light absorption. We find that inter-helical hydrogen bonds mediated by a key arginine group largely govern the dynamics of the protein and water groups coordinating the chloride ion.

Behavioral flexibility is increased by optogenetic inhibition of neurons in the nucleus accumbens shell during specific time segments.

Behavioral flexibility is vital for survival in an environment of changing contingencies. The nucleus accumbens may play an important role in behavioral flexibility, representing learned stimulus-reward associations in neural activity during response selection and learning from results. To investigate the role of nucleus accumbens neural activity in behavioral flexibility, we used light-activated halorhodopsin to inhibit nucleus accumbens shell neurons during specific time segments of a bar-pressing task requiring a win-stay/lose-shift strategy. We found that optogenetic inhibition during action selection in the time segment preceding a lever press had no effect on performance. However, inhibition occurring in the time segment during feedback of results--whether rewards or nonrewards--reduced the errors that occurred after a change in contingency. Our results demonstrate critical time segments during which nucleus accumbens shell neurons integrate feedback into subsequent responses. Inhibiting nucleus accumbens shell neurons in these time segments, during reinforced performance or after a change in contingencies, increases lose-shift behavior. We propose that the activity of nucleus shell accumbens shell neurons in these time segments plays a key role in integrating knowledge of results into subsequent behavior, as well as in modulating lose-shift behavior when contingencies change.

His166 is the Schiff base proton acceptor in attractant phototaxis receptor sensory rhodopsin I.

Photoactivation of attractant phototaxis receptor sensory rhodopsin I (SRI) in Halobacterium salinarum entails transfer of a proton from the retinylidene chromophore's Schiff base (SB) to an unidentified acceptor residue on the cytoplasmic half-channel, in sharp contrast to other microbial rhodopsins, including the closely related repellent phototaxis receptor SRII and the outward proton pump bacteriorhodopsin, in which the SB proton acceptor is an aspartate residue salt-bridged to the SB in the extracellular (EC) half-channel. His166 on the cytoplasmic side of the SB in SRI has been implicated in the SB proton transfer reaction by mutation studies, and mutants of His166 result in an inverted SB proton release to the EC as well as inversion of the protein's normally attractant phototaxis signal to repellent. Here we found by difference Fourier transform infrared spectroscopy the appearance of Fermi-resonant X-H stretch modes in light-minus-dark difference spectra; their assignment with (15)N labeling and site-directed mutagenesis demonstrates that His166 is the SB proton acceptor during the photochemical reaction cycle of the wild-type SRI-HtrI complex.

Optogenetic inhibition of chemically induced hypersynchronized bursting in mice.

Synchronized activity is common during various physiological operations but can culminate in seizures and consequently in epilepsy in pathological hyperexcitable conditions in the brain. Many types of seizures are not possible to control and impose significant disability for patients with epilepsy. Such intractable epilepsy cases are often associated with degeneration of inhibitory interneurons in the cortical areas resulting in impaired inhibitory drive onto the principal neurons. Recently emerging optogenetic technique has been proposed as an alternative approach to control such seizures but whether it may be effective in situations where inhibitory processes in the brain are compromised has not been addressed. Here we used pharmacological and optogenetic techniques to block inhibitory neurotransmission and induce epileptiform activity in vitro and in vivo. We demonstrate that NpHR-based optogenetic hyperpolarization and thereby inactivation of a principal neuronal population in the hippocampus is effectively attenuating seizure activity caused by disconnected network inhibition both in vitro and in vivo. Our data suggest that epileptiform activity in the hippocampus caused by impaired inhibition may be controlled by optogenetic silencing of principal neurons and potentially can be developed as an alternative treatment for epilepsy.

Optical recording of action potentials in mammalian neurons using a microbial rhodopsin.

Reliable optical detection of single action potentials in mammalian neurons has been one of the longest-standing challenges in neuroscience. Here we achieved this goal by using the endogenous fluorescence of a microbial rhodopsin protein, Archaerhodopsin 3 (Arch) from Halorubrum sodomense, expressed in cultured rat hippocampal neurons. This genetically encoded voltage indicator exhibited an approximately tenfold improvement in sensitivity and speed over existing protein-based voltage indicators, with a roughly linear twofold increase in brightness between -150 mV and +150 mV and a sub-millisecond response time. Arch detected single electrically triggered action potentials with an optical signal-to-noise ratio >10. Arch(D95N) lacked endogenous proton pumping and had 50% greater sensitivity than wild type but had a slower response (41 ms). Nonetheless, Arch(D95N) also resolved individual action potentials. Microbial rhodopsin-based voltage indicators promise to enable optical interrogation of complex neural circuits and electrophysiology in systems for which electrode-based techniques are challenging.

