Driving forces of proton-pumping rhodopsins.

Proton-pumping rhodopsins are light-driven proton transporters that have been discovered from various microbiota. They are categorized into two groups, outward-directed and inward-directed proton pumps. Although the directions of transport are opposite, they are active proton transporters that create a H+ gradient across a membrane. Here, we aimed to study the driving force of the proton-pumping rhodopsins, and the effect of ΔΨ and ΔpH on their pumping functions. We systematically characterized the H+ transport properties of nine different rhodopsins, six outward-directed H+ pumps, and three inward-directed pumps by patch clamp measurements after expressing them in mammalian cells. The driving force of each pump was estimated from the slope of the current-voltage relations (I-V plot). Notably, among the tested rhodopsins, we found a large variation in driving forces, ranging from 83 to 399 mV. The driving force and decay rate of each pump current exhibited a good correlation. We determined driving forces under various pHs. pH dependency was less than predicted by the Nersnt potential in most of the rhodopsins. Our study demonstrates that the H+-pumping rhodopsins from different organisms exhibit various pumping properties in terms of driving force, kinetics, and pH dependency, which could be evolutionarily derived from adaptations to their environments.

The high-light-sensitivity mechanism and optogenetic properties of the bacteriorhodopsin-like channelrhodopsin GtCCR4.

Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), GtCCR4 has higher light sensitivity than typical channelrhodopsins. Furthermore, GtCCR4 shows superior properties as an optogenetic tool, such as minimal desensitization. Our structural analyses of GtCCR2 and GtCCR4 revealed that GtCCR4 has an outwardly bent transmembrane helix, resembling the conformation of activated G-protein-coupled receptors. Spectroscopic and electrophysiological comparisons suggested that this helix bend in GtCCR4 omits channel recovery time and contributes to high light sensitivity. An electrophysiological comparison of GtCCR4 and the well-characterized optogenetic tool ChRmine demonstrated that GtCCR4 has superior current continuity and action-potential spike generation with less invasiveness in neurons. We also identified highly active mutants of GtCCR4. These results shed light on the diverse structures and dynamics of microbial rhodopsins and demonstrate the strong optogenetic potential of GtCCR4.

Optogenetic manipulation of neuronal and cardiomyocyte functions in zebrafish using microbial rhodopsins and adenylyl cyclases.

Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control cell and tissue functions in vivo. We exploited GtCCR4 and KnChR, cation channelrhodopsins from algae, BeGC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria (OaPAC) or bacteria (bPAC), to control cell functions in zebrafish. Optical activation of GtCCR4 and KnChR in the hindbrain reticulospinal V2a neurons, which are involved in locomotion, induced swimming behavior at relatively short latencies, whereas activation of BeGC1 or PACs achieved it at long latencies. Activation of GtCCR4 and KnChR in cardiomyocytes induced cardiac arrest, whereas activation of bPAC gradually induced bradycardia. KnChR activation led to an increase in intracellular Ca2+ in the heart, suggesting that depolarization caused cardiac arrest. These data suggest that these optogenetic tools can be used to reveal the function and regulation of zebrafish neurons and cardiomyocytes.

Light-Driven Chloride and Sulfate Pump with Two Different Transport Modes.

Ion pumps are membrane proteins that actively translocate ions by using energy. All known pumps bind ions in the resting state, and external energy allows ion transport through protein structural changes. The light-driven sodium-ion pump Krokinobacter eikastus rhodopsin 2 (KR2) is an exceptional case in which ion binding follows the energy input. In this study, we report another case of this unusual transport mode. The NTQ rhodopsin from Alteribacter aurantiacus (AaClR) is a natural light-driven chloride pump, in which the chloride ion binds to the resting state. AaClR is also able to pump sulfate ions, though the pump efficiency is much lower for sulfate ions than for chloride ions. Detailed spectroscopic analysis revealed no binding of the sulfate ion to the resting state of AaClR, indicating that binding of the substrate (sulfate ion) to the resting state is not necessary for active transport. This property of the AaClR sulfate pump is similar to that of the KR2 sodium pump. Photocycle dynamics of the AaClR sulfate pump resemble a non-functional cycle in the absence of anions. Despite this, flash photolysis and difference Fourier transform infrared spectroscopy suggest transient binding of the sulfate ion to AaClR. The molecular mechanism of this unusual active transport by AaClR is discussed.

