Rod Pigment Research Articles

1P-221 桿体・錐体視物質における活性中間体のpH依存性の比較研究(光生物-視覚・光受容,第47回日本生物物理学会年会)

1P-221 桿体・錐体視物質における活性中間体のpH依存性の比較研究(光生物-視覚・光受容,第47回日本生物物理学会年会)

Quantal noise from human red cone pigment

The rod pigment, rhodopsin, shows spontaneous isomerization activity. This quantal noise produces a dark light of approximately 0.01 photons s(-1) rod(-1) in human, setting the threshold for rod vision. The spontaneous isomerization activity of human cone pigments has long remained a mystery because the effect of a single isomerized pigment molecule in cones, unlike that in rods, is small and beyond measurement. We have now overcome this problem by expressing human red cone pigment transgenically in mouse rods in order to exploit their large single-photon response, especially after genetic removal of a key negative-feedback regulation. Extrapolating the measured quantal noise of transgenic cone pigment to native human red cones, we obtained a dark rate of approximately 10 false events s(-1) cone(-1), almost 10(3)-fold lower than the overall dark transduction noise previously reported in primate cones. Our measurements provide a rationale for why mammalian red, green and blue cones have comparable sensitivities, unlike their amphibian counterparts.

Amino acid residues controlling the properties and functions of rod and cone visual pigments.

The visual transduction processes in rod and cone photoreceptor cells are initiated by photon absorption by the different types of visual pigments. In relation to the functional difference between these cells, cone visual pigments in chicken retinas exhibit faster regeneration from 11-cis-retinal and opsin and faster decay of physiologically active intermediate (Meta II) than rod visual pigment, rhodopsin. Replacement of the amino acid residue at position 122 of chicken rhodopsin by the residues present in the respective cone pigments dramatically changes both the decay rate of Meta II and the rate of regeneration into those of the cone pigment-type, indicating that the residue at this position is a major determinant controlling these properties. Thus, the single replacement of amino acid residue at this position would be one of the key steps of the divergence into twilight and daylight vision.

The visual pigments of a deep-sea teleost, the pearl eyeScopelarchus analis

The eyes of deep-sea fish have evolved to function under vastly reduced light conditions compared to those that inhabit surface waters. This has led to a bathochromatic shift in the spectral location of maximum absorbance (lambda(max)) of their rod (RH1) pigments and the loss of cone photoreceptors. There are exceptions to this, however, as demonstrated by the deep-sea pearl eye Scopelarchus analis. Here we show the presence of two RH1 pigments (termed RH1A and RH1B) and a cone RH2 pigment. This is therefore the first time that the presence of a cone pigment in a deep-sea fish has been confirmed by molecular analysis. The lambda(max) values of the RH1A and RH1B pigments at 486 and 479 nm, respectively, have been determined by in vitro expression of the recombinant opsins and show the typical short-wave shifts of fish that live in deep water compared to surface dwellers. RH1B, however, is expressed only in more adult fish and lacks key residues for phosphorylation, indicating that it may not be involved in image formation. In contrast, the RH2 pigment has additional residues near the C terminus that may be involved in phosphorylation and does not show temporal changes in expression. The distribution of these pigments within the multiple retinae of S. analis is discussed.

Spectral absorbance, structure, and population density of photoreceptors in the retina of the lake sturgeon (Acipenser fulvescens)

Lake sturgeon ( Acipenser fulvescens Rafinesque, 1817) photoreceptors were studied with scanning electron microscopy and microspectrophotometry. The retina contains both rods and cones, with cones estimated composing about 30% of the photoreceptor population. Only large single cones were identified and they are similar to those found in other species of the order Acipenseriformes. The rods are large, with long, broad outer segments, and are similar to the dominant rod found in other sturgeons and the North American paddlefish ( Polyodon spathula (Walbaum, 1792)). Mean (SD) rod packing density at 22 624 ± 3 509 rods/mm2 is low compared with those of other animals that function primarily in dim light. The visual pigment of the rods has a mean (SD) peak absorbance (λmax) at 541 ± 2 nm. Three different cone populations were identified: a long wavelength sensitive cone containing a visual pigment with λmax at 619 ± 3 nm; middle wavelength sensitive cone with λmax at 538 ± 1 nm; and short wavelength sensitive cone with λmax at 448 ± 1 nm. All the visual pigments are based on the vitamin A2 chromophore.