Optogenetic-guided cortical plasticity after nerve injury.

Peripheral nerve injury causes sensory dysfunctions that are thought to be attributable to changes in neuronal activity occurring in somatosensory cortices both contralateral and ipsilateral to the injury. Recent studies suggest that distorted functional response observed in deprived primary somatosensory cortex (S1) may be the result of an increase in inhibitory interneuron activity and is mediated by the transcallosal pathway. The goal of this study was to develop a strategy to manipulate and control the transcallosal activity to facilitate appropriate plasticity by guiding the cortical reorganization in a rat model of sensory deprivation. Since transcallosal fibers originate mainly from excitatory pyramidal neurons somata situated in laminae III and V, the excitatory neurons in rat S1 were engineered to express halorhodopsin, a light-sensitive chloride pump that triggers neuronal hyperpolarization. Results from electrophysiology, optical imaging, and functional MRI measurements are concordant with that within the deprived S1, activity in response to intact forepaw electrical stimulation was significantly increased by concurrent illumination of halorhodopsin over the healthy S1. Optogenetic manipulations effectively decreased the adverse inhibition of deprived cortex and revealed the major contribution of the transcallosal projections, showing interhemispheric neuroplasticity and thus, setting a foundation to develop improved rehabilitation strategies to restore cortical functions.

A pH-sensitive closed-loop nanomachine to control hyperexcitability at the single neuron level.

Epilepsy affects 1% of the general population and 30% of patients are resistant to antiepileptic drugs. Although optogenetics is an efficient antiepileptic strategy, the difficulty of illuminating deep brain areas poses translational challenges. Thus, the search of alternative light sources is strongly needed. Here, we develop pH-sensitive inhibitory luminopsin (pHIL), a closed-loop chemo-optogenetic nanomachine composed of a luciferase-based light generator, a fluorescent sensor of intracellular pH (E2GFP), and an optogenetic actuator (halorhodopsin) for silencing neuronal activity. Stimulated by coelenterazine, pHIL experiences bioluminescence resonance energy transfer between luciferase and E2GFP which, under conditions of acidic pH, activates halorhodopsin. In primary neurons, pHIL senses the intracellular pH drop associated with hyperactivity and optogenetically aborts paroxysmal activity elicited by the administration of convulsants. The expression of pHIL in hippocampal pyramidal neurons is effective in decreasing duration and increasing latency of pilocarpine-induced tonic-clonic seizures upon in vivo coelenterazine administration, without affecting higher brain functions. The same treatment is effective in markedly decreasing seizure manifestations in a murine model of genetic epilepsy. The results indicate that pHIL represents a potentially promising closed-loop chemo-optogenetic strategy to treat drug-refractory epilepsy.

Role of Thr218 in the light-driven anion pump halorhodopsin from Natronomonas pharaonis.

Halorhodopsin (HR) is an inward-directed light-driven halogen ion pump, and NpHR is a HR from Natronomonas pharaonis. Unphotolyzed NpHR binds halogen ion in the vicinity of the Schiff base, which links retinal to Lys256. This halogen ion is transported during the photocycle. We made various mutants of Thr218, which is located one half-turn up from the Schiff base to the cytoplasm (CP) channel, and analyzed the photocycle using a sequential irreversible model. Four photochemically defined intermediates (P(i), i = 1-4) were adequate to describe the photocycle. The third component, P3, was a quasi-equilibrium complex between the N and O intermediates, where a N ↔ O + Cl⁻ equilibrium was attained. The K(d,N↔O) values of this equilibrium for various mutants were determined, and the value of Thr (wild type) was the highest. The partial molar volume differences between N and O, ΔV(N→O), were estimated from the pressure dependence of K(d,N↔O). A comparison between K(d,N↔O) and ΔV(N→O) led to the conclusion that water entry by the F-helix opening at O may occur, which may increase K(d,N↔O). For some mutants, however, large ΔV(N→O) values were found, whereas the K(d,N↔O) values were small. This suggests that the special coordination of a water molecule with the OH group of Thr is necessary for the increase in K(d,N↔O). Mutants with a small K(d,N↔O) showed low pumping activities in the presence of inside negative membrane potential, while the mutant activities were not different in the absence of membrane potential. The effect of the mutation on the pumping activities is discussed.