A Novel Color Switch of Microbial Rhodopsin.

Function of animal and microbial rhodopsins starts by light absorption of the retinal chromophore. The absorption maximum wavelength (λmax) of rhodopsins is determined by the energy gap between the electronically ground (S0) and first excited (S1) state of the retinal chromophore, and the color tuning mechanism is one of the central topics in rhodopsin research. "Color switches", color-determining residues, are red- and blue-shifting amino acids at the same position in two rhodopsins, whose exchange causes spectral blue- and red-shifts, respectively, in each rhodopsin. As mutation easily destroys elaborate chromophore-protein interactions, the known color switches in microbial rhodopsins are limited; the L/Q switch in C-helix (TM3), the A/TS switch in G-helix (TM7), and the G/P switch in F-helix (TM6). Here, we report a novel color switch of microbial rhodopsins, which is located in D-helix (TM4). In this color switch, the red- and blue-shifting amino acids are Asn (N) and Leu (L)/Ile (I), respectively. As Asn and Leu/Ile are polar and nonpolar amino acids, respectively, and the position is located near the ß-ionone ring, the N/LI switch matches the general rule of color tuning by polarity. The N/LI switch is also useful for optogenetics, as many ion-transporting rhodopsins contain blue-shifting amino acids, such as L and I, at that position.

A light-gated cation channel with high reactivity to weak light.

The cryptophyte algae, Guillardia theta, possesses 46 genes that are homologous to microbial rhodopsins. Five of them are functionally light-gated cation channelrhodopsins (GtCCR1-5) that are phylogenetically distinct from chlorophyte channelrhodopsins (ChRs) such as ChR2 from Chlamydomonas reinhardtii. In this study, we report the ion channel properties of these five CCRs and compared them with ChR2 and other ChRs widely used in optogenetics. We revealed that light sensitivity varied among GtCCR1-5, in which GtCCR1-3 exhibited an apparent EC50 of 0.21-1.16 mW/mm2, similar to that of ChR2, whereas GtCCR4 and GtCCR5 possess two EC50s, one of which is significantly small (0.025 and 0.032 mW/mm2). GtCCR4 is able to trigger action potentials in high temporal resolution, similar to ChR2, but requires lower light power, when expressed in cortical neurons. Moreover, a high light-sensitive response was observed when GtCCR4 was introduced into blind retina ganglion cells of rd1, a mouse model of retinitis pigmentosa. Thus, GtCCR4 provides optogenetic neuronal activation with high light sensitivity and temporal precision.

Unusual Photoisomerization Pathway in a Near-Infrared Light Absorbing Enzymerhodopsin.

Microbial and animal rhodopsins possess retinal chromophores which capture light and normally photoisomerize from all-trans to 13-cis and from 11-cis to all-trans-retinal, respectively. Here, we show that a near-infrared light-absorbing enzymerhodopsin from Obelidium mucronatum (OmNeoR) contains the all-trans form in the dark but isomerizes into the 7-cis form upon illumination. The photoproduct (λmax = 372 nm; P372) possesses a deprotonated Schiff base, and the system exhibits a bistable nature. The photochemistry of OmNeoR was arrested at <270 K, indicating the presence of a potential barrier in the excited state. Formation of P372 is accompanied by protonation changes of protonated carboxylic acids and peptide backbone changes of an α-helix. Photoisomerization from the all-trans to 7-cis retinal conformation rarely occurs in any solvent and protein environments; thus, the present study reports on a novel photochemistry mediated by a microbial rhodopsin, leading from the all-trans to 7-cis form selectively.

Proton-transporting heliorhodopsins from marine giant viruses.

