M opsin phosphorylation in intact mammalian retinas

The deactivation of visual pigments involved in phototransduction is critical for recovering sensitivity after exposure to light in rods and cones of the vertebrate retina. In rods, phosphorylation of rhodopsin by rhodopsin kinase (GRK1) and the subsequent binding of visual arrestin completely terminates phototransduction. Although signal termination in cones is predicted to occur via a similar mechanism as in rods, there may be differences due to the expression of related but distinct gene products. While rods only express GRK1, cones in some species express only GRK1 or GRK7 and others express both GRKs. In the mouse, cone opsin is phosphorylated by GRK1, but this has not been demonstrated in mammals that express GRK7 in cones. We compared cone opsin phosphorylation in intact retinas from the 13-lined ground squirrel (GS) and pig, cone- and rod-dominant mammals, respectively, which both express GRK7. M opsin phosphorylation increased during continuous exposure to light, then declined between 3 and 6 min. In contrast, rhodopsin phosphorylation continued to increase during this time period. In GS retina homogenates, anti-GS GRK7 antibody blocked M opsin phosphorylation by 73%. In pig retina homogenates, only 20% inhibition was observed, possibly due to phosphorylation by GRK1 released from rods during homogenization. Our results suggest that GRK7 phosphorylates M opsin in both of these mammals. Using an in vitro GTPgammaS binding assay, we also found that the ability of recombinant M opsin to activate G(t) was greatly reduced by phosphorylation. Therefore, phosphorylation may participate directly in the termination of phototransduction in cones by decreasing the activity of M opsin.     

BiP prevents rod opsin aggregation

Mutations in rod opsin-the light-sensitive protein of rod cells-cause retinitis pigmentosa. Many rod opsin mutations lead to protein misfolding, and therefore it is important to understand the role of molecular chaperones in rod opsin biogenesis. We show that BiP (HSPA5) prevents the aggregation of rod opsin. Cleavage of BiP with the subtilase cytotoxin SubAB results in endoplasmic reticulum (ER) retention and ubiquitylation of wild-type (WT) rod opsin (WT-green fluorescent protein [GFP]) at the ER. Fluorescence recovery after photobleaching reveals that WT-GFP is usually mobile in the ER. By contrast, depletion of BiP activity by treatment with SubAB or coexpression of a BiP ATPase mutant, BiP(T37G), decreases WT-GFP mobility to below that of the misfolding P23H mutant of rod opsin (P23H-GFP), which is retained in the ER and can form cytoplasmic ubiquitylated inclusions. SubAB treatment of P23H-GFP-expressing cells decreases the mobility of the mutant protein further and leads to ubiquitylation throughout the ER. Of interest, BiP overexpression increases the mobility of P23H-GFP, suggesting that it can reduce mutant rod opsin aggregation. Therefore inhibition of BiP function results in aggregation of rod opsin in the ER, which suggests that BiP is important for maintaining the solubility of rod opsin in the ER.     

A radioimmunoassay specific for opsin

A radioimmunoassay is developed for bovine opsin using a rabbit antiserum against bovine rod outer segment membranes. The assay is specific for opsin. Rhodopsin, bacteriorhodopsin and hemoglobin do not show cross-reaction. It can be carried out rapidly, has a sensitivity of 0.01 pmol bovine opsin and gives accurate results, even in the presence of a large excess of rhodopsin. Under the conditions described, the assay can be used to measure bovine opsin and rhodopsin in each other's presence by running a sample before and after illumination, with a sensitivity 2000-times higher than with spectrophotometric methods. The opsin content of rather crude preparations such as bovine retina homogenates can be accurately determined. Rabbit and mouse opsin can also be assayed with a reasonable degree of accuracy using the same rabbit antiserum.     

Immunoreactivity of rhodopsin and opsin

An examination by a radioimmunoassay of the relative affinity of opsin and rhodopsin for rabbit antibody raised against bovine rhodopsin revealed that opsin was the preferred antigen. About 10-fold greater amounts of rhodopsin than opsin were required to achieve 50% inhibition of binding of 125I-labeled ligand in the RIA. Opsin was more reactive when examined in the light or dark, compared to rhodopsin incubated in the dark. Mixtures of opsin and rhodopsin (prepared by partial bleaching of rhodopsin or synthetic mixtures) exhibited increased reactivity with increasing mole fraction of opsin. This response was nonlinear, with small increases in opsin producing relatively large increases in reactivity. A partial fractionation of the antibody into two groups showing differential reactivities toward opsin and rhodopsin was achieved by affinity chromatography on opsin-Sepharose. However, with both groups, opsin was still the preferred antigen. Scatchard analysis of 125I-labeled rhodopsin and opsin produced nonlinear plots, indicating the presence of multiple species of antibody. The affinities and binding capacities were similar for both labeled antigens. In competitive binding studies, the antibody showed a strong preference for either labeled ligand (rhodopsin or opsin) as compared to the unlabeled material. These latter observations indicate that altering rhodopsin either by bleaching or iodination produced changes in the relative immunoreactivity of the molecule.     

Retinal release from opsin in molecular dynamics simulations

The crystal structures of opsin in the ligand-free and the G-protein-interacting states showed two inter-helical openings between transmembrane (TM) helices TM1 and TM7 and between TM5 and TM6 near the extracellular side that were thought to serve as the retinal uptake and release gates. However, it is unclear which opening is for 11-cis-retinal uptake or all-trans-retinal release although speculations have been proposed based on the structural features of opsin and retinal. In this work, we simulated the exit process of all-trans-retinal from the ligand-free opsin structure by the classical molecular dynamics (MD) and random acceleration molecular dynamics (RAMD). In the 64 ns classical MD simulation, retinal remained in the receptor but moved significantly toward the TM5-TM6 opening and almost inserted into the opening after 50 ns. Complete exit was observed in 114 out of 160 RAMD trajectories with the TM5-TM6 opening being the predominant egress gate while egress from the TM1-TM7 opening was observed in only a few trajectories when relatively large acceleration was applied and large structural alteration of the protein resulted. These results suggest that photolyzed all-trans-retinal is likely released through the TM5-TM6 opening. Based on the unidirectional mechanism of retinal exchange suggested by experiment, we speculate that the TM1-TM7 opening serves as the 11-cis-retinal uptake gate. The spatial occupancy maps of retinal computed from the 160 RAMD trajectories further indicated that retinal experienced significant interactions with the receptor during the exit process. The implications of these findings for disease mechanisms of rhodopsin mutants are discussed.     

Reduced opsin gene expression in a cave-dwelling fish

Regressive evolution of structures associated with vision in cave-dwelling organisms is the focus of intense research. Most work has focused on differences between extreme visual phenotypes: sighted, surface animals and their completely blind, cave-dwelling counterparts. We suggest that troglodytic systems, comprising multiple populations that vary along a gradient of visual function, may prove critical in understanding the mechanisms underlying initial regression in visual pathways. Gene expression assays of natural and laboratory-reared populations of the Atlantic molly (Poecilia mexicana) revealed reduced opsin expression in cave-dwelling populations compared with surface-dwelling conspecifics. Our results suggest that the reduction in opsin expression in cave-dwelling populations is not phenotypically plastic but reflects a hardwired system not rescued by exposure to light during retinal ontogeny. Changes in opsin gene expression may consequently represent a first evolutionary step in the regression of eyes in cave organisms.     

Shedding new light on opsin evolution

Opsin proteins are essential molecules in mediating the ability of animals to detect and use light for diverse biological functions. Therefore, understanding the evolutionary history of opsins is key to understanding the evolution of light detection and photoreception in animals. As genomic data have appeared and rapidly expanded in quantity, it has become possible to analyse opsins that functionally and histologically are less well characterized, and thus to examine opsin evolution strictly from a genetic perspective. We have incorporated these new data into a large-scale, genome-based analysis of opsin evolution. We use an extensive phylogeny of currently known opsin sequence diversity as a foundation for examining the evolutionary distributions of key functional features within the opsin clade. This new analysis illustrates the lability of opsin protein-expression patterns, site-specific functionality (i.e. counterion position) and G-protein binding interactions. Further, it demonstrates the limitations of current model organisms, and highlights the need for further characterization of many of the opsin sequence groups with unknown function.     

Multiple origins of the green-sensitive opsin genes in fish

Vertebrate opsins are divided into four major groups: RH1 (rhodopsins), RH2 (rhodopsinlike with various absorption sensitivities), SWS (short-wavelength sensitive), and LWS/MWS (long and middle-wavelength sensitive) groups. The green opsin genes (g101 _Af and g101 _Af ) in a Mexican characin Astyanax fasciatus belong to the LWS/MWS group, whereas those in goldfish belong to the RH2 group (Yokoyama 1994, Mol Biol Evol 11:32–39). A newly isolated opsin gene (rh11 _Af ) from A. fasciatus contains five exons and four introns, spanning 4.2 kilobases from start to stop codons. This gene is most closely related to the two green opsin genes of goldfish and belongs to the RH2 group. In the LWS/MWS group, gene duplication of the ancestral red and green opsin genes predates the speciation between A. fasciatus and goldfish, suggesting that goldfish also has an additional gene which is orthologous to g101 _Af and g103 _Af .Correspondence to: S. Yokoyama     

Acid-base properties of rhodopsin and opsin

Purified preparations of cattle rhodopsin have been titrated to various pH, irradiated, and the pH changes followed thereafter until completed. In this way we have obtained the titration curves of rhodopsin, of the immediate product of irradiation, measured within 30 seconds; and of the final product of irradiation (opsin). The rhodopsin preparations display about 54 titratable groups per mole of pigment: about 34 base-binding and 20 acid-binding groups. In default of an absolute purification, one cannot be sure that all of these go with rhodopsin itself. Exposure to light induces an immediate rise of pH between pH 2 and 8, maximal at about pH 5. This—followed by its slow partial or complete reversal—is the only change of pH in the physiological range (6–7). It involves the exposure of 1 new acid-binding group per mole of rhodopsin with pK about 6.6, close therefore to that of the imidazole group of histidine. At acid and alkaline pH this immediate change is followed by slower changes, occupying up to 40 minutes at 20°C. These changes are always in the direction of neutrality. They involve increases of 5 to 6 moles acid bound at acid pH, and 7 moles base bound at alkaline pH. They are associated with the irreversible denaturation of opsin in acid and alkaline solution, as evidenced by loss of its capacity to regenerate rhodopsin. Such frank denaturation procedures as the exposure of rhodopsin to alkali or heat in the dark result in comparable acid-base changes.     

The Drosophila ninaE gene encodes an opsin

The Drosophila ninaE gene was isolated by a multistep protocol on the basis of its homology to bovine opsin cDNA. The gene encodes the major visual pigment protein (opsin) contained in Drosophila photoreceptor cells R1-R6. The coding sequence is interrupted by four short introns. The positions of three introns are conserved with respect to positions in mammalian opsin genes. The nucleotide sequence has intermittent regions of homology to bovine opsin coding sequences. The deduced amino acid sequence reveals significant homology to vertebrate opsins; there is strong conservation of the retinal binding site and two other regions. The predicted protein secondary structure strikingly resembles that of mammalian opsins. We conclude the Drosophila and vertebrate opsin genes are derived from a common ancestor.     

An opsin mutant with increased thermal stability

This report describes the biochemical characterization of a double mutant of rhodopsin (N2C,D282C) in which Cys residues engineered into the protein at positions 2 (in the amino-terminal extracellular domain) and 282 (in the extracellular loop between transmembrane helices 6 and 7) are shown to form a disulfide bond and increase the thermal stability of the unliganded or opsin form of the protein. Wild-type opsin does not survive detergent solubilization and purification at pH 7.5 and 25 degrees C. In contrast, the N2C,D282C mutant opsin survives the purification protocol and loses less than 50% activity after incubation for 20 days under the same conditions. Less than 5% is lost after 20 days at 4 degrees C. While the disulfide bond clearly has a dramatic effect on protein stability, it has a minor impact on the activity of the pigment. The MII lifetime of the mutant (6.6 min) is similar to that of the wild type (7.9 min), and the specific activity of the light-activated mutant for activation of transducin is within 20% of the wild-type activity. Therefore, it seems likely that the disulfide bond does not perturb greatly the structure of the protein. For these reasons, we anticipate that the mutant may be of use in detailed kinetic and mechanistic investigations of the ligand binding reaction and for crystallization trials involving recombinant rhodopsin, especially the unliganded opsin form of the protein.     

Testing sensory drive speciation in cichlid fish: Linking light conditions to opsin expression,opsin genotype and female mate preference

Ecological speciation is facilitated when divergent adaptation has direct effects on selective mating. Divergent sensory adaptation could generate such direct effects, by mediating both ecological performance and mate selection. In aquatic environments, light attenuation creates distinct photic environments, generating divergent selection on visual systems. Consequently, divergent sensory drive has been implicated in the diversification of several fish species. Here, we experimentally test whether divergent visual adaptation explains the divergence of mate preferences in Haplochromine cichlids. Blue and red Pundamilia co‐occur across south‐eastern Lake Victoria. They inhabit different photic conditions and have distinct visual system properties. Previously, we documented that rearing fish under different light conditions influences female preference for blue versus red males. Here, we examine to what extent variation in female mate preference can be explained by variation in visual system properties, testing the causal link between visual perception and preference. We find that our experimental light manipulations influence opsin expression, suggesting a potential role for phenotypic plasticity in optimizing visual performance. However, variation in opsin expression does not explain species differences in female preference. Instead, female preference covaries with allelic variation in the long‐wavelength‐sensitive opsin gene (LWS), when assessed under broad‐spectrum light. Taken together, our study presents evidence for environmental plasticity in opsin expression and confirms the important role of colour perception in shaping female mate preferences in Pundamilia. However, it does not constitute unequivocal evidence for the direct effects of visual adaptation on assortative mating.     

Paralogous origin of the rhodopsinlike opsin genes in lizards

Rhodopsinlike opsins constitute a distinct phylogenetic group (Yokoyama 1994, Mol. Biol. Evol. 11:32–39). This RH2 group includes the green-sensitive opsins in chicken and goldfish and the blue-sensitive opsin in a nocturnal lizard gecko. In the present study, we isolated and sequenced the genomic DNA clones for the RH2 opsin gene, rh2 _Ac , of the diurnal lizard Anolis carolinensis. This single-copy gene spans 18.3 kb from start to stop codons, making it the longest opsin gene known in vertebrates. Phylogenetic analysis strongly suggests that rh2 _Ac is more closely related to the chicken green opsin gene than to the gecko blue opsin gene. This gene tree differs from the organismal tree, where the two lizard species should be most closely related, implying that rh2 _Ac and the gecko blue-sensitive opsin genes have been derived from duplicate ancestral genes.Correspondence to: S. Yukiyama     

Time course of opsin expression in developing rod photoreceptors

We have investigated the time course of rod photoreceptor determination in the goldfish retina. Rod precursor cells located in the outer nuclear layer of the mature retina continuously generate rod photoreceptors. In this study, we asked when rod precursor cells begin to express opsin, which would signal their commitment to the rod pathway of differentiation. There are three possibilities: a rod precursor could express opsin while still mitotic, at or shortly after the terminal mitosis but before differentiation, or during differentiation. We used immunocytochemistry with antibodies against bromodeoxyuridine, BrdU (a thymidine analogue) and against opsin to determine when during the mitotic history of a cell the expression of opsin first occurred, taking a double labelled cell to be evidence of commitment to the rod cell fate. We found that the first double labelled cells appeared at 4 days after BrdU injection. The number of double labelled cells increased to peak at 10 days, and then fell. These results support the hypothesis that dividing rod precursor cells are probably multipotent stem cells not committed to the rod cell fate.     

A photoswitchable GPCR-based opsin for presynaptic inhibition

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The microbial opsin family of optogenetic tools

The capture and utilization of light is an exquisitely evolved process. The single-component microbial opsins, although more limited than multicomponent cascades in processing, display unparalleled compactness and speed. Recent advances in understanding microbial opsins have been driven by molecular engineering for optogenetics and by comparative genomics. Here we provide a Primer on these light-activated ion channels and pumps, describe a group of opsins bridging prior categories, and explore the convergence of molecular engineering and genomic discovery for the utilization and understanding of these remarkable molecular machines.     

Constitutive activity of a UV cone opsin

Vertebrate visual pigment proteins contain a conserved carboxylic acid residue in the third transmembrane helix. In rhodopsin, Glu113 serves as a counterion to the positively charged protonated Schiff base formed by 11-cis retinal attached to Lys296. Activation involves breaking of this ion pair. In UV cone pigments, the retinyl Schiff base is unprotonated, and hence such a salt bridge is not present; yet the pigment is inactive in the dark. Mutation of Glu108, which corresponds to rhodopsin's Glu113, to Gln yields a pigment that remains inactive in the dark. The apoproteins of both the wild-type and mutant, however, are constitutively active with the mutant being of significantly higher activity. Thus, one important role for preserving the negatively charged glutamate in the third helix of UV pigments is to maintain a less active opsin in a manner similar to rhodopsin. Ligand binding itself in the absence of a salt bridge is sufficient for deactivation.