Optical inhibition of motor nerve and muscle activity in vivo.

INTRODUCTION: There is no therapeutic approach that provides precise and rapidly reversible inhibition of motor nerve and muscle activity for treatment of spastic hypertonia. METHODS: We used optogenetics to demonstrate precise and rapidly reversible light-mediated inhibition of motor nerve and muscle activity in vivo in transgenic Thy1::eNpHR2.0 mice. RESULTS: We found optical inhibition of motor nerve and muscle activity to be effective at all muscle force amplitudes and determined that muscle activity can be modulated by changing light pulse duration and light power density. CONCLUSIONS: This demonstration of optical inhibition of motor nerves is an important advancement toward novel optogenetics-based therapies for spastic hypertonia.

Multimodal fast optical interrogation of neural circuitry.

Our understanding of the cellular implementation of systems-level neural processes like action, thought and emotion has been limited by the availability of tools to interrogate specific classes of neural cells within intact, living brain tissue. Here we identify and develop an archaeal light-driven chloride pump (NpHR) from Natronomonas pharaonis for temporally precise optical inhibition of neural activity. NpHR allows either knockout of single action potentials, or sustained blockade of spiking. NpHR is compatible with ChR2, the previous optical excitation technology we have described, in that the two opposing probes operate at similar light powers but with well-separated action spectra. NpHR, like ChR2, functions in mammals without exogenous cofactors, and the two probes can be integrated with calcium imaging in mammalian brain tissue for bidirectional optical modulation and readout of neural activity. Likewise, NpHR and ChR2 can be targeted together to Caenorhabditis elegans muscle and cholinergic motor neurons to control locomotion bidirectionally. NpHR and ChR2 form a complete system for multimodal, high-speed, genetically targeted, all-optical interrogation of living neural circuits.

Optogenetic investigation of neuropsychiatric diseases.

Optogenetic technology, also known as optogenetics, is a novel multidisciplinary field in biotechnology that integrates genetic engineering, electrophysiology, and optical and electronic engineering. This recently developed technology has evolved rapidly and generated considerable excitement in neuroscience research. This technology successfully solves the severe problem of achieving both high temporal and spatial precision within intact neural tissues of animals that electrical stimulation and pharmacological methods cannot achieve. It allows neurons to express light-sensitive genes that enable the identification, dissection, and manipulation of specific neural populations and their connections in the tissues and organs of awake animals with unprecedented spatial and temporal precision. Light-sensitive genes chiefly including the genetically targeted light-gated channels channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR) cause intracellular ion flow during optical illumination. Subsequently, the neurons undergo a series of changes resulting from membrane depolarization or hyperpolarization. To date, there are many published research articles and reviews that describe this new technology; however, few of the reports concern its application to neuropsychiatric diseases. In this review, we summarize the most recent optogenetic research in these diseases, including Parkinson's disease (PD), epilepsy, schizophrenia, anxiety, fear, reward behaviors, and sleep disorders. We propose that novel optogenetics technology creates excellent opportunities for innovative treatment strategies of neuropsychiatric diseases.

Key determinants for signaling in the sensory rhodopsin II/transducer complex are different between Halobacterium salinarum and Natronomonas pharaonis.

The cell membrane of Halobacterium salinarum contains a retinal-binding photoreceptor, sensory rhodopsin II (HsSRII), coupled with its cognate transducer (HsHtrII), allowing repellent phototaxis behavior for shorter wavelength light. Previous studies on SRII from Natronomonas pharaonis (NpSRII) pointed out the importance of the hydrogen bonding interaction between Thr204NpSRII and Tyr174NpSRII in signal transfer from SRII to HtrII. Here, we investigated the effect on phototactic function by replacing residues in HsSRII corresponding to Thr204NpSRII and Tyr174NpSRII . Whereas replacement of either residue altered the photocycle kinetics, introduction of any mutations at Ser201HsSRII and Tyr171HsSRII did not eliminate negative phototaxis function. These observations imply the possibility of the presence of an unidentified molecular mechanism for photophobic signal transduction differing from NpSRII-NpHtrII.