Rhodopsins convert light into signals and energy in animals and microbes. Heliorhodopsins (HeRs), a recently discovered new rhodopsin family, are widely present in archaea, bacteria, unicellular eukaryotes, and giant viruses, but their function remains unknown. Here, we report that a viral HeR from Emiliania huxleyi virus 202 (V2HeR3) is a light-activated proton transporter. V2HeR3 absorbs blue-green light, and the active intermediate contains the deprotonated retinal Schiff base. Site-directed mutagenesis study revealed that E191 in TM6 constitutes the gate together with the retinal Schiff base. E205 and E215 form a PAG of the Schiff base, and mutations at these positions converted the protein into an outward proton pump. Three environmental viral HeRs from the same group as well as a more distantly related HeR exhibited similar proton-transport activity, indicating that HeR functions might be diverse similarly to type-1 microbial rhodopsins. Some strains of E. huxleyi contain one HeR that is related to the viral HeRs, while its viruses EhV-201 and EhV-202 contain two and three HeRs, respectively. Except for V2HeR3 from EhV-202, none of these proteins exhibit ion transport activity. Thus, when expressed in the E. huxleyi cell membranes, only V2HeR3 has the potential to depolarize the host cells by light, possibly to overcome the host defense mechanisms or to prevent superinfection. The neuronal activity generated by V2HeR3 suggests that it can potentially be used as an optogenetic tool, similarly to type-1 microbial rhodopsins.

Optogenetic reprogramming of carbon metabolism using light-powering microbial proton pump systems.

In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. We first demonstrated a photo-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing already-known delta rhodopsin (dR) in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the rhodopsin-activator, all-trans-retinal. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.

Ion transport activity and optogenetics capability of light-driven Na+-pump KR2.

KR2 from marine bacteria Krokinobacter eikastus is a light-driven Na+ pumping rhodopsin family (NaRs) member that actively transports Na+ and/or H+ depending on the ionic state. We here report electrophysiological studies on KR2 to address ion-transport properties under various electrochemical potentials of Δ[Na+], ΔpH, membrane voltage and light quality, because the contributions of these on the pumping activity were less understood so far. After transient expression of KR2 in mammalian cultured cells (ND7/23 cells), photocurrents were measured by whole-cell patch clamp under various intracellular Na+ and pH conditions. When KR2 was continuously illuminated with LED light, two distinct time constants were obtained depending on the Na+ concentration. KR2 exhibited slow ion transport (τoff of 28 ms) below 1.1 mM NaCl and rapid transport (τoff of 11 ms) above 11 mM NaCl. This indicates distinct transporting kinetics of H+ and Na+. Photocurrent amplitude (current density) depends on the intracellular Na+ concentration, as is expected for a Na+ pump. The M-intermediate in the photocycle of KR2 could be transferred into the dark state without net ion transport by blue light illumination on top of green light. The M intermediate was stabilized by higher membrane voltage. Furthermore, we assessed the optogenetic silencing effect of rat cortical neurons after expressing KR2. Light power dependency revealed that action potential was profoundly inhibited by 1.5 mW/mm2 green light illumination, confirming the ability to apply KR2 as an optogenetics silencer.

Remote control of neural function by X-ray-induced scintillation.

Scintillators emit visible luminescence when irradiated with X-rays. Given the unlimited tissue penetration of X-rays, the employment of scintillators could enable remote optogenetic control of neural functions at any depth of the brain. Here we show that a yellow-emitting inorganic scintillator, Ce-doped Gd3(Al,Ga)5O12 (Ce:GAGG), can effectively activate red-shifted excitatory and inhibitory opsins, ChRmine and GtACR1, respectively. Using injectable Ce:GAGG microparticles, we successfully activated and inhibited midbrain dopamine neurons in freely moving mice by X-ray irradiation, producing bidirectional modulation of place preference behavior. Ce:GAGG microparticles are non-cytotoxic and biocompatible, allowing for chronic implantation. Pulsed X-ray irradiation at a clinical dose level is sufficient to elicit behavioral changes without reducing the number of radiosensitive cells in the brain and bone marrow. Thus, scintillator-mediated optogenetics enables minimally invasive, wireless control of cellular functions at any tissue depth in living animals, expanding X-ray applications to functional studies of biology and medicine.

TAT Rhodopsin Is an Ultraviolet-Dependent Environmental pH Sensor.

