RESUMO
Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the ß-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.
Assuntos
Rodopsinas Microbianas/química , Thermoplasmales/química , Bacteriorodopsinas/química , Sítios de Ligação , Cristalografia por Raios X , Microscopia de Força Atômica , Modelos Moleculares , Dobramento de Proteína , Multimerização Proteica , Retinaldeído/química , Rodopsinas Microbianas/ultraestruturaRESUMO
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.
Assuntos
Flavobacteriaceae/química , Bombas de Íon/química , Bombas de Íon/efeitos da radiação , Luz , Rodopsina/química , Rodopsina/efeitos da radiação , Sódio/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Bombas de Íon/genética , Bombas de Íon/metabolismo , Transporte de Íons/genética , Transporte de Íons/efeitos da radiação , Modelos Biológicos , Modelos Moleculares , Mutagênese/genética , Optogenética , Potássio/metabolismo , Conformação Proteica , Engenharia de Proteínas , Retinaldeído/química , Retinaldeído/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Bases de Schiff , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Gadolínio/metabolismo , Rodopsina/metabolismo , Estreptófitas/metabolismo , Cátions/química , Cátions/metabolismo , Gadolínio/química , Transporte de Íons , Rodopsina/química , Estreptófitas/químicaRESUMO
All-optical methods that provide deeper understanding of neural activity are currently being developed. Optogenetics is a biological technique useful to control neuronal activity or life phenomena using light. Microbial rhodopsins are light-activated membrane proteins used as optogenetic tools. Microbial rhodopsins such as channelrhodopsin2 (ChR2) consist of seven-pass transmembrane proteins with a covalently bound retinal. Light absorption is followed by photoisomerization of the all-trans retinal to a 13-cis configuration and subsequent conformational changes in the molecule, with consequent permeability of the channel structure to ions. Recent studies have reported the discovery of microbial rhodopsins with novel functions. Microbial rhodopsin diversity has also increased. We describe the characteristics of microbial rhodopsins used as optogenetic tools and the latest research in this domain.
Assuntos
Optogenética , Optogenética/métodos , Humanos , Animais , Luz , Channelrhodopsins/metabolismo , Channelrhodopsins/genética , Rodopsinas Microbianas/metabolismo , Rodopsina/metabolismo , Rodopsina/genéticaRESUMO
Microbial rhodopsins are photoreceptive membrane proteins found from diverse microorganisms such as archaea, eubacteria, eukaryotes and viruses. Many microbial rhodopsins possess ion-transport activity by light, such as channels and pumps, and ion-transporting rhodopsins are important tools in optogenetics that control animal behavior by light. Historically, molecular mechanism of rhodopsins has been studied by spectroscopic methods for purified proteins. On the other hand, ion-transport function has to be studied by different methods. This chapter introduces two methods of functional assay of ion-transporting rhodopsins by light. One is a patch clamp method using mammalian cells, and another is an ion-transport assay using pH electrode and microbial cells. These functional assay provides fundamental data of ion-transporting rhodopsins, and thus contributes to evaluation for optogenetic tools.
Assuntos
Rodopsina , Rodopsinas Microbianas , Animais , Rodopsina/química , Rodopsina/metabolismo , Transporte de Íons , Rodopsinas Microbianas/química , Archaea/metabolismo , Bactérias/metabolismo , Mamíferos/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Parada Cardíaca , Miócitos Cardíacos , Animais , Adenilil Ciclases/genética , Peixe-Zebra , Rodopsinas Microbianas , Optogenética , NeurôniosRESUMO
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.
Assuntos
Luz , Fotofobia , Camundongos , Animais , Channelrhodopsins , Cátions/metabolismo , Células Ganglionares da Retina/metabolismo , OptogenéticaRESUMO
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.
Assuntos
Vírus Gigantes , Prótons , Animais , Transporte de Íons , Rodopsina/genética , Rodopsinas Microbianas/genética , Bases de SchiffRESUMO
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.
Assuntos
Flavobacteriaceae/metabolismo , Íons/metabolismo , Luz , Neurônios/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Linhagem Celular , Neurônios/citologia , RatosRESUMO
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.
