Convergent mechanism underlying the acquisition of vertebrate scotopic vision.

High sensitivity of scotopic vision (vision in dim light conditions) is achieved by the rods' low background noise, which is attributed to a much lower thermal activation rate (kth) of rhodopsin compared with cone pigments. Frogs and nocturnal geckos uniquely possess atypical rods containing noncanonical cone pigments that exhibit low kth, mimicking rhodopsin. Here, we investigated the convergent mechanism underlying the low kth of rhodopsins and noncanonical cone pigments. Our biochemical analysis revealed that the kth of canonical cone pigments depends on their absorption maximum (λmax). However, rhodopsin and noncanonical cone pigments showed a substantially lower kth than predicted from the λmax dependency. Given that the λmax is inversely proportional to the activation energy of the pigments in the Hinshelwood distribution-based model, our findings suggest that rhodopsin and noncanonical cone pigments have convergently acquired low frequency of spontaneous-activation attempts, including thermal fluctuations of the protein moiety, in the molecular evolutionary processes from canonical cone pigments, which contributes to highly sensitive scotopic vision.

Chromophore Structure in an Inactive State of a Novel Photosensor Protein Opn5L1: Resonance Raman Evidence for the Formation of a Deprotonated Adduct at the 11th Carbon Atom.

Opsins are photosensitive G protein-coupled receptor proteins and are classified into visual and nonvisual receptors. Opn5L1 is a nonvisual opsin that binds all-trans retinal as a chromophore. A unique feature of Opn5L1 is that the protein exhibits a photocyclic reaction upon photoexcitation. Determining the chromophore structures of intermediates in the photocycle is essential for understanding the functional mechanism of Opn5L1. A previous study revealed that a long-lived intermediate in the photocycle cannot activate the G protein and forms a covalent bond between the retinal chromophore and a nearby cysteine residue. However, the position of this covalent bond in the chromophore remains undetermined. Here, we report a resonance Raman study on isotopically labeled samples in combination with density functional theory calculations and reveal that the 11th carbon atom of the chromophore of the intermediate forms a covalent linkage to the cysteine residue. Furthermore, vibrational assignments based on the isotopic substitutions and density functional theory calculations suggested that the Schiff base of the intermediate is deprotonated. The chromophore structure determined in the present study well explains the mechanism of the photocyclic reaction, which is crucial to the photobiological function of Opn5L1.

Role of Monomer/Tetramer Equilibrium of Rod Visual Arrestin in the Interaction with Phosphorylated Rhodopsin.

The phototransduction cascade in vertebrate rod visual cells is initiated by the photoactivation of rhodopsin, which enables the activation of the visual G protein transducin. It is terminated by the phosphorylation of rhodopsin, followed by the binding of arrestin. Here we measured the solution X-ray scattering of nanodiscs containing rhodopsin in the presence of rod arrestin to directly observe the formation of the rhodopsin/arrestin complex. Although arrestin self-associates to form a tetramer at physiological concentrations, it was found that arrestin binds to phosphorylated and photoactivated rhodopsin at 1:1 stoichiometry. In contrast, no complex formation was observed for unphosphorylated rhodopsin upon photoactivation, even at physiological arrestin concentrations, suggesting that the constitutive activity of rod arrestin is sufficiently low. UV-visible spectroscopy demonstrated that the rate of the formation of the rhodopsin/arrestin complex well correlates with the concentration of arrestin monomer rather than the tetramer. These findings indicate that arrestin monomer, whose concentration is almost constant due to the equilibrium with the tetramer, binds to phosphorylated rhodopsin. The arrestin tetramer would act as a reservoir of monomer to compensate for the large changes in arrestin concentration in rod cells caused by intense light or adaptation.

Convergent evolutionary counterion displacement of bilaterian opsins in ciliary cells.