Introduction of a rod pigment aromatic cluster does not improve the structural stability of the human green cone pigment

In the course of our studies on the structure/function relationship of visual pigments, we have expressed the human green cone pigment in the baculovirus/insect cell expression system. Purification of the human green cone pigment, however, has so far proven to be severely hampered by the low thermal stability of this receptor in a solubilized state. In order to overcome this problem, we tested a variety of chemical compounds that have been described to improve protein stability in various applications. The presence of glycerol, sucrose, trehalose and lipids during extraction improved the thermal stability of the recombinant green cone pigment up to twofold. We also analyzed the effect of mutation of residues Met208, Cys212 and Cys273 into Phe in all combinations. These mutants were designed in an attempt to increase the thermal stability by replacing weakly interacting side chains in the green pigment with their counterparts in rhodopsin with strong aromatic stacking interaction. All mutants produced wild-type levels of functional pigment, but none showed an increase in thermal stability.

Visual Cycle: Dependence of Retinol Production and Removal on Photoproduct Decay and Cell Morphology

The visual cycle is a chain of biochemical reactions that regenerate visual pigment following exposure to light. Initial steps, the liberation of all-trans retinal and its reduction to all-trans retinol by retinol dehydrogenase (RDH), take place in photoreceptors. We performed comparative microspectrophotometric and microfluorometric measurements on a variety of rod and cone photoreceptors isolated from salamander retinae to correlate the rates of photoproduct decay and retinol production. Metapigment decay rate was spatially uniform within outer segments and 50–70 times faster in the cells that contained cone-type pigment (SWS2 and M/LWS) compared to cells with rod-type pigment (RH1). Retinol production rate was strongly position dependent, fastest at the base of outer segments. Retinol production rate was 10–40 times faster in cones with cone pigments (SWS2 and M/LWS) than in the basal OS of rods containing rod pigment (RH1). Production rate was approximately five times faster in rods containing cone pigment (SWS2) than the rate in basal OS of rods containing the rod pigment (RH1). We show that retinol production is defined either by metapigment decay rate or RDH reaction rate, depending on cell type or outer segment region, whereas retinol removal is defined by the surface-to-volume ratio of the outer segment and the availability of retinoid binding protein (IRBP). The more rapid rates of retinol production in cones compared to rods are consistent with the more rapid operation of the visual cycle in these cells.

Spectral sensitivity of mollies: comparing surface‐ and cave‐dwelling Atlantic mollies,Poecilia mexicana

The visual pigments of cones and rods in three species of mollies,Poecilia mexicana,Poecilia latipinnaand their asexual hybridPoecilia formosa, were examined using microspectrophotometry. InP. mexicana, populations from extreme photic habitats were used: one population originated from a clear water habitat, one from a milky water habitat and another from a completely dark cave. Ultraviolet‐sensitive cones were found in all species. Differences in the λmaxvalues of the visual pigments were small between species and among the threeP. mexicanapopulations, but dark‐reared cave fishes showed appreciably higher variance. The hybrid speciesP. formosashowed a highly variable long wavelength cone absorbance, ranging from 528·9 to 598·5 nm, suggesting multiple opsin expression or chromophore mixing.

Visual pigments of marine carnivores: pinnipeds, polar bear, and sea otter

Rod and cone visual pigments of 11 marine carnivores were evaluated. Rod, middle/long-wavelength sensitive (M/L) cone, and short-wavelength sensitive (S) cone opsin (if present) sequences were obtained from retinal mRNA. Spectral sensitivity was inferred through evaluation of known spectral tuning residues. The rod pigments of all but one of the pinnipeds were similar to those of the sea otter, polar bear, and most other terrestrial carnivores with spectral peak sensitivities (lambda(max)) of 499 or 501 nm. Similarly, the M/L cone pigments of the pinnipeds, polar bear, and otter had inferred lambda(max) of 545 to 560 nm. Only the rod opsin sequence of the elephant seal had sensitivity characteristic of adaptation for vision in the marine environment, with an inferred lambda(max) of 487 nm. No evidence of S cones was found for any of the pinnipeds. The polar bear and otter had S cones with inferred lambda(max) of approximately 440 nm. Flicker-photometric ERG was additionally used to examine the in situ sensitivities of three species of pinniped. Despite the use of conditions previously shown to evoke cone responses in other mammals, no cone responses could be elicited from any of these pinnipeds. Rod photoreceptor responses for all three species were as predicted by the genetic data.