Assuntos
Channelrhodopsins/metabolismo , Clorófitas/metabolismo , Ativação do Canal Iônico , Sequência de Aminoácidos , Animais , Linhagem Celular , Channelrhodopsins/química , Channelrhodopsins/genética , Channelrhodopsins/efeitos da radiação , Clorófitas/genética , Clorófitas/efeitos da radiação , Ativação do Canal Iônico/efeitos da radiação , Cinética , Luz , Potenciais da Membrana , Camundongos , Optogenética , Domínios Proteicos , Ratos , Relação Estrutura-AtividadeRESUMO
Optogenetics is a growing technique which allows manipulation of biological events simply by illumination. The technique is appreciated especially in the neuroscience field because of its availability in controlling neuronal functions. A light-gated cation channel, Cr_ChR2 from Chlamydomonas reinhardtii, is the first and mostly applied to optogenetics for activating neuronal excitability. In addition, the molecular mechanism of Cr_ChR2 has been intensively studied by electrophysiology, spectroscopy, X-ray structural studies, etc. Novel cation channelrhodopsins from Guillardia theta, namely, Gt_CCR1-4, were discovered in 2016 and 2017. These channelrhodopsins are more homologous to haloarchaeal rhodopsins, particularly the proton pumps. Thus these cryptophyte-type light-gated cation channels are structurally and mechanistically distinct from chlorophyte channelrhodopsin such as Cr_ChR2. We here compared the photocurrent properties, cation selectivity, and kinetics between well-known Cr_ChR2 and Gt_CCR4. The light sensitivity of Gt_CCR4 is significantly higher than that of Cr_ChR2, while the channel open lifetime is in the same range as that of Cr_ChR2. Gt_CCR4 shows high Na+ selectivity in which the selectivity ratio for Na+ was 37-fold larger than that for Cr_ChR2, which primarily conducts H+. On the other hand, Gt_CCR4 conducted almost no H+ and no Ca2+ under physiological conditions. Other unique features and the applicability of Gt_CCR4 for optogenetics were discussed.
RESUMO
Microbial rhodopsin is a large family of membrane proteins having seven transmembrane helices (TM1-7) with an all-trans retinal (ATR) chromophore that is covalently bound to Lys in the TM7. The Trp residue in the middle of TM3, which is homologous to W86 of bacteriorhodopsin (BR), is highly conserved among microbial rhodopsins with various light-driven functions. However, the significance of this Trp for the ion transport function of microbial rhodopsins has long remained unknown. Here, we replaced the W163 (BR W86 counterpart) of a channelrhodopsin (ChR), C1C2/ChRWR, which is a chimera between ChR1 and 2, with a smaller aromatic residue, Phe to verify its role in the ion transport. Under whole-cell patch clamp recordings from the ND7/23 cells that were transfected with the DNA plasmid coding human codon optimized C1C2/ChRWR (hWR) or its W163F mutant (hWR-W163F), the photocurrents were evoked by a pulsatile light at 475 nm. The ion-transporting activity of hWR was strongly altered by the W163F mutation in 3 points: (1) the H+ leak at positive membrane potential (V m) and its light-adaptation, (2) the attenuation of cation channel activity and (3) the manifestation of outward H+ pump activity. All of these results strongly suggest that W163 has a role in stabilizing the structure involved in the gating-on and -off of the cation channel, the role of "gate keeper". We can attribute the attenuation of cation channel activity to the incomplete gating-on and the H+ leak to the incomplete gating-off.
RESUMO
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.
Assuntos
Archaea/metabolismo , Ativação do Canal Iônico/efeitos da radiação , Bombas de Próton/metabolismo , Rodopsina/metabolismo , Archaea/genética , Membrana Celular/metabolismo , Imunofluorescência , Luz , Modelos Moleculares , Família Multigênica , Mutação , Conformação Proteica , Bombas de Próton/química , Bombas de Próton/genética , Rodopsina/química , Rodopsina/genética , Espectroscopia de Infravermelho com Transformada de Fourier , Relação Estrutura-AtividadeRESUMO
Channelrhodopsin-1 and 2 (ChR1 and ChR2) form cation channels that are gated by light through an unknown mechanism. We tested the DC-gate hypothesis that C167 and D195 are involved in the stabilization of the cation-permeable state of ChRWR/C1C2 which consists of TM1-5 of ChR1 and TM6-7 of ChR2 and ChRFR which consists of TM1-2 of ChR1 and TM3-7 of ChR2. The cation permeable state of each ChRWR and ChRFR was markedly prolonged in the order of several tens of seconds when either C167 or D195 position was mutated to alanine (A). Therefore, the DC-gate function was conserved among these chimeric ChRs. We next investigated the kinetic properties of the ON/OFF response of these bi-stable ChR mutants as they are important in designing the photostimulation protocols for the optogenetic manipulation of neuronal activities. The turning-on rate constant of each photocurrent followed a linear relationship to 0-0.12 mW mm(-2) of blue LED light or to 0-0.33 mW mm(-2) of cyan LED light. Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance. As the magnitude of the photocurrent was mostly determined by the turning-on rate constant and the irradiation time, the minimal irradiance that effectively evoked an action potential (threshold irradiance) was decreased with time only if the neuron, which expresses bi-stable ChRs, has a certain large membrane time constant (eg. τm > 20 ms). On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time. Based on these quantitative data, we would propose that these bi-stable ChRs would be most suitable for enhancing the intrinsic activity of excitatory pyramidal neurons at a minimal magnitude of irradiance.
Assuntos
Ativação do Canal Iônico/genética , Transtornos de Fotossensibilidade/genética , Células Piramidais/metabolismo , Sequência de Aminoácidos , Animais , Channelrhodopsins , Potenciais Evocados Visuais , Ativação do Canal Iônico/fisiologia , Cinética , Luz , Mutação , Células Piramidais/fisiologia , RatosRESUMO
Non-invasive remote control technologies designed to manipulate neural functions have been long-awaited for the comprehensive and quantitative understanding of neuronal network in the brain as well as for the therapy of neurological disorders. Recently, it has become possible for the neuronal activity to be optically manipulated using biological photo-reactive molecules such as channelrhodopsin (ChR)-2. However, ChR2 and its relatives are mostly reactive to visible light, which does not effectively penetrate through biological tissues. In contrast, near-infrared (NIR) light (650-1450 nm) penetrates deep into the tissues because biological systems are almost transparent to light within this so-called 'imaging window'. Here we used lanthanide nanoparticles (LNPs), composed of rare-earth elements, as luminous bodies to activate ChRs since they absorb low-energy NIR light to emit high-energy visible light (up-conversion). Here, we created a new type of optogenetic system which consists of the donor LNPs and the acceptor ChRs. The NIR laser irradiation emitted visible light from LNPs, then induced the photo-reactive responses in the near-by cells that expressed ChRs. However, there remains room for large improvements in the energy efficiency of the LNP-ChR system.
Assuntos
Raios Infravermelhos , Optogenética , Animais , Elementos da Série dos Lantanídeos , Nanopartículas Metálicas , Camundongos , Neurônios/fisiologia , Ratos , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
The calyx-type synapse of chick ciliary ganglion (CG) has been intensively studied for decades as a model system for the synaptic development, morphology and physiology. Despite recent advances in optogenetics probing and/or manipulation of the elementary steps of the transmitter release such as membrane depolarization and Ca(2+) elevation, the current gene-manipulating methods are not suitable for targeting specifically the calyx-type presynaptic terminals. Here, we evaluated a method for manipulating the molecular and functional organization of the presynaptic terminals of this model synapse. We transfected progenitors of the Edinger-Westphal (EW) nucleus neurons with an EGFP expression vector by in ovo electroporation at embryonic day 2 (E2) and examined the CG at E8-14. We found that dozens of the calyx-type presynaptic terminals and axons were selectively labeled with EGFP fluorescence. When a Brainbow construct containing the membrane-tethered fluorescent proteins m-CFP, m-YFP and m-RFP, was introduced together with a Cre expression construct, the color coding of each presynaptic axon facilitated discrimination among inter-tangled projections, particularly during the developmental re-organization period of synaptic connections. With the simultaneous expression of one of the chimeric variants of channelrhodopsins, channelrhodopsin-fast receiver (ChRFR), and R-GECO1, a red-shifted fluorescent Ca(2+)-sensor, the Ca(2+) elevation was optically measured under direct photostimulation of the presynaptic terminal. Although this optically evoked Ca(2+) elevation was mostly dependent on the action potential, a significant component remained even in the absence of extracellular Ca(2+). It is suggested that the photo-activation of ChRFR facilitated the release of Ca(2+) from intracellular Ca(2+) stores directly or indirectly. The above system, by facilitating the molecular study of the calyx-type presynaptic terminal, would provide an experimental platform for unveiling the molecular mechanisms underlying the morphology, physiology and development of synapses.