Opsins are universal photoreceptive proteins in animals. Vertebrate rhodopsin in ciliary photoreceptor cells photo-converts to a metastable active state to regulate cyclic nucleotide signaling. This active state cannot photo-convert back to the dark state, and thus vertebrate rhodopsin is categorized as a mono-stable opsin. By contrast, mollusk and arthropod rhodopsins in rhabdomeric photoreceptor cells photo-convert to a stable active state to stimulate IP3/calcium signaling. This active state can photo-convert back to the dark state, and thus these rhodopsins are categorized as bistable opsins. Moreover, the negatively charged counterion position crucial for the visible light sensitivity is different between vertebrate rhodopsin (Glu113) and mollusk and arthropod rhodopsins (Glu181). This can be explained by an evolutionary scenario where vertebrate rhodopsin newly acquired Glu113 as a counterion, which is thought to have led to higher signaling efficiency of vertebrate rhodopsin. However, the detailed evolutionary steps which led to the higher efficiency in vertebrate rhodopsin still remain unknown. Here, we analyzed the xenopsin group, which is phylogenetically distinct from vertebrate rhodopsin and functions in protostome ciliary cells. Xenopsins are blue-sensitive bistable opsins that regulate cAMP signaling. We found that a bistable xenopsin of Leptochiton asellus had Glu113 as a counterion but did not exhibit elevated signaling efficiency. Therefore, our results show that vertebrate rhodopsin and L. asellus xenopsin regulate cyclic nucleotide signaling in ciliary cells and displaced the counterion position from Glu181 to Glu113 via convergent evolution, whereas subsequently only vertebrate rhodopsin elevated its signaling efficiency by acquiring the mono-stable property.

Creation of photocyclic vertebrate rhodopsin by single amino acid substitution.

Opsins are universal photoreceptive proteins in animals and can be classified into three types based on their photoreaction properties. Upon light irradiation, vertebrate rhodopsin forms a metastable active state, which cannot revert back to the original dark state via either photoreaction or thermal reaction. By contrast, after photoreception, most opsins form a stable active state which can photoconvert back to the dark state. Moreover, we recently found a novel type of opsins whose activity is regulated by photocycling. However, the molecular mechanism underlying this diversification of opsins remains unknown. In this study, we showed that vertebrate rhodopsin acquired the photocyclic and photoreversible properties upon introduction of a single mutation at position 188. This revealed that the residue at position 188 contributes to the diversification of photoreaction properties of opsins by its regulation of the recovery from the active state to the original dark state.

Amino acid residue at position 188 determines the UV-sensitive bistable property of vertebrate non-visual opsin Opn5.

Opsins are G protein-coupled receptors specialized for photoreception in animals. Opn5 is categorized in an independent opsin group and functions for various non-visual photoreceptions. Among vertebrate Opn5 subgroups (Opn5m, Opn5L1 and Opn5L2), Opn5m and Opn5L2 bind 11-cis retinal to form a UV-sensitive resting state, which is inter-convertible with the all-trans retinal bound active state by photoreception. Thus, these opsins are characterized as bistable opsins. To assess the molecular basis of the UV-sensitive bistable property, we introduced comprehensive mutations at Thr188, which is well conserved among these opsins. The mutations in Opn5m drastically hampered 11-cis retinal incorporation and the bistable photoreaction. Moreover, T188C mutant Opn5m exclusively bound all-trans retinal and thermally self-regenerated to the original form after photoreception, which is similar to the photocyclic property of Opn5L1 bearing Cys188. Therefore, the residue at position 188 underlies the UV-sensitive bistable property of Opn5m and contributes to the diversification of vertebrate Opn5 subgroups.

Retinal ganglion cell topography and spatial resolving power of eye in the native chicken of Bangladesh / Topografía de células ganglionares de la retina y poder de resolución espacial del ojo en el pollo nativo de Bangladesh

SUMMARY: The distribution of retinal ganglion cells (RGCs) was observed in the retinal wholemount of native chicken (Gallus gallus domestricus) of Bangladesh by using light microscopy. We considered five different anatomic regions (central, nasal, temporal, dorsal, and ventral) of Nissl stained wholemount, and the RGCs were counted, plotted, and measured accordingly. The average area of the retina was 431.75 mm2 while the total number of ganglion cells was 2124431 on average. Only the central area of the retina was the peak density (10400 cells/mm2) area, signifying the acute visual area, whilst the maximum spatial resolving power was 11 cycles/degree. The overall concentration of RGCs gradually declined towards the periphery but the size of cells generally decreased towards centrally. The size of ganglion cell was not uniform (12 to 180 µm2), specifically the central retina, just above the optic disc was packed with tiny-sized cells. The number, topographic distribution, and size of RGCs in native chicken signified their domesticated or terrestrial characters, uneven visual acuteness, and possibly only the central retina was the area for fine vision as the function of RGCs.

RESUMEN: En este studio se observó la distribución de las células ganglionares en la retina (CGR) de pollo nativo (Gallus gallus domesticus) de Bangladesh mediante el uso de microscopía óptica. Consideramos cinco regiones anatómicas diferentes (central, nasal, temporal, dorsal y ventral). Las muestras de CGR se tiñeron con Nissl, posteriormente, se midieron y contó el número de células totales. El área promedio de la retina fue de 431,75 mm2, mientras que el promedio del número total de células ganglionares fue de 2124431. El área central de la retina fue el área de densidad máxima (10400 células / mm2), señalando el área visual aguda, mientras que el poder de resolución espacial máximo fue de 11 ciclos / grado. La concentración general de CGR disminuyó gradualmente hacia la periferia, sin embargo, el tamaño de las células disminuyó hacia el centro. El tamaño de las CGR no fue uniforme (12 a 180 mm2), específicamente en la retina central, por encima del disco óptico, aumentaron significativamente las células pequeñas. El número, la distribución topográfica y el tamaño de las CGR en pollos nativos determinaron las características domésticas o terrestres, agudeza visual desigual y, posiblemente, la función de las CGR, en la retina central era el área de visión fina.

Evolutionary adaptation of visual pigments in geckos for their photic environment.

Vertebrates generally have a single type of rod for scotopic vision and multiple types of cones for photopic vision. Noteworthily, nocturnal geckos transmuted ancestral photoreceptor cells into rods containing not rhodopsin but cone pigments, and, subsequently, diurnal geckos retransmuted these rods into cones containing cone pigments. High sensitivity of scotopic vision is underlain by the rod's low background noise, which originated from a much lower spontaneous activation rate of rhodopsin than of cone pigments. Here, we revealed that nocturnal gecko cone pigments decreased their spontaneous activation rates to mimic rhodopsin, whereas diurnal gecko cone pigments recovered high rates similar to those of typical cone pigments. We also identified amino acid residues responsible for the alterations of the spontaneous activation rates. Therefore, we concluded that the switch between diurnality and nocturnality in geckos required not only morphological transmutation of photoreceptors but also adjustment of the spontaneous activation rates of visual pigments.

Rapid Oxidation Following Photoreduction in the Avian Cryptochrome4 Photocycle.

Avian magnetoreception is assumed to occur in the retina. Although its molecular mechanism is unclear, magnetic field-dependent formation and the stability of radical-containing photointermediate(s) are suggested to play key roles in a hypothesis called the radical pair mechanism. Chicken cryptochrome4 (cCRY4) has been identified as a candidate magnetoreceptive molecule due to its expression in the retina and its ability to form stable flavin neutral radicals (FADH●) upon blue light absorption. Herein, we used millisecond flash photolysis to investigate the cCRY4 photocycle, in both the presence and absence of dithiothreitol (DTT); detecting the anion radical form of FAD (FAD●-) under both conditions. Using spectral data obtained during flash photolysis and UV-visible photospectroscopy, we estimated the absolute absorbance spectra of the photointermediates, thus allowing us to decompose each spectrum into its individual components. Notably, in the absence of DTT, approximately 37% and 63% of FAD●- was oxidized to FADOX and protonated to form FADH●, respectively. Singular value decomposition analysis suggested the presence of two FAD●- molecular species, each of which was destined to be oxidized to FADOX or protonated to FADH●. A tyrosine neutral radical was also detected; however, it likely decayed concomitantly with the oxidation of FAD●-. On the basis of these results, we considered the occurrence of bifurcation prior to FAD●- generation, or during FAD●- oxidization, and discussed the potential role played by the tyrosine radical in the radical pair mechanism.

Synthesis of One Double Bond-Inserted Retinal Analogs and Their Binding Experiments with Opsins: Preparation of Novel Red-Shifted Channelrhodopsin Variants.

In optogenetics, red-shifted channelrhodopsins (ChRs) are eagerly sought. We prepared six kinds of new chromophores with one double bond inserted into the polyene side chain of retinal (A1) or 3,4-didehydroretinal (A2), and examined their binding efficiency with opsins (ReaChR and ChrimsonR). All analogs bound with opsins to afford new ChRs. Among them, A2-10ex (an extra double bond is inserted at the C10-C11 position of A2) showed the greatest red-shift in the absorption spectrum of ChrimsonR, with a maximum absorbance at 654 nm (67 nm red-shifted from that of A1-ChrimsonR). Moreover, a long-wavelength spectral boundary of A2-10ex-ChrimsonR was extended to 756 nm, which reached into the far-red region (710-850 nm).

Regulation of Photocycle Kinetics of Photoactive Yellow Protein by Modulating Flexibility of the ß-Turn.

The role of the significant flexibility of the ß-turn in photoactive yellow protein (PYP) due to Gly115 was studied. G115A and G115P mutations were observed to accelerate the photocycle and shift the equilibrium between the late photocycle intermediate (pB) and its precursor (pR) toward pR. Thermodynamic analysis of dark-state recovery from pB demonstrated that the transition state (pB⧧) has a negative change in transition heat capacity, suggesting that an exposed hydrophobic surface of pB is buried in pB⧧. Fourier transform infrared spectroscopy showed that the structural ensemble of pB is populated by the compact structure in G115P. Taken together, the rigid structure induced by mutation of Gly115 facilitates its transition to pB⧧, which adopts a substantially more compact structure as opposed to the ensemble-averaged structure of pB. The photocycle kinetics of PYP may be fine-tuned by modulating the flexibility of the 115 loop to activate an appropriate number of transducer proteins.

Conformational Differences among Metarhodopsin I, Metarhodopsin II, and Opsin Probed by Wide-Angle X-ray Scattering.

Among the photoproducts of vertebrate rhodopsin, only metarhodopsin II (Meta-II) preferentially adopts the active structure in which transmembrane helices are rearranged. Light-induced helical rearrangement of rhodopsin in membrane-embedded form was directly monitored by wide-angle X-ray scattering (WAXS) using nanodiscs. The change in the WAXS curve for the formation of Meta-II was characterized by a peak at 0.2 Å-1 and a valley at 0.6 Å-1, which were not observed in metarhodopsin I and opsin. However, acid-induced active opsin (Opsin*) showed a 0.2 Å-1 peak, but no 0.6 Å-1 valley. Analyses using the model structures based on the crystal structures of dark state and Meta-II suggest that the outward movement of helix VI occurred in Opsin*. However, the displaced helices III and V in Meta-II resulting from the disruption of cytoplasmic ionic lock were restored in Opsin*, which is likely to destabilize the G-protein-activating structure of opsin.

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

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

Shift in Conformational Equilibrium Induces Constitutive Activity of G-Protein-Coupled Receptor, Rhodopsin.

Constitutively active mutants (CAMs) of G-protein-coupled receptors (GPCRs) cause various kinds of diseases. Rhodopsin, a light-absorbing GPCR in animal retinas, has retinal as an endogenous ligand; only very low levels of activation of G-protein can be obtained with the ligand-free opsin. However, the CAM of opsin activates G-protein much more efficiently than the wild type, but the mechanism underlying this remains unclear. The present work revisits the constitutive activity of rhodopsin from the standpoint of conformational dynamics. Single-molecule observation of the M257Y mutant of bovine rhodopsin demonstrated that the switch between active and inactive conformations frequently occurred in M257Y opsin, and frequent generation of the active state results in the population shift toward the active state, which accounts for the constitutive activity of M257Y opsin. Our findings demonstrate that the protein function has a direct connection with the structural dynamics.

Opn5L1 is a retinal receptor that behaves as a reverse and self-regenerating photoreceptor.

Most opsins are G protein-coupled receptors that utilize retinal both as a ligand and as a chromophore. Opsins' main established mechanism is light-triggered activation through retinal 11-cis-to-all-trans photoisomerization. Here we report a vertebrate non-visual opsin that functions as a Gi-coupled retinal receptor that is deactivated by light and can thermally self-regenerate. This opsin, Opn5L1, binds exclusively to all-trans-retinal. More interestingly, the light-induced deactivation through retinal trans-to-cis isomerization is followed by formation of a covalent adduct between retinal and a nearby cysteine, which breaks the retinal-conjugated double bond system, probably at the C11 position, resulting in thermal re-isomerization to all-trans-retinal. Thus, Opn5L1 acts as a reverse photoreceptor. We conclude that, like vertebrate rhodopsin, Opn5L1 is a unidirectional optical switch optimized from an ancestral bidirectional optical switch, such as invertebrate rhodopsin, to increase the S/N ratio of the signal transduction, although the direction of optimization is opposite to that of vertebrate rhodopsin.

Adaptation of cone pigments found in green rods for scotopic vision through a single amino acid mutation.

Most vertebrate retinas contain a single type of rod for scotopic vision and multiple types of cones for photopic and color vision. The retinas of certain amphibian species uniquely contain two types of rods: red rods, which express rhodopsin, and green rods, which express a blue-sensitive cone pigment (M1/SWS2 group). Spontaneous activation of rhodopsin induced by thermal isomerization of the retinal chromophore has been suggested to contribute to the rod's background noise, which limits the visual threshold for scotopic vision. Therefore, rhodopsin must exhibit low thermal isomerization rate compared with cone visual pigments to adapt to scotopic condition. In this study, we determined whether amphibian blue-sensitive cone pigments in green rods exhibit low thermal isomerization rates to act as rhodopsin-like pigments for scotopic vision. Anura blue-sensitive cone pigments exhibit low thermal isomerization rates similar to rhodopsin, whereas Urodela pigments exhibit high rates like other vertebrate cone pigments present in cones. Furthermore, by mutational analysis, we identified a key amino acid residue, Thr47, that is responsible for the low thermal isomerization rates of Anura blue-sensitive cone pigments. These results strongly suggest that, through this mutation, anurans acquired special blue-sensitive cone pigments in their green rods, which could form the molecular basis for scotopic color vision with normal red rods containing green-sensitive rhodopsin.