Late stages of visual pigment photolysis in situ: Cones vs. rods

Slow photolysis reactions and the regeneration of the dark pigment constitute the mechanisms of dark adaptation whereby photoreceptor cells restore their sensitivity after bright illumination. We present data on the kinetics of the late stages of the photolysis of the visual pigment in intact rods and red- and green-sensitive cones of the goldfish retina. Measurements were made on single photoreceptors by means of a fast-scanning dichroic microspectrophotometer. We show that in cones the hydrolysis of the opsin–all- trans 3-dehydroretinal linkage proceeds with a half-time of ≈5 s at 20 °C that is almost two orders of magnitude faster than in rods. 3-Dehydroretinol in cones is produced ≈3-fold faster than retinol in amphibian rhodopsin rods; the rate of the reaction is limited by the speed of retinal reduction catalyzed by retinoldehydrogenase. The fast hydrolysis of the 3-dehydroretinal/opsin Schiff base and the correspondingly fast appearance of the substrates for dark visual pigment regeneration (free opsin and 3-dehydroretinol) provide essential conditions for faster dark adaptation of cone (diurnal) as compared to rod (nocturnal) vision.

Scaffold Proteins and the Regeneration of Visual Pigments†

CRALBP, cellular retinaldehyde-binding protein, is a retinoid-binding protein necessary for efficient regeneration of rod and cone visual pigments. The C terminus of CRALBP binds to the PDZ domains of EBP50/NHERF-1, which in turn bind to ezrin and actin, proteins localized to the apical processes of the retinal pigment epithelium. In this study, we examined structural features associated with the interaction of the two proteins. The C-terminal amino-acid sequence of 11 orthologous CRALBPs is either ENTAL, ENTAF or EDTAL. Peptides ending in each of these sequences inhibited the interaction of CRALBP and EBP50/NHERF-1 with the use of an overlay assay. Molecular modeling showed that both NTAL and NTAF formed similar networks of H bonds with PDZ1 of EBP50/ NHERF-1, and the side chains of both C-terminal Leu and Phe fit into the peptide-binding groove of PDZ1x CRALBP.11-cis-retinal and EBP50/NHERF-1 migrated as single components when analyzed individually by gel filtration and as a complex when mixed together before gel filtration. Complex formation was abolished by preincubation of EBP50/NHERF-1 with peptide EVENTAL. The ligand absorption spectrum of the complex was identical with that of CRALBP x 11-cis-retinal, demonstrating that complex formation did not perturb the ligand-binding domain of CRALBP.

Retinoid cycles in the cone-dominated chicken retina

In past decades, the role of retinoids in support of rod photopigment regeneration has been extensively characterized. In the rhodopsin cycle, retinal chromophore from bleached rod pigments is reduced to retinol and transferred to the retinal pigment epithelium (RPE) to store as all-trans retinyl ester. This ester pool is subsequently utilized for visual pigment regeneration. However, there is a lack of information on the putative cone visual cycle. In the present study, we provide experimental evidence in support of a novel retinoid cycle for cone photopigment regeneration. In the cone-rich chicken, light exposure resulted in the accumulation of 11-cis retinyl esters to the retina and all-trans retinyl esters to the RPE. Both the rate of increase and the amount of 11-cis retinyl esters in the retina far exceeded those of the all-trans retinyl esters in the RPE. In response to dark adaptation, this 11-cis retinyl ester pool in the retina depletes at a rate several times faster than the all-trans retinyl ester pool in the RPE. In vitro, isolated, dark-adapted retinas devoid of RPE show both an accumulation of 11-cis retinyl ester and a concomitant reduction of 11-cis retinal chromophore in response to light exposure. Finally, we provide experimental results to elucidate a cone visual cycle in chicken by relating the change in retinoids (retinal and retinyl ester) with time during light and dark adaptation. Our results support a new paradigm for cone photopigment regeneration in which the 11-cis retinyl ester pool in the retina serves as the primary source of visual chromophore for cone pigment regeneration.

Colour vision and speciation in Lake Victoria cichlids of the genus Pundamilia

Lake Victoria cichlids are one of the most speciose groups of vertebrates. Selection on coloration is likely playing an important role in their rapid speciation. To test the hypothesis that sensory biases could explain species differences in mating preferences and nuptial coloration, we studied seven populations of four closely related species of the genus Pundamilia that differ in visual environment and male nuptial colour. Microspectrophotometry determined that the wavelength of maximum absorption (lambdamax) of the rod pigment and three cone pigments were similar in all four species. Only the long wavelength sensitive (LWS) pigment varied among species, with 3-4 nm shifts in lambdamax that correlated with differences in the LWS opsin sequence. These subtle shifts in lambdamax coincided with large shifts in male body colour, with red species having longer LWS pigments than blue species. Furthermore, we observed within and between species a correlation between water transparency and the proportion of red/red vs. red/green double cones. Individuals from turbid water had more red/red double cones than individuals from clear water. The variation in LWS lambdamax and in the proportion of red/red double cones could lead to differences in perceived brightness that may explain the evolution of variation in male coloration. However, other factors, such as chromophore shifts and higher order neural processing, should also be investigated to fully understand the physiological basis of differential responses to male mating hues in cichlid fish.

Molecular properties of rod and cone visual pigments from purified chicken cone pigments to mouse rhodopsin in situ

We have investigated the molecular properties of rod and cone visual pigments to elucidate the differences in the molecular mechanism(s) of the photoresponses between rod and cone photoreceptor cells. We have found that the cone pigments exhibit a faster pigment regeneration and faster decay of meta-II and meta-III intermediates than the rod pigment, rhodopsin. Mutagenesis experiments have revealed that the amino acid residues at positions 122 and 189 in the opsins are the determinants for these differences. In order to study the relationship between the molecular properties of visual pigments and the physiology of rod photoreceptors, we used mouse rhodopsin as a model pigment because, by gene-targeting, the spectral properties of the pigment can be directly correlated to the physiology of the cells. In the present paper, we summarize the spectroscopic properties of cone pigments and describe our studies with mouse rhodopsin utilizing a high performance charge coupled device (CCD) spectrophotometer.

Photoreceptors and photopigments in a subterranean rodent, the pocket gopher (Thomomys bottae)

Pocket gophers (Thomomys bottae) are rodents that spend much of their lives in near-lightless subterranean burrows. The visual adaptations associated with this extreme environment were investigated by making anatomical observations of retinal organization and by recording retinal responses to photic stimulation. The size of the eye is within the normal range for rodents, the lens transmits light well down into the ultraviolet, and the retina conforms to the normal mammalian plan. Electroretinogram recording revealed the presence of three types of photopigments, a rod pigment with a spectral peak of about 495 nm and two types of cone pigment with respective peak values of about 367 nm (UV) and 505 nm (medium-wavelength sensitive). Both in terms of responsivity to lights varying in temporal frequency and in response recovery following intense light adaptation, the cone responses of the pocket gopher are similar to those of other rodents. Labeling experiments indicate an abundance of cones that reach densities in excess of 30,000 mm-2. Cones containing UV opsin are found throughout the retina, but those containing medium-wavelength sensitive opsin are mostly restricted to the dorsal retina where coexpression of the two photopigments is apparently the rule. Rod densities are lower than those typical for nocturnal mammals.

Characterization of a novel form of X-linked incomplete achromatopsia

X-linked incomplete achromatopsia (XIA), also called blue-cone monochromacy (BCM), is a rare cone disorder that most commonly results either from one of two conditions. The first condition is a deletion of the locus control region (LCR) which is a critical DNA element that lies upstream of the L and M photopigment gene array on the X-chromosome and is necessary for expression of the photopigment genes. The second condition is an inactivating point mutation within the coding sequence of the remaining photopigment gene in an array from which all but one gene has been deleted. Many previous studies have concluded that affected individuals either have only rods and S-cones (Blackwell & Blackwell, 1957, 1961; Daw & Enoch, 1973; Hess et al., 1989) or have rods, S-cones, and another cone type that contains the rod pigment (Pokorny et al., 1970; Alpern et al., 1971). However, Smith et al. (1983) described individuals with XIA who had residual L-cone function. Here we report results for a subject with XIA who appears to have residual M-cone function. Genetic analysis revealed that he had apparently normal genes for M-cone photopigment thus leaving open the possibility that he has a contribution to vision based on expression of these genes at a very low level.

Role of the 9-methyl group of retinal in cone visual pigments.

In rhodopsin, the 9-methyl group of retinal has previously been identified as being critical in linking the ligand isomerization with the subsequent protein conformational changes that result in the activation of its G protein, transducin. Here, we report studies on the role of this methyl group in the salamander rod and cone pigments. Pigments were generated by combining proteins expressed in COS cells with 11-cis 9-demethyl retinal, where the 9-methyl group on the polyene chain has been deleted. The absorption spectra of all pigments were blue-shifted. The red cone and blue cone/green rod pigments were unstable to hydroxylamine; whereas, the rhodopsin and UV cone pigments were stable. The lack of the 9-methyl group of the chromophore did not affect the ability of the red cone and blue cone/green rod pigments to activate transducin. On the other hand, with the rhodopsin and UV cone pigments, activation was diminished. Interestingly, the red cone pigment containing the retinal analogue remained active longer than the native pigment. Thus, the 9-methyl group of retinal is not important in the activation pathway of the red cone and blue cone/green rod pigments. However, for the red cone pigment, the 9-methyl group of retinal appears to be critical in the deactivation pathway.

The rod opsin pigments from two marsupial species, the South American bare-tailed woolly opossum and the Australian fat-tailed dunnart

Rod visual pigment genes have been studied in a wide range of vertebrates including a number of mammalian species. However, no marsupials have yet been examined. To correct this omission, we have studied the rod pigments in two marsupial species, the nocturnal and frugivorous bare-tailed woolly opossum, Caluromys philander, from Central and South America, and the arhythmic and insectivorous fat-tailed dunnart, Sminthopsis crassicaudata, from Australia. Phylogenetic analysis establishes that the cloned opsin sequences are orthologues of rod opsin genes from other vertebrate species. The deduced amino acid sequences show that both possess glutamate at residue 122, a feature of rod opsins, and the corresponding gene follows the typical vertebrate rod opsin pattern of five exons separated by four introns. Compared to other vertebrates, a stretch of five residues near the C-terminus is deleted in the rod opsin of both marsupials and all eutherian mammals. From microspectrophotometric measurements, the pigments in the two species show an 8 nm difference in peak absorbance; the molecular basis for this spectral shift is discussed and two candidate substitutions are identified.

Role of visual pigment properties in rod and cone phototransduction.

Retinal rods and cones share a phototransduction pathway involving cyclic GMP. Cones are typically 100 times less photosensitive than rods and their response kinetics are several times faster, but the underlying mechanisms remain largely unknown. Almost all proteins involved in phototransduction have distinct rod and cone variants. Differences in properties between rod and cone pigments have been described, such as a 10-fold shorter lifetime of the meta-II state (active conformation) of cone pigment and its higher rate of spontaneous isomerization, but their contributions to the functional differences between rods and cones remain speculative. We have addressed this question by expressing human or salamander red cone pigment in Xenopus rods, and human rod pigment in Xenopus cones. Here we show that rod and cone pigments when present in the same cell produce light responses with identical amplification and kinetics, thereby ruling out any difference in their signalling properties. However, red cone pigment isomerizes spontaneously 10,000 times more frequently than rod pigment. This high spontaneous activity adapts the native cones even in darkness, making them less sensitive and kinetically faster than rods. Nevertheless, additional factors are probably involved in these differences.

The thermal contribution to photoactivation in A2 visual pigments studied by temperature effects on spectral properties.

Effects of temperature on the spectral properties of visual pigments were measured in the physiological range (5-28 degrees C) in photoreceptor cells of bullfrog (Rana catesbeiana) and crucian carp (Carassius carassius). Absorbance spectra recorded by microspectrophotometry (MSP) in single cells and sensitivity spectra recorded by electroretinography (ERG) across the isolated retina were combined to yield accurate composite spectra from ca. 400 nm to 800 nm. The four photoreceptor types selected for study allowed three comparisons illuminating the properties of pigments using the dehydroretinal (A2) chromophore: (1) the two members of an A1/A2 pigment pair with the same opsin (porphyropsin vs. rhodopsin in bullfrog "red" rods); (2) two A2 pigments with similar spectra (porphyropsin rods of bullfrog and crucian carp); and (3) two A2 pigments with different spectra (rods vs. long-wavelength-sensitive (L-) cones of crucian carp). Qualitatively, the temperature effects on A2 pigments were similar to those described previously for the A1 pigment of toad "red" rods. Warming caused an increase in relative sensitivities at very long wavelengths but additionally a small shift of lambdamax toward shorter wavelengths. The former effect was used for estimating the minimum energy required for photoactivation (Ea) of the pigment. Bullfrog rod opsin with A2 chromophore had Ea = 44.2 +/- 0.9 kcal/mol, significantly lower (one-tailed P < 0.05) than the value Ea = 46.5 +/- 0.8 kcal/mol for the same opsin coupled to A1. The A2 rod pigment of crucian carp had Ea = 42.3 +/- 0.6 kcal/mol, which is significantly higher (one-tailed P < 0.01) than that of the L-cones in the same retina (Ea = 38.3 +/- 0.4 kcal/mol), whereas the difference compared with the bullfrog A2 rod pigment is not statistically significant (two-tailed P = 0.13). No strict connection between lambdamax and Ea appears to exist among A2 pigments any more than among A1 pigments. Still, the A1 --> A2 chromophore substitution in bullfrog opsin causes three changes correlated as originally hypothesized by Barlow (1957): a red-shift of lambdamax, a decrease in Ea, and an increase in thermal noise.