Cone visual pigments

Cone visual pigments are visual opsins that are present in vertebrate cone photoreceptor cells and act as photoreceptor molecules responsible for photopic vision. Like the rod visual pigment rhodopsin, which is responsible for scotopic vision, cone visual pigments contain the chromophore 11-cis-retinal, which undergoes cis–trans isomerization resulting in the induction of conformational changes of the protein moiety to form a G protein-activating state. There are multiple types of cone visual pigments with different absorption maxima, which are the molecular basis of color discrimination in animals. Cone visual pigments form a phylogenetic sister group with non-visual opsin groups such as pinopsin, VA opsin, parapinopsin and parietopsin groups. Cone visual pigments diverged into four groups with different absorption maxima, and the rhodopsin group diverged from one of the four groups of cone visual pigments. The photochemical behavior of cone visual pigments is similar to that of pinopsin but considerably different from those of other non-visual opsins. G protein activation efficiency of cone visual pigments is also comparable to that of pinopsin but higher than that of the other non-visual opsins. Recent measurements with sufficient time-resolution demonstrated that G protein activation efficiency of cone visual pigments is lower than that of rhodopsin, which is one of the molecular bases for the lower amplification of cones compared to rods. In this review, the uniqueness of cone visual pigments is shown by comparison of their molecular properties with those of non-visual opsins and rhodopsin. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.     

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.     

Developmental Changes in the Visual Pigments of the Yellowfin Tuna, Thunnus Albacares

Previous studies have suggested that adult tunas have only two visual pigments in their retinas - a rod pigment with a wavelength at maximum absorbance ( λmax ) around 485 nm and one with similar λmax in both twin and single cones inferred from extraction data. Using microspectrophotometry we confirm the presence of a λmax 483 nm visual pigment in the rods of adult yellowfin tuna and a λmax 485 nm pigment in both members of the twin cones. However, all single cones contain a previously undetected violet visual pigment with λmax 426 nm making the adult yellowfin tuna a photopic dichromat. The situation for larvae and early juveniles is different from that of the adults. The all single-cone retina of preflexion larvae shows a wide distribution in individual cone absorbances suggesting not only mixtures of the two adult cone pigments, but the presence of at least a third visual pigment with λmax greater than 560 nm. With growth, the variation in cone absorbances decreases with convergence to the adult condition coincident with cone twinning. The significance of λmax variability, multiple visual pigment expression and age-related differences are discussed in terms of the visual ecology of larval, juvenile and adult tunas.     

Photoreceptors and visual pigments in three species of newts

Photoreceptor composition and retinal visual pigments in three newt (Caudata, Salamandridae, Pleurodelinae) species (Pleurodeles waltl, Lissotriton (Triturus) vulgaris, and Cynops orientalis) were studied by light microscopy and single-cell microspectrophotometry. Retinas of all three species contain “red” (rhodopsin/porphyropsin) rods, large and small single cones, and double cones. Large single cones and both components of double cones contain red-sensitive (presumably LWS) visual pigment whose absorption spectrum peaks between 593 and 611 nm. Small single cones are either blue- (SWS2, maximum absorption between 470 and 489 nm) or UV-sensitive (SWS1, maximum absorption between 340 and 359 nm). Chromophore composition of visual pigments (A₁ vs. A₂) was assessed both from template fitting of absorption spectra and by the method of selective bleaching. All pigments contained a mixture of A₁ (11-cis retinal) and A₂ (11-cis-3,4-dehydroretinal) chromophore in the proportion depending on the species and cell type. In all cases, A₂ was dominant. However, in C. orientalis rods the fraction of A₁ could reach 45%, while in P. waltl and L. vulgaris cones it did not exceed 5%. Remarkably, the absorption of the newt blue-sensitive visual pigment was shifted by up to 45 nm toward the longer wavelength, as compared with all other amphibian SWS2-pigments. We found no “green” rods typical of retinas of Anura and some Caudata (ambystomas) in the three newt species studied.     

Activity of long-wavelength cones under scotopic conditions in the cyprinid fish Danio aequipinnatus

In carp (Cyprinus) and goldfish (Carassius), long-wavelength cones are reported to be active under scotopic conditions. Using the electroretinogram (ERG), we tested another cyprinid fish, Danio aequipinnatus, which contains A₁-based visual pigments and for which we had previously measured the spectral sensitivities of individual cones. Dark adaptation curves show a rod/cone break at about 45 min. When thoroughly dark-adapted, the spectral sensitivity function is broader than can be accounted for by self-screening of rhodopsin, but it can be modeled by an additive combination of rods and the 560-nm cones. Dim, red background light causes adaptation of rods and a broadening of the spectral sensitivity function, which can be simulated by increasing the proportion of cones in the model. Brighter red backgrounds adapt the 560-nm cones. Because of the effect of red adapting lights, the ERG evidence for the participation of long-wavelength cones close to visual threshold appears to be different in Danio than in the goldfish Carassius. Accepted: 14 June 1997     

The visual system of the alligator

The eye tissues and liver of the alligator contain vitamin A₁ alone. The retina contains rhodopsin, typical in absorption spectrum (λ_max 500 mµ); but synthesized in solution from neo-b retinene and opsin much more rapidly than are the frog, mammalian, or chicken rhodopsins previously examined. In this regard alligator rhodopsin resembles the rhodopsins and porphyropsins of fishes, all of which so far investigated are synthesized rapidly in solution. The rates of synthesis in vitro of frog and alligator rhodopsins are matched closely by the rates of rod dark adaptation in living frogs and alligators, measured electrophysiologically at the same temperature. Alligator rods dark-adapt, and alligator rhodopsin is synthesized in solution, at rates characteristically associated with cones and cone pigments in frogs, mammals, and birds.     

Amino acid residues responsible for the meta-III decay rates in rod and cone visual pigments

Vertebrate retinas have two types of photoreceptor cells, rods and cones, which contain visual pigments with different molecular properties. These pigments diverged from a common ancestor, and their difference in molecular properties originates from the difference in their amino acid residues. We previously reported that the difference in decay times of G protein-activating meta-II intermediates between the chicken rhodopsin and green-sensitive cone (chicken green) pigments is about 50 times. This difference only originates from the differences of two residues at positions 122 and 189 (Kuwayama, S., Imai, H., Hirano, T., Terakita, A., and Shichida, Y. (2002) Biochemistry 41, 15245-15252). Here we show that the meta-III intermediates exhibit about 700 times difference in decay times between the two pigments, and the faster decay in chicken green can be converted to the slower decay in rhodopsin by replacing the residues in chicken green with the corresponding rhodopsin residues. However, the inverse directional conversion did not occur when the two residues in rhodopsin were replaced by those of chicken green. Analysis using chimerical mutants derived from these pigments has demonstrated that amino acid residues responsible for the slow rhodopsin meta-III decay are situated at several positions throughout the C-terminal half of rhodopsin. Considering that rhodopsins evolved from cone pigments, it has been suggested that the molecular properties of rhodopsin have been optimized by mutations at several positions, and the chicken green mutants at two positions could be rhodopsin-like pigments transiently produced in the course of molecular evolution.     

Developmental Changes in the Visual Pigments of the Yellowfin Tuna,Thunnus Albacares

Previous studies have suggested that adult tunas have only two visual pigments in their retinas - a rod pigment with a wavelength at maximum absorbance (u max) around 485 nm and one with similar u max in both twin and single cones inferred from extraction data. Using microspectrophotometry we confirm the presence of a u max 483 nm visual pigment in the rods of adult yellowfin tuna and a u max 485 nm pigment in both members of the twin cones. However, all single cones contain a previously undetected violet visual pigment with u max 426 nm making the adult yellowfin tuna a photopic dichromat. The situation for larvae and early juveniles is different from that of the adults. The all single-cone retina of preflexion larvae shows a wide distribution in individual cone absorbances suggesting not only mixtures of the two adult cone pigments, but the presence of at least a third visual pigment with u max greater than 560 nm. With growth, the variation in cone absorbances decreases with convergence to the adult condition coincident with cone twinning. The significance of u max variability, multiple visual pigment expression and age-related differences are discussed in terms of the visual ecology of larval, juvenile and adult tunas.     

Visual pigments, oil droplets, ocular media and cone photoreceptor distribution in two species of passerine bird: the blue tit (Parus caeruleus L.) and the blackbird (Turdus merula L.)

The spectral absorption characteristics of the retinal photoreceptors of the blue tit (Parus caeruleus) and blackbird (Turdus merula) were investigated using microspectrophotometry. The retinae of both species contained rods, double cones and four spectrally distinct types of single cone. Whilst the visual pigments and cone oil droplets in the other receptor types are very similar in both species, the wavelength of maximum sensitivity (λ_max) of long-wavelength-sensitive single and double cone visual pigment occurs at a shorter wavelength (557 nm) in the blackbird than in the blue tit (563 nm). Oil droplets located in the long-wavelength-sensitivesingle cones of both species cut off wavelengths below 570–573 nm, theoretically shifting cone peak spectral sensitivity some 40 nm towards the long-wavelength end of the spectrum. This raises the possibility that the precise λ_max of the long-wavelength-sensitive visual pigment is optimised for the visual function of the double cones. The distribution of cone photoreceptors across the retina, determined using conventional light and fluorescence microscopy, also varies between the two species and may reflect differences in their visual ecology. Accepted: 8 January 2000     

Scanning electron microscopy and microspectrophotometry of the photoreceptors of ictalurid catfishes

The retinal photoreceptors from larval channel catfish (Ictalurus punctatus) were studied using single cell, in situ microspectrophotometry. Rods appear at 5 days after hatch; cones are present from day one. The rods contain a visual pigment which absorbs light maximally at 540 nm. The cones contain either a green sensitive visual pigment with peak absorbance at 535 nm or a red sensitive visual pigment with peak absorbance at 608 nm. All pigments are based on vitamin A₂. Visual pigment complement does not change with age, as photoreceptors from adultI. punctatus, I. catus andI. melas contain visual pigments virtually identical to those of the larvalI. punctatus. Regardless of age, no visual pigment with peak absorbance in the short wavelength region of the spectrum was ever observed. Scanning electron microscopy of adultI. punctatus retinas showed large rods with long, cylindrical outer segments and smaller cones with short, tapered outer segments. The myoids of both rods and cones are extensable. The rods, embedded in a granular tapetal material, comprise from 50 to 60% of the photoreceptors. Only single cones are present. The data are consistent with the idea that the ictalurid catfishes spend their entire lives in an environment deficient in blue light.     

The photoreceptors and visual pigments of two species of Acipenseriformes, the shovelnose sturgeon (Scaphirhynchus platorynchus ) and the paddlefish (Polyodon spathula )

Scanning electron microscopy, microspectrophotometry, and spectrophotometry of digitonin extracts were employed to characterize the photoreceptors and visual pigments of two freshwater Acipenseriformes. The retinas of the shovelnose sturgeon, Scaphirhynchus platorynchus (Acipenseridae), and the paddlefish, Polyodon spathula (Polyodontidae) are dominated by large rods with long, broad outer segments. A second rod, rare and much narrower than the dominant rod, is present in Scaphirhynchus but not seen in Polyodon. The absorbance maximum of the visual pigment in the rods of Polyodon is near 540 nm; that of Scaphirhynchus near 534 nm. The retinas of both species contain substantial numbers of large, single cones, about 33% of the photoreceptors in Scaphirhynchus; 37% in Polyodon. Scaphirhynchus cone pigments have absorbance maxima near 610 nm, 521 nm and 470 nm, respectively. Polyodon cone pigments absorb maximally near 607 nm and 535 nm, respectively. All visual pigments are based on vitamin A₂. The data are compared to those from other Acipenseriformes and are discussed in terms of lifestyle and behavior. Accepted: 7 October 1998     

Functional Comparison of Rod and Cone Gαt on the Regulation of Light Sensitivity

The signaling cascades mediated by G protein-coupled receptors (GPCRs) exhibit a wide spectrum of spatial and temporal response properties to fulfill diverse physiological demands. However, the mechanisms that shape the signaling response of the GPCR are not well understood. In this study, we replaced cone transducin α (cTα) for rod transducin α (rTα) in rod photoreceptors of transgenic mice, which also express S opsin, to evaluate the role of Gα subtype on signal amplification from different GPCRs in the same cell; such analysis may explain functional differences between retinal rod and cone photoreceptors. We showed that ectopically expressed cTα 1) forms a heterotrimeric complex with rod Gβ₁γ₁, 2) substitutes equally for rTα in generating photoresponses initiated by either rhodopsin or S-cone opsin, and 3) exhibited similar light-activated translocation as endogenous rTα in rods and endogenous cTα in cones. Thus, rTα and cTα appear functionally interchangeable. Interestingly, light sensitivity appeared to correlate with the concentration of cTα when expression is reduced below 35% of normal. However, quantification of endogenous cTα concentration in cones showed a higher level to rTα in rods. Thus, reduced sensitivity in cones cannot be explained by reduced coupling efficiency between the GPCR and G protein or a lower concentration of G protein in cones versus rods.     

Physiological properties of rod photoreceptor cells in green-sensitive cone pigment knock-in mice

Rod and cone photoreceptor cells that are responsible for scotopic and photopic vision, respectively, exhibit photoresponses different from each other and contain similar phototransduction proteins with distinctive molecular properties. To investigate the contribution of the different molecular properties of visual pigments to the responses of the photoreceptor cells, we have generated knock-in mice in which rod visual pigment (rhodopsin) was replaced with mouse green-sensitive cone visual pigment (mouse green). The mouse green was successfully transported to the rod outer segments, though the expression of mouse green in homozygous retina was approximately 11% of rhodopsin in wild-type retina. Single-cell recordings of wild-type and homozygous rods suggested that the flash sensitivity and the single-photon responses from mouse green were three to fourfold lower than those from rhodopsin after correction for the differences in cell volume and levels of several signal transduction proteins. Subsequent measurements using heterozygous rods expressing both mouse green and rhodopsin E122Q mutant, where these pigments in the same rod cells can be selectively irradiated due to their distinctive absorption maxima, clearly showed that the photoresponse of mouse green was threefold lower than that of rhodopsin. Noise analysis indicated that the rate of thermal activations of mouse green was 1.7 x 10(-7) s(-1), about 860-fold higher than that of rhodopsin. The increase in thermal activation of mouse green relative to that of rhodopsin results in only 4% reduction of rod photosensitivity for bright lights, but would instead be expected to severely affect the visual threshold under dim-light conditions. Therefore, the abilities of rhodopsin to generate a large single photon response and to retain high thermal stability in darkness are factors that have been necessary for the evolution of scotopic vision.     

Low Activation and Fast Inactivation of Transducin in Carp Cones

Cone photoreceptors show lower light sensitivity and briefer light responses than rod photoreceptors. The light detection signal in these cells is amplified through a phototransduction cascade. The first step of amplification in the cascade is the activation of a GTP-binding protein, transducin (Tr), by light-activated visual pigment (R*). We quantified transducin activation by measuring the binding of GTPγS in purified carp rod and cone membrane preparations with the use of a rapid quench apparatus and found that transducin activation by an R* molecule is ∼5 times less efficient in cones than in rods. Transducin activation terminated in less than 1 s in cones, more quickly than in rods. The rate of GTP hydrolysis in Tr*, and thus the rate of Tr* inactivation, was ∼25 times higher in cones than in rods. This faster inactivation of Tr* ensures briefer light responses in cones. The expression level of RGS9 was found to be ∼20 times higher in cones than in rods, which explains higher GTP hydrolytic activity and, thus, faster Tr* inactivation in cones than in rods. Although carp rods and cones express rod- or cone-versions of visual pigment and transducin, these molecules themselves do not seem to induce the differences significantly in the transducin activation and Tr* inactivation in rods and cones. Instead, the differences seem to be brought about in a rod or cone cell-type specific manner.     

Visual Pigments of Goldfish Cones : Spectral Properties and Dichroism

Freshly isolated retinal photoreceptors of goldfish were studied microspectrophotometrically. Absolute absorptance spectra obtained from dark-adapted cone outer segments reaffirm the existence of three spectrally distinct cone types with absorption maxima at 455 ± 3,530 ± 3, and 625 ± 5 nm. These types were found often recognizable by gross cellular morphology. Side-illuminated cone outer segments were dichroic. The measured dichroic ratio for the main absorption band of each type was 2–3:1. Rapidly bleached cells revealed spectral and dichroic transitions in regions near 400–410, 435–455, and 350–360 nm. These photoproducts decay about fivefold as fast as the intermediates in frog rods. The spectral maxima of photoproducts, combined with other evidence, indicate that retinene₂ is the chromophore of all three cone pigments. The average specific optical density for goldfish cone outer segments was found to be 0.0124 ± 0.0015/µm. The spectra of the blue-, and green-absorbing cones appeared to match porphyropsin standards with half-band width Δν = 4,832 ± 100 cm^–1. The red-absorbing spectrum was found narrower, having Δν = 3,625 ± 100 cm^–1. The results are consistent with the notion that visual pigment concentration within the outer segments is about the same for frog rods and goldfish cones, but that the blue-, and green-absorbing pigments possess molar extinctions of 30,000 liter/mol cm. The red-absorbing pigment was found to have extinction of 40,000 liter/mol cm, assuming invariance of oscillator strength among the three cone spectra.     

Identification of small-molecule allosteric modulators that act as enhancers/disrupters of rhodopsin oligomerization

The elongated cilia of the outer segment of rod and cone photoreceptor cells can contain concentrations of visual pigments of up to 5 mM. The rod visual pigments, G protein–coupled receptors called rhodopsins, have a propensity to self-aggregate, a property conserved among many G protein–coupled receptors. However, the effect of rhodopsin oligomerization on G protein signaling in native cells is less clear. Here, we address this gap in knowledge by studying rod phototransduction. As the rod outer segment is known to adjust its size proportionally to overexpression or reduction of rhodopsin expression, genetic perturbation of rhodopsin cannot be used to resolve this question. Therefore, we turned to high-throughput screening of a diverse library of 50,000 small molecules and used a novel assay for the detection of rhodopsin dimerization. This screen identified nine small molecules that either disrupted or enhanced rhodopsin dimer contacts in vitro. In a subsequent cell-free binding study, we found that all nine compounds decreased intrinsic fluorescence without affecting the overall UV-visible spectrum of rhodopsin, supporting their actions as allosteric modulators. Furthermore, ex vivo electrophysiological recordings revealed that a disruptive, hit compound #7 significantly slowed down the light response kinetics of intact rods, whereas compound #1, an enhancing hit candidate, did not substantially affect the photoresponse kinetics but did cause a significant reduction in light sensitivity. This study provides a monitoring tool for future investigation of the rhodopsin signaling cascade and reports the discovery of new allosteric modulators of rhodopsin dimerization that can also alter rod photoreceptor physiology.     

Characterization of photoreceptor cell types in the little brown bat Myotis lucifugus (Vespertilionidae)

We report the expression of three visual opsins in the retina of the little brown bat (Myotis lucifugus, Vespertilionidae). Gene sequences for a rod-specific opsin and two cone-specific opsins were cloned from cDNA derived from bat eyes. Comparative sequence analyses indicate that the two cone opsins correspond to an ultraviolet short-wavelength opsin (SWS1) and a long-wavelength opsin (LWS). Immunocytochemistry using antisera to visual opsins revealed that the little brown bat retina contains two types of cone photoreceptors within a rod-dominated background. However, unlike other mammalian photoreceptors, M. lucifugus cones and rods are morphologically indistinguishable by light microscopy. Both photoreceptor types have a thin, elongated outer segment. Using microspectrophotometry we classified the absorption spectrum for the ubiquitous rods. Similar to other mammals, bat rhodopsin has an absorption peak near 500 nm. Although we were unable to confirm a spectral range, cellular and molecular analyses indicate that M. lucifugus expresses two types of cone visual pigments located within the photoreceptor layer. This study provides important insights into the visual capacity of a nocturnal microchiropteran species.     

An Alternative Pathway Mediates the Mouse and Human Cone Visual Cycle

One of the fundamental mysteries of the human visual system is the continuous function of cone photoreceptors in bright daylight. As visual pigment is destroyed, or bleached, by light [1], cones require its rapid regeneration, which in turn involves rapid recycling of the pigment's chromophore. The canonical visual cycle for rod and cone pigments involves recycling of their chromophore from all-trans retinol to 11-cis retinal in the pigment epithelium, adjacent to photoreceptors [2]. However, shortcomings of this pathway indicate the function of a second, cone-specific, mechanism for chromophore recycling [3]. Indeed, biochemical [3], [4], [5], [6] and [7] and physiological [8] studies on lower species have described a cone-specific visual cycle in addition to the long-known pigment epithelium pathway. Two important questions remain, however: what is the role of this pathway in the function of mammalian cones, and is it present in higher mammals, including humans? Here, we show that mouse, primate, and human neural retinas promote pigment regeneration and dark adaptation selectively in cones, but not in rods. This pathway supports rapid dark adaptation of mammalian cones and extends their dynamic range in background light independently of the pigment epithelium. This pigment-regeneration mechanism is essential for our daytime vision and appears to be evolutionarily conserved.     

Visual cycle and dark adaptation: a new approach in research

Visual cycle is the series of reactions that support regeneration of the visual pigmen after its photolysis in retinal rods and cones. Inherited or acquired deficiencies of the visual cycle impair dark adaptation and lead to a series of visual disorders. The paper describes a new approach to study of the visual cycle that uses fast dichroic microspectrophotometer. The method allows studying interconversion of bleaching products in single intact photoreceptors in condition approaching the situation in vivo. Using this approach, we established a complete scheme of transitions between metarhodopsins, retinal and retinol in amphibian rods. It appeared that the decay of metarhodopsins controls both the time course of rod dark adaptation following small bleaches and the production of retinol that is the substrate for rhodopsin regeneration. We also obtained novel data on kinetics of the decay of cone metapigments that was found to be by an order of magnitude faster than in rods. Possible application of the method for further study of the visual cycle in normal and pathological conditions is discussed.     

Regulation of Mammalian Cone Phototransduction by Recoverin and Rhodopsin Kinase

Cone photoreceptors function under daylight conditions and are essential for color perception and vision with high temporal and spatial resolution. A remarkable feature of cones is that, unlike rods, they remain responsive in bright light. In rods, light triggers a decline in intracellular calcium, which exerts a well studied negative feedback on phototransduction that includes calcium-dependent inhibition of rhodopsin kinase (GRK1) by recoverin. Rods and cones share the same isoforms of recoverin and GRK1, and photoactivation also triggers a calcium decline in cones. However, the molecular mechanisms by which calcium exerts negative feedback on cone phototransduction through recoverin and GRK1 are not well understood. Here, we examined this question using mice expressing various levels of GRK1 or lacking recoverin. We show that although GRK1 is required for the timely inactivation of mouse cone photoresponse, gradually increasing its expression progressively delays the cone response recovery. This surprising result is in contrast with the known effect of increasing GRK1 expression in rods. Notably, the kinetics of cone responses converge and become independent of GRK1 levels for flashes activating more than ∼1% of cone pigment. Thus, mouse cone response recovery in bright light is independent of pigment phosphorylation and likely reflects the spontaneous decay of photoactivated visual pigment. We also find that recoverin potentiates the sensitivity of cones in dim light conditions but does not contribute to their capacity to function in bright light.