Evolutionary steps involving counterion displacement in a tunicate opsin.

Ci-opsin1 is a visible light-sensitive opsin present in the larval ocellus of an ascidian, Ciona intestinalis This invertebrate opsin belongs to the vertebrate visual and nonvisual opsin groups in the opsin phylogenetic tree. Ci-opsin1 contains candidate counterions (glutamic acid residues) at positions 113 and 181; the former is a newly acquired position in the vertebrate visual opsin lineage, whereas the latter is an ancestral position widely conserved among invertebrate opsins. Here, we show that Glu113 and Glu181 in Ci-opsin1 act synergistically as counterions, which imparts molecular properties to Ci-opsin1 intermediate between those of vertebrate- and invertebrate-type opsins. Synergy between the counterions in Ci-opsin1 was demonstrated by E113Q and E181Q mutants that exhibit a pH-dependent spectral shift, whereas only the E113Q mutation in vertebrate rhodopsin yields this spectral shift. On absorbing light, Ci-opsin1 forms an equilibrium between two intermediates with protonated and deprotonated Schiff bases, namely the MI-like and MII-like intermediates, respectively. Adding G protein caused the equilibrium to shift toward the MI-like intermediate, indicating that Ci-opsin1 has a protonated Schiff base in its active state, like invertebrate-type opsins. Ci-opsin1's G protein activation efficiency is between the efficiencies of vertebrate- and invertebrate-type opsins. Interestingly, the E113Y and E181S mutations change the molecular properties of Ci-opsin1 into those resembling invertebrate-type or bistable opsins and vertebrate ancient/vertebrate ancient-long or monostable opsins, respectively. These results strongly suggest that acquisition of counterion Glu113 changed the molecular properties of visual opsin in a vertebrate/tunicate common ancestor as a crucial step in the evolution of vertebrate visual opsins.

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

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

Diversity of Active States in TMT Opsins.

Opn3/TMT opsins belong to one of the opsin groups with vertebrate visual and non-visual opsins, and are widely distributed in eyes, brains and other internal organs in various vertebrates and invertebrates. Vertebrate Opn3/TMT opsins are further classified into four groups on the basis of their amino acid identities. However, there is limited information about molecular properties of these groups, due to the difficulty in preparing the recombinant proteins. Here, we successfully expressed recombinant proteins of TMT1 and TMT2 opsins of medaka fish (Oryzias latipes) in cultured cells and characterized their molecular properties. Spectroscopic and biochemical studies demonstrated that TMT1 and TMT2 opsins functioned as blue light-sensitive Gi/Go-coupled receptors, but exhibited spectral properties and photo-convertibility of the active state different from each other. TMT1 opsin forms a visible light-absorbing active state containing all-trans-retinal, which can be photo-converted to 7-cis- and 9-cis-retinal states in addition to the original 11-cis-retinal state. In contrast, the active state of TMT2 opsin is a UV light-absorbing state having all-trans-retinal and does not photo-convert to any other state, including the original 11-cis-retinal state. Thus, TMT opsins are diversified so as to form a different type of active state, which may be responsible for their different functions.

Helical rearrangement of photoactivated rhodopsin in monomeric and dimeric forms probed by high-angle X-ray scattering.

Light-induced helical rearrangement of vertebrate visual rhodopsin was directly monitored by high-angle X-ray scattering (HAXS), ranging from Q (= 4π sin θ/λ) = 0.03 Å(-1) to Q = 1.5 Å(-1). HAXS of nanodiscs containing a single rhodopsin molecule was performed before and after photoactivation of rhodopsin. The intensity difference curve obtained by HAXS agreed with that calculated from the crystal structure of dark state rhodopsin and metarhodopsin II, indicating that the conformational change of monomeric rhodopsin in the membrane is consistent with that occurring in the crystal. On the other hand, the HAXS intensity difference curve of nanodiscs containing two rhodopsin molecules was significantly reduced, similar to that calculated from the crystal structure of the deprotonated intermediate, without a large conformational change. These results suggest that rhodopsin is dimerized in the membrane and that the interaction between rhodopsin molecules modulates structural changes.