Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments.     

Spectral tuning in salamander visual pigments studied with dihydroretinal chromophores.

In visual pigments, opsin proteins regulate the spectral absorption of a retinal chromophore by mechanisms that change the energy level of the excited electronic state relative to the ground state. We have studied these mechanisms by using photocurrent recording to measure the spectral sensitivities of individual red rods and red (long-wavelength-sensitive) and blue (short-wavelength-sensitive) cones of salamander before and after replacing the native 3-dehydro 11-cis retinal chromophore with retinal analogs: 11-cis retinal, 3-dehydro 9-cis retinal, 9-cis retinal, and 5,6-dihydro 9-cis retinal. The protonated Schiff's bases of analogs with unsaturated bonds in the ring had broader spectra than the same chromophores bound to opsins. Saturation of the bonds in the ring reduced the spectral bandwidths of the protonated Schiff's bases and the opsin-bound chromophores and made them similar to each other. This indicates that torsion of the ring produces spectral broadening and that torsion is limited by opsin. Saturating the 5,6 double bond in retinal reduced the perturbation of the chromophore by opsin in red and in blue cones but not in red rods. Thus an interaction between opsin and the chromophoric ring shifts the spectral maxima of the red and blue cone pigments, but not that of the red rod pigment.     

Multiple visual pigments in a photoreceptor of the salamander retina

Although a given retina typically contains several visual pigments, each formed from a retinal chromophore bound to a specific opsin protein, single photoreceptor cells have been thought to express only one type of opsin. This design maximizes a cell''s sensitivity to a particular wavelength band and facilitates wavelength discrimination in retinas that process color. We report electrophysiological evidence that the ultraviolet-sensitive cone of salamander violates this rule. This cell contains three different functional opsins. The three opsins could combine with the two different chromophores present in salamander retina to form six visual pigments. Whereas rods and other cones of salamander use both chromophores, they appear to express only one type of opsin per cell. In visual pigment absorption spectra, the bandwidth at half-maximal sensitivity increases as the pigment''s wavelength maximum decreases. However, the bandwidth of the UV-absorbing pigment deviates from this trend; it is narrow like that of a red-absorbing pigment. In addition, the UV-absorbing pigment has a high apparent photosensitivity when compared with that of red- and blue-absorbing pigments and rhodopsin. These properties suggest that the mechanisms responsible for spectrally tuning visual pigments separate two absorption bands as the wavelength of maximal sensitivity shifts from UV to long wavelengths.     

Halorhodopsin and sensory rhodopsin contain a C6-C7 s-trans retinal chromophore.

Halorhodopsin (HR) and sensory rhodopsin (SR) have been regenerated with retinal analogues that are covalently locked in the 6-s-cis or 6-s-trans conformations. Both pigments regenerate more completely with the locked 6-s-trans retinal and produce analogue pigments with absorption maxima (577 nm for HR and 592 nm for SR) nearly identical to those of the native pigments (577 and 587 nm). This indicates that HR and SR bind retinal in the 6-s-trans conformation. The opsin shift for the locked 6-s-trans analogue in HR is 1,200 cm-1 less than that for the native chromophore (5,400 cm-1). The opsin shift for the 6-s-trans analogue in SR is 1,100 cm-1 less than that for the native retinal (5,700 cm-1). This demonstrates that approximately 20% of the opsin shift in these pigments arises from a protein-induced change in the chromophore conformation from twisted 6-s-cis in solution to planar 6-s-trans in the protein. The reduced opsin shift observed for the locked 6-s-cis analogue pigments compared with the locked 6-s-trans pigments may be due to a positive electrostatic perturbation near C7.     

Opsin shift in an aldolase antibody.

An antibody-retinal assembly that mimics the opsin shift (OS) of the naturally occurring visual pigments is reported. Both experiments and calculations show that the aldolase antibody 33F12 covalently binds all-trans retinal via a protonated Schiff base with a lysine residue. This chromophore, which exhibits a remarkable opsin red shift (140 nm), represents a useful model system for studying the factors that contribute to the OS.     

Spectral tuning by opsin coexpression in retinal regions that view different parts of the visual field

Vision frequently mediates critical behaviours, and photoreceptors must respond to the light available to accomplish these tasks. Most photoreceptors are thought to contain a single visual pigment, an opsin protein bound to a chromophore, which together determine spectral sensitivity. Mechanisms of spectral tuning include altering the opsin, changing the chromophore and incorporating pre-receptor filtering. A few exceptions to the use of a single visual pigment have been documented in which a single mature photoreceptor coexpresses opsins that form spectrally distinct visual pigments, and in these exceptions the functional significance of coexpression is unclear. Here we document for the first time photoreceptors coexpressing spectrally distinct opsin genes in a manner that tunes sensitivity to the light environment. Photoreceptors of the cichlid fish, Metriaclima zebra, mix different pairs of opsins in retinal regions that view distinct backgrounds. The mixing of visual pigments increases absorbance of the corresponding background, potentially aiding the detection of dark objects. Thus, opsin coexpression may be a novel mechanism of spectral tuning that could be useful for detecting prey, predators and mates. However, our calculations show that coexpression of some opsins can hinder colour discrimination, creating a trade-off between visual functions.     

The opsins

The photosensitive molecule rhodopsin and its relatives consist of a protein moiety - an opsin - and a non-protein moiety - the chromophore retinal. Opsins, which are G-protein-coupled receptors (GPCRs), are found in animals, and more than a thousand have been identified so far. Detailed molecular phylogenetic analyses show that the opsin family is divided into seven subfamilies, which correspond well to functional classifications within the family: the vertebrate visual (transducin-coupled) and non-visual opsin subfamily, the encephalopsin/tmt-opsin subfamily, the G_q-coupled opsin/melanopsin subfamily, the G_o-coupled opsin subfamily, the neuropsin subfamily, the peropsin subfamily and the retinal photoisomerase subfamily. The subfamilies diversified before the deuterostomes (including vertebrates) split from the protostomes (most invertebrates), suggesting that a common animal ancestor had multiple opsin genes. Opsins have a seven-transmembrane structure similar to that of other GPCRs, but are distinguished by a lysine residue that is a retinal-binding site in the seventh helix. Accumulated evidence suggests that most opsins act as pigments that activate G proteins in a light-dependent manner in both visual and non-visual systems, whereas a few serve as retinal photoisomerases, generating the chