Activating an invertebrate bistable opsin with the all-trans 6.11 retinal analog.

Animal vision depends on opsins, a category of G protein-coupled receptor (GPCR) that achieves light sensitivity by covalent attachment to retinal. Typically binding as an inverse agonist, 11-cis retinal photoisomerizes to the all-trans isomer and activates the receptor, initiating downstream signaling cascades. Retinal bound to bistable opsins isomerizes back to the 11-cis state after absorption of a second photon, inactivating the receptor. Bistable opsins are essential for invertebrate vision and nonvisual light perception across the animal kingdom. While crystal structures are available for bistable opsins in the inactive state, it has proven difficult to form homogeneous populations of activated bistable opsins either via illumination or reconstitution with all-trans retinal. Here, we show that a nonnatural retinal analog, all-trans retinal 6.11 (ATR6.11), can be reconstituted with the invertebrate bistable opsin, Jumping Spider Rhodopsin-1 (JSR1). Biochemical activity assays demonstrate that ATR6.11 functions as a JSR1 agonist. ATR6.11 binding also enables complex formation between JSR1 and signaling partners. Our findings demonstrate the utility of retinal analogs for biophysical characterization of bistable opsins, which will deepen our understanding of light perception in animals.

Whale shark rhodopsin adapted to deep-sea lifestyle by a substitution associated with human disease.

Spectral tuning of visual pigments often facilitates adaptation to new environments, and it is intriguing to study the visual ecology of pelagic sharks with secondarily expanded habitats. The whale shark, which dives into the deep sea of nearly 2,000 meters besides near-surface filter feeding, was previously shown to possess the 'blue-shifted' rhodopsin (RHO), which is a signature of deep-sea adaptation. In this study, our spectroscopy of recombinant whale shark RHO mutants revealed that this blue shift is caused dominantly by an unprecedented spectral tuning site 94. In humans, the mutation at the site causes congenital stationary night blindness (CSNB) by reducing the thermal stability of RHO. Similarly, the RHO of deep-diving whale shark has reduced thermal stability, which was experimentally shown to be achieved by site 178 and 94. RHOs having the natural substitution at site 94 are also found in some Antarctic fishes, suggesting that the blue shift by the substitution at the CSNB site associated with the reduction in thermal stability might be allowed in cold-water deep-sea habitats.

High-performance optical control of GPCR signaling by bistable animal opsins MosOpn3 and LamPP in a molecular property-dependent manner.

Optical control of G protein-coupled receptor (GPCR) signaling is a highly valuable approach for comprehensive understanding of GPCR-based physiologies and controlling them precisely. However, optogenetics for GPCR signaling is still developing and requires effective and versatile tools with performance evaluation from their molecular properties. Here, we systematically investigated performance of two bistable opsins that activate Gi/Go-type G protein (mosquito Opn3 (MosOpn3) and lamprey parapinopsin (LamPP)) in optical control in vivo using Caenorhabditis elegans. Transgenic worms expressing MosOpn3, which binds 13-cis retinal to form photopigments, in nociceptor neurons showed light-induced avoidance responses in the presence of all-trans retinal, a retinal isomer ubiquitously present in every tissue, like microbial rhodopsins and unlike canonical vertebrate opsins. Remarkably, transgenic worms expressing MosOpn3 were ~7,000 times more sensitive to light than transgenic worms expressing ChR2 in this light-induced behavior, demonstrating the advantage of MosOpn3 as a light switch. LamPP is a UV-sensitive bistable opsin having complete photoregenerative ability by green light. Accordingly, transgenic worms expressing LamPP in cholinergic motor neurons stopped moving upon violet light illumination and restored coordinate movement upon green light illumination, demonstrating color-dependent control of behavior using LamPP. Furthermore, we applied molecular engineering to produce MosOpn3-based tools enabling light-dependent upregulation of cAMP or Ca2+ levels and LamPP-based tool enabling clamping cAMP levels color dependently and context independently, extending their usability. These findings define the capacity of two bistable opsins with similar retinal requirement as ChR2, providing numerous strategies for optical control of various GPCR-based physiologies as well as GPCR signaling itself.

Investigating the mechanism of photoisomerization in jellyfish rhodopsin with the counterion at an atypical position.

The position of the counterion in animal rhodopsins plays a crucial role in maintaining visible light sensitivity and facilitating the photoisomerization of their retinal chromophore. The counterion displacement is thought to be closely related to the evolution of rhodopsins, with different positions found in invertebrates and vertebrates. Interestingly, box jellyfish rhodopsin (JelRh) acquired the counterion in transmembrane 2 independently. This is a unique feature, as in most animal rhodopsins, the counterion is found in a different location. In this study, we used Fourier Transform Infrared spectroscopy to examine the structural changes that occur in the early photointermediate state of JelRh. We aimed to determine whether the photochemistry of JelRh is similar to that of other animal rhodopsins by comparing its spectra to those of vertebrate bovine rhodopsin (BovRh) and invertebrate squid rhodopsin (SquRh). We observed that the N-D stretching band of the retinal Schiff base was similar to that of BovRh, indicating the interaction between the Schiff base and the counterion is similar in both rhodopsins, despite their different counterion positions. Furthermore, we found that the chemical structure of the retinal in JelRh is similar to that in BovRh, including the changes in the hydrogen-out-of-plane band that indicates a retinal distortion. Overall, the protein conformational changes induced by the photoisomerization of JelRh yielded spectra that resemble an intermediate between BovRh and SquRh, suggesting a unique spectral property of JelRh, and making it the only animal rhodopsin with a counterion in TM2 and an ability to activate Gs protein.

Functional characterization of four opsins and two G alpha subtypes co-expressed in the molluscan rhabdomeric photoreceptor.

BACKGROUND: Rhabdomeric photoreceptors of eyes in the terrestrial slug Limax are the typical invertebrate-type but unique in that three visual opsins (Gq-coupled rhodopsin, xenopsin, Opn5A) and one retinochrome, all belonging to different groups, are co-expressed. However, molecular properties including spectral sensitivity and G protein selectivity of any of them are not determined, which prevents us from understanding an advantage of multiplicity of opsin properties in a single rhabdomeric photoreceptor. To gain insight into the functional role of the co-expression of multiple opsin species in a photoreceptor, we investigated the molecular properties of the visual opsins in the present study. RESULTS: First, we found that the fourth member of visual opsins, Opn5B, is also co-expressed in the rhabdomere of the photoreceptor together with previously identified three opsins. The photoreceptors were also demonstrated to express Gq and Go alpha subunits. We then determined the spectral sensitivity of the four visual opsins using biochemical and spectroscopic methods. Gq-coupled rhodopsin and xenopsin exhibit maximum sensitivity at ~ 456 and 475 nm, respectively, and Opn5A and Opn5B exhibit maximum sensitivity at ~ 500 and 470 nm, respectively, with significant UV sensitivity. Notably, in vitro experiments revealed that Go alpha was activated by all four visual opsins, in contrast to the specific activation of Gq alpha by Gq-coupled rhodopsin, suggesting that the eye photoreceptor of Limax uses complex G protein signaling pathways. CONCLUSIONS: The eye photoreceptor in Limax expresses as many as four different visual opsin species belonging to three distinct classes. The combination of opsins with different spectral sensitivities and G protein selectivities may underlie physiological properties of the ocular photoreception, such as a shift in spectral sensitivity between dark- and light-adapted states. This may be allowed by adjustment of the relative contribution of the four opsins without neural networks, enabling a simple strategy for fine-tuning of vision.

Difference FTIR Spectroscopy of Jumping Spider Rhodopsin-1 at 77 K.

Animal visual rhodopsins can be classified into monostable and bistable rhodopsins, which are typically found in vertebrates and invertebrates, respectively. The former example is bovine rhodopsin (BovRh), whose structures and functions have been extensively studied. On the other hand, those of bistable rhodopsins are less known, despite their importance in optogenetics. Here, low-temperature Fourier-transform infrared (FTIR) spectroscopy was applied to jumping spider rhodopsin-1 (SpiRh1) at 77 K, and the obtained light-induced spectral changes were compared with those of squid rhodopsin (SquRh) and BovRh. Although chromophore distortion of the resting state monitored by HOOP vibrations is not distinctive between invertebrate and vertebrate rhodopsins, distortion of the all-trans chromophore after photoisomerization is unique for BovRh, and the distortion was localized at the center of the chromophore in SpiRh1 and SquRh. Highly conserved aspartate (D83 in BovRh) does not change the hydrogen-bonding environment in invertebrate rhodopsins. Thus, present FTIR analysis provides specific structural changes, leading to activation of invertebrate and vertebrate rhodopsins. On the other hand, the analysis of O-D stretching vibrations in D2O revealed unique features of protein-bound water molecules. Numbers of water bands in SpiRh1 and SquRh were less and more than those in BovRh. The X-ray crystal structure of SpiRh1 observed a bridged water molecule between the protonated Schiff base and its counterion (E194), but strongly hydrogen-bonded water molecules were never detected in SpiRh1, as well as SquRh and BovRh. Thus, absence of strongly hydrogen-bonded water molecules is substantial for animal rhodopsins, which is distinctive from microbial rhodopsins.

Crystal structure of jumping spider rhodopsin-1 as a light sensitive GPCR.

Light-sensitive G protein-coupled receptors (GPCRs)-rhodopsins-absorb photons to isomerize their covalently bound retinal, triggering conformational changes that result in downstream signaling cascades. Monostable rhodopsins release retinal upon isomerization as opposed to the retinal in bistable rhodopsins that "reisomerize" upon absorption of a second photon. Understanding the mechanistic differences between these light-sensitive GPCRs has been hindered by the scarcity of recombinant models of the latter. Here, we reveal the high-resolution crystal structure of a recombinant bistable rhodopsin, jumping spider rhodopsin-1, bound to the inverse agonist 9-cis retinal. We observe a water-mediated network around the ligand hinting toward the basis of their bistable nature. In contrast to bovine rhodopsin (monostable), the transmembrane bundle of jumping spider rhodopsin-1 as well that of the bistable squid rhodopsin adopts a more "activation-ready" conformation often observed in other nonphotosensitive class A GPCRs. These similarities suggest the role of jumping spider rhodopsin-1 as a potential model system in the study of the structure-function relationship of both photosensitive and nonphotosensitive class A GPCRs.

Insights into the evolutionary origin of the pineal color discrimination mechanism from the river lamprey.

BACKGROUND: Pineal-related organs in cyclostomes, teleosts, amphibians, and reptiles exhibit color opponency, generating antagonistic neural responses to different wavelengths of light and thereby sensory information about its "color". Our previous studies suggested that in zebrafish and iguana pineal-related organs, a single photoreceptor cell expressing both UV-sensitive parapinopsin and green-sensitive parietopsin generates color opponency in a "one-cell system." However, it remains unknown to what degree these opsins and the single cell-based mechanism in the pineal color opponency are conserved throughout non-mammalian vertebrates. RESULTS: We found that in the lamprey pineal organ, the two opsins are conserved but that, in contrast to the situation in other vertebrate pineal-related organs, they are expressed in separate photoreceptor cells. Intracellular electrophysiological recordings demonstrated that the parietopsin-expressing photoreceptor cells with Go-type G protein evoke a depolarizing response to visible light. Additionally, spectroscopic analyses revealed that parietopsin with 11-cis 3-dehydroretinal has an absorption maximum at ~570 nm, which is in approximate agreement with the wavelength (~560 nm) that produces the maximum rate of neural firing in pineal ganglion cells exposed to visible light. The vesicular glutamate transporter is localized at both the parietopsin- and parapinopsin-expressing photoreceptor terminals, suggesting that both types of photoreceptor cells use glutamate as a transmitter. Retrograde tracing of the pineal ganglion cells revealed that the terminal of the parietopsin-expressing cells is located close enough to form a neural connection with the ganglion cells, which is similar to our previous observation for the parapinopsin-expressing photoreceptor cells and the ganglion cells. In sum, our observations point to a "two-cell system" in which parietopsin and parapinopsin, expressed separately in two different types of photoreceptor cells,  contribute to the generation of color opponency in the pineal ganglion cells. CONCLUSION: Our results indicate that the jawless vertebrate, lamprey, employs a system for color opponency that differes from that described previously in jawed vertebrates. From a physiological viewpoint, we propose an evolutionary insight, the emergence of pineal "one-cell system" from the ancestral "multiple (two)-cell system," showing the opposite evolutionary direction to that of the ocular color opponency.

Optogenetic Potentials of Diverse Animal Opsins: Parapinopsin, Peropsin, LWS Bistable Opsin.

Animal opsin-based pigments are light-activated G-protein-coupled receptors (GPCRs), which drive signal transduction cascades via G-proteins. Thousands of animal opsins have been identified, and molecular phylogenetic and biochemical analyses have revealed the unexpected diversity in selectivity of G-protein activation and photochemical property. Here we discuss the optogenetic potentials of diverse animal opsins, particularly recently well-characterized three non-canonical opsins, parapinopsin, peropsin, and LWS bistable opsin. Unlike canonical opsins such as vertebrate visual opsins that have been conventionally used for optogenetic applications, these opsins are bistable; opsin-based pigments do not release the chromophore retinal after light absorption, and the stable photoproducts revert to their original dark states upon subsequent light absorption. Parapinopsins have a "complete photoregeneration ability," which allows a clear color-dependent regulation of signal transductions. On the other hand, peropsins serve as a "dark-active and light-inactivated" GPCR to regulate signal transductions in the opposite way compared with usual opsins. In addition, an LWS bistable opsin from a butterfly was revealed to be the longest wavelength-sensitive animal opsin with its absorption maximum at ~570 nm. The property-dependent optical regulations of signal transductions were demonstrated in mammalian cultured cells, showing potentials of new optogenetic tools.

Color opponency with a single kind of bistable opsin in the zebrafish pineal organ.

Lower vertebrate pineal organs discriminate UV and visible light. Such color discrimination is typically considered to arise from antagonism between two or more spectrally distinct opsins, as, e.g., human cone-based color vision relies on antagonistic relationships between signals produced by red-, green-, and blue-cone opsins. Photosensitive pineal organs contain a bistable opsin (parapinopsin) that forms a signaling-active photoproduct upon UV exposure that may itself be returned to the signaling-inactive "dark" state by longer-wavelength light. Here we show the spectrally distinct parapinopsin states (with antagonistic impacts on signaling) allow this opsin alone to provide the color sensitivity of this organ. By using calcium imaging, we show that single zebrafish pineal photoreceptors held under a background light show responses of opposite signs to UV and visible light. Both such responses are deficient in zebrafish lacking parapinopsin. Expressing a UV-sensitive cone opsin in place of parapinopsin recovers UV responses but not color opponency. Changes in the spectral composition of white light toward enhanced UV or visible wavelengths respectively increased vs. decreased calcium signal in parapinopsin-sufficient but not parapinopsin-deficient photoreceptors. These data reveal color opponency from a single kind of bistable opsin establishing an equilibrium-like mixture of the two states with different signaling abilities whose fractional concentrations are defined by the spectral composition of incident light. As vertebrate visual color opsins evolved from a bistable opsin, these findings suggest that color opponency involving a single kind of bistable opsin might have been a prototype of vertebrate color opponency.

Convergent evolution of tertiary structure in rhodopsin visual proteins from vertebrates and box jellyfish.

Box jellyfish and vertebrates are separated by >500 million years of evolution yet have structurally analogous lens eyes that employ rhodopsin photopigments for vision. All opsins possess a negatively charged residue-the counterion-to maintain visible-light sensitivity and facilitate photoisomerization of their retinaldehyde chromophore. In vertebrate rhodopsins, the molecular evolution of the counterion position-from a highly conserved distal location in the second extracellular loop (E181) to a proximal location in the third transmembrane helix (E113)-is established as a key driver of higher fidelity photoreception. Here, we use computational biology and heterologous action spectroscopy to determine whether the appearance of the advanced visual apparatus in box jellyfish was also accompanied by changes in the opsin tertiary structure. We found that the counterion in an opsin from the lens eye of the box jellyfish Carybdea rastonii (JellyOp) has also moved to a unique proximal location within the transmembrane bundle-E94 in TM2. Furthermore, we reveal that this Schiff base/counterion system includes an additional positive charge-R186-that has coevolved with E94 to functionally separate E94 and E181 in the chromophore-binding pocket of JellyOp. By engineering this pocket-neutralizing R186 and E94, or swapping E94 with the vertebrate counterion E113-we can recreate versions of the invertebrate and vertebrate counterion systems, respectively, supporting a relatively similar overall architecture in this region of animal opsins. In summary, our data establish the third only counterion site in animal opsins and reveal convergent evolution of tertiary structure in opsins from distantly related species with advanced visual systems.

The Two-Photon Reversible Reaction of the Bistable Jumping Spider Rhodopsin-1.

Bistable opsins are photopigments expressed in both invertebrates and vertebrates. These light-sensitive G-protein-coupled receptors undergo a reversible reaction upon illumination. A first photon initiates the cis to trans isomerization of the retinal chromophore-attached to the protein through a protonated Schiff base-and a series of transition states that eventually results in the formation of the thermally stable and active Meta state. Excitation by a second photon reverts this process to recover the original ground state. On the other hand, monostable opsins (e.g., bovine rhodopsin) lose their chromophore during the decay of the Meta II state (i.e., they bleach). Spectroscopic studies on the molecular details of the two-photon cycle in bistable opsins are limited. Here, we describe the successful expression and purification of recombinant rhodopsin-1 from the jumping spider Hasarius adansoni (JSR1). In its natural configuration, spectroscopic characterization of JSR1 is hampered by the similar absorption spectra in the visible spectrum of the inactive and active states. We solved this issue by separating their absorption spectra by replacing the endogenous 11-cis retinal chromophore with the blue-shifted 9-cis JSiR1. With this system, we used time-resolved ultraviolet-visible spectroscopy after pulsed laser excitation to obtain kinetic details of the rise and decay of the photocycle intermediates. We also used resonance Raman spectroscopy to elucidate structural changes of the retinal chromophore upon illumination. Our data clearly indicate that the protonated Schiff base is stable throughout the entire photoreaction. We additionally show that the accompanying conformational changes in the protein are different from those of monostable rhodopsin, as recorded by light-induced FTIR difference spectroscopy. Thus, we envisage JSR1 as becoming a model system for future studies on the reaction mechanisms of bistable opsins, e.g., by time-resolved x-ray crystallography.

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins.

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.

Homologs of vertebrate Opn3 potentially serve as a light sensor in nonphotoreceptive tissue.

Most opsins selectively bind 11-cis retinal as a chromophore to form a photosensitive pigment, which underlies various physiological functions, such as vision and circadian photoentrainment. Recently, opsin 3 (Opn3), originally called encephalopsin or panopsin, and its homologs were identified in various tissues including brain, eye, and liver in both vertebrates and invertebrates, including human. Because Opn3s are mainly expressed in tissues that are not considered to contain sufficient amounts of 11-cis retinal to form pigments, the photopigment formation ability of Opn3 has been of interest. Here, we report the successful expression of Opn3 homologs, pufferfish teleost multiple tissue opsin (PufTMT) and mosquito Opn3 (MosOpn3) and show that these proteins formed functional photopigments with 11-cis and 9-cis retinals. The PufTMT- and MosOpn3-based pigments have absorption maxima in the blue-to-green region and exhibit a bistable nature. These Opn3 homolog-based pigments activate Gi-type and Go-type G proteins light dependently, indicating that they potentially serve as light-sensitive Gi/Go-coupled receptors. We also demonstrated that mammalian cultured cells transfected with the MosOpn3 or PufTMT became light sensitive without the addition of 11-cis retinal and the photosensitivity retained after the continuous light exposure, showing a reusable pigment formation with retinal endogenously contained in culture medium. Interestingly, we found that the MosOpn3 also acts as a light sensor when constituted with 13-cis retinal, a ubiquitously present retinal isomer. Our findings suggest that homologs of vertebrate Opn3 might function as photoreceptors in various tissues; furthermore, these Opn3s, particularly the mosquito homolog, could provide a promising optogenetic tool for regulating cAMP-related G protein-coupled receptor signalings.

Diversification of non-visual photopigment parapinopsin in spectral sensitivity for diverse pineal functions.

BACKGROUND: Recent genome projects of various animals have uncovered an unexpectedly large number of opsin genes, which encode protein moieties of photoreceptor molecules, in most animals. In visual systems, the biological meanings of this diversification are clear; multiple types of visual opsins with different spectral sensitivities are responsible for color vision. However, the significance of the diversification of non-visual opsins remains uncertain, in spite of the importance of understanding the molecular mechanism and evolution of varied non-visual photoreceptions. RESULTS: Here, we investigated the diversification of the pineal photopigment parapinopsin, which serves as the UV-sensitive photopigment for the pineal wavelength discrimination in the lamprey, linking it with other pineal photoreception. Spectroscopic analyses of the recombinant pigments of the two teleost parapinopsins PP1 and PP2 revealed that PP1 is a UV-sensitive pigment, similar to lamprey parapinopsin, but PP2 is a blue-sensitive pigment, with an absorption maximum at 460-480 nm, showing the diversification of non-visual pigment with respect to spectral sensitivity. We also found that PP1 and PP2 exhibit mutually exclusive expressions in the pineal organs of three teleost species. By using transgenic zebrafish in which these parapinopsin-expressing cells are labeled, we found that PP1-expressing cells basically possess neuronal processes, which is consistent with their involvement in wavelength discrimination. Interestingly, however, PP2-expressing cells rarely possess neuronal processes, raising the possibility that PP2 could be involved in non-neural responses rather than neural responses. Furthermore, we found that PP2-expressing cells contain serotonin and aanat2, the key enzyme involved in melatonin synthesis from serotonin, whereas PP1-expressing cells do not contain either, suggesting that blue-sensitive PP2 is instead involved in light-regulation of melatonin secretion. CONCLUSIONS: In this paper, we have clearly shown the different molecular properties of duplicated non-visual opsins by demonstrating the diversification of parapinopsin with respect to spectral sensitivity. Moreover, we have shown a plausible link between the diversification and its physiological impact by discovering a strong candidate for the underlying pigment in light-regulated melatonin secretion in zebrafish; the diversification could generate a new contribution of parapinopsin to pineal photoreception. Current findings could also provide an opportunity to understand the "color" preference of non-visual photoreception.

Intramolecular interactions that induce helical rearrangement upon rhodopsin activation: light-induced structural changes in metarhodopsin IIa probed by cysteine S-H stretching vibrations.

Rhodopsin undergoes rearrangements of its transmembrane helices after photon absorption to transfer a light signal to the G-protein transducin. To investigate the mechanism by which rhodopsin adopts the transducin-activating conformation, the local environmental changes in the transmembrane region were probed using the cysteine S-H group, whose stretching frequency is well isolated from the other protein vibrational modes. The S-H stretching modes of cysteine residues introduced into Helix III, which contains several key residues for the helical movements, and of native cysteine residues were measured by Fourier transform infrared spectroscopy. This method was applied to metarhodopsin IIa, a precursor of the transducin-activating state in which the intramolecular interactions are likely to produce a state ready for helical movements. No environmental change was observed near the ionic lock between Arg-135 in Helix III and Glu-247 in Helix VI that maintains the inactive conformation. Rather, the cysteine residues that showed environmental changes were located around the chromophore, Ala-164, His-211, and Phe-261. These findings imply that the hydrogen bond between Helix III and Helix V involving Glu-122 and His-211 and the hydrophobic packing between Helix III and Helix VI involving Gly-121, Leu-125, Phe-261, and Trp-265 are altered before the helical rearrangement leading toward the active conformation.