In many rhodopsins, the retinal Schiff base pKa remains very high, ensuring Schiff base protonation captures visible light. Nevertheless, recently we found that TAT rhodopsin contains protonated and unprotonated forms at physiological pH. The protonated form displays a unique photochemical behavior in which the primary K intermediate returns to the original state within 10-5 s, and the lack of photocycle completion poses questions about the functional role of TAT rhodopsin. Here we studied the molecular properties of the protonated and unprotonated forms of the Schiff base in TAT rhodopsin. We confirmed no photointermediate formation at >10-5 s for the protonated form of TAT rhodopsin in microenvironments such as detergents, nanodiscs, and liposomes. In contrast, the unprotonated form features a very long photocycle with a time constant of 15 s. A low-temperature study revealed that the primary reaction of the unprotonated form is all-trans to 13-cis photoisomerization, which is usual, but with a proton transfer reaction occurring at 77 K, which is unusual. The active intermediate contains the unprotonated Schiff base as well as the resting state. Electrophysiological measurements excluded ion-transport activity for TAT rhodopsin, while transient outward proton movement only at an alkaline extracellular pH indicates that TAT rhodopsin senses the extracellular pH. On the basis of the findings presented here, we propose that TAT rhodopsin is an ultraviolet (UV)-dependent environmental pH sensor in marine bacteria. At acidic pH, absorbed visible light energy is quickly dissipated into heat without any function. In contrast, when the environmental pH becomes high, absorption of UV/blue light yields formation of the long-lived intermediates, possibly driving the signal transduction cascade in marine bacteria.

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.

Molecular Properties and Optogenetic Applications of Enzymerhodopsins.

The cyclic nucleotides cAMP and cGMP are ubiquitous secondary messengers that regulate multiple biological functions including gene expression, differentiation, proliferation, and cell survival. In sensory neurons, cyclic nucleotides are responsible for signal modulation, amplification, and encoding. For spatial and temporal manipulation of cyclic nucleotide dynamics, optogenetics have a great advantage over pharmacological approaches. Enzymerhodopsins are a unique family of microbial rhodopsins. These molecules are made up of a membrane-embedded rhodopsin domain, which binds an all trans-retinal to form a chromophore, and a cytoplasmic water-soluble catalytic domain. To date, three kinds of molecules have been identified from lower eukaryotes such as fungi, algae, and flagellates. Among these, histidine kinase rhodopsin (HKR) is a light-inhibited guanylyl cyclase. Rhodopsin GC (Rh-GC) functions as a light-activated guanylyl cyclase, while rhodopsin PDE (Rh-PDE) functions as a light-activated phosphodiesterase that degrades cAMP and cGMP. These enzymerhodopsins have great potential in optogenetic applications for manipulating the intracellular cyclic nucleotide dynamics of living cells. Here we introduce the molecular function and applicability of these molecules.

Structural insights into the mechanism of rhodopsin phosphodiesterase.

Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase and photosensitive adenylyl cyclase. Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The crystal structure of the rhodopsin domain at 2.6 Å resolution revealed a new topology of rhodopsins, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the rhodopsin domain (3.5 Å) and PDE domain (2.1 Å) with their connecting linkers, which showed a rough sketch of the full-length Rh-PDE. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.

Molecular Properties of New Enzyme Rhodopsins with Phosphodiesterase Activity.

The choanoflagellate Salpingoeca rosetta contains a chimeric rhodopsin protein composed of an N-terminal rhodopsin (Rh) domain and a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. The Rh-PDE enzyme (SrRh-PDE), which decreases the concentrations of cyclic nucleotides such as cGMP and cAMP in light, is a useful tool in optogenetics. Recently, eight additional Rh-PDE enzymes were found in choanoflagellate species, four from Choanoeca flexa and the other four from other species. In this paper, we studied the molecular properties of these new Rh-PDEs, which were compared with SrRh-PDE. Upon expression in HEK293 cells, four Rh-PDE proteins, including CfRh-PDE2 and CfRh-PDE3, exhibited no PDE activity when assessed by in-cell measurements and in vitro HPLC analysis. On the other hand, CfRh-PDE1 showed light-dependent PDE activity toward cGMP, which absorbed maximally at 491 nm. Therefore, CfRh-PDE1 is presumably responsible for colony inversion in C. flexa by absorbing blue-green light. The molecular properties of MrRh-PDE were similar to those of SrRh-PDE, although the λmax of MrRh-PDE (516 nm) was considerably red-shifted from that of SrRh-PDE (492 nm). One Rh-PDE, AsRh-PDE, did not contain the retinal-binding Lys at TM7 and showed cAMP-specific PDE activity both in the dark and light. These results provide mechanistic insight into rhodopsin-mediated, light-dependent regulation of second-messenger levels in eukaryotic microbes.

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.