Diversity of animal opsin-based pigments and their optogenetic potential.

Most animal opsin-based pigments are typical G protein-coupled receptors (GPCR) and consist of a protein moiety, opsin, and 11-cis retinal as a chromophore. More than several thousand opsins have been identified from a wide variety of animals, which have multiple opsin genes. Accumulated evidence reveals the molecular property of opsin-based pigments, particularly non-conventional visual pigments including non-visual pigments. Opsin-based pigments are generally a bistable pigment having two stable and photointerconvertible states and therefore are bleach-resistant and reusable, unlike vertebrate visual pigments which become bleached. The opsin family contains Gt-coupled, Gq-coupled, Go-coupled, Gs-coupled, Gi-coupled, and Gi/Go-coupled opsins, indicating the existence of a large diversity of light-driven GPCR-signaling cascades. It is suggested that these molecular properties might contribute to different physiologies. In addition, various opsin based-pigments, especially nonconventional visual pigments having different molecular characteristics would facilitate the design and development of promising optogenetic tools for modulating GPCR-signaling, which is involved in a wide variety of physiological responses. We here introduce molecular and functional properties of various kinds of opsins and discuss their physiological function and also their potentials for optogenetic applications. This article is part of a Special Issue entitled: Retinal proteins - you can teach an old dog new tricks.

Functional properties of opsins and their contribution to light-sensing physiology.

Many animals have developed systems for sensing environmental conditions during evolution. In sensory cells, receptor molecules are responsible for their sensing abilities. In light sensing, most animals capture light information via rhodopsin-like photoreceptive proteins known as opsin-based pigments. A body of evidence from comparisons of amino acid sequences and in vitro experiments demonstrates that opsins have phylogenetically and functionally diversified during evolution and suggests that the phylogenetic diversity in opsins correlates with the variety of molecular properties of opsin-based pigments. In this review, we discuss the various molecular properties of opsin-based pigments and their contribution to light-sensing ability by providing two examples: i) contribution of photoregeneration ability and Chromophore retinal binding property of an Opn3 homolog to non-visual photoreception, and ii) contribution of an absorption characteristic of a visual pigment to depth perception in jumping spiders.

Multiple opsins in a reef-building coral, Acropora millepora.

Opsins, light-sensitive G protein-coupled receptors, have been identified in corals but their properties are largely unknown. Here, we identified six opsin genes (acropsins 1-6) from a coral species Acropora millepora, including three novel opsins (acropsins 4-6), and successfully characterized the properties of four out of the six acropsins. Acropsins 1 and 6 exhibited light-dependent cAMP increases in cultured cells, suggesting that the acropsins could light-dependently activate Gs-type G protein like the box jellyfish opsin from the same opsin group. Spectral sensitivity curves having the maximum sensitivities at ~ 472 nm and ~ 476 nm were estimated for acropsins 1 and 6, respectively, based on the light wavelength-dependent cAMP increases in these opsins-expressing cells (heterologous action spectroscopy). Acropsin 2 belonging to the same group as acropsins 1 and 6 did not induce light-dependent cAMP or Ca2+ changes. We then successfully estimated the acropsin 2 spectral sensitivity curve having its maximum value at ~ 471 nm with its chimera mutant which possessed the third cytoplasmic loop of the Gs-coupled jellyfish opsin. Acropsin 4 categorized as another group light-dependently induced intracellular Ca2+ increases but not cAMP changes. Our results uncovered that the Acropora coral possesses multiple opsins coupling two distinct cascades, cyclic nucleotide and Ca2+signaling light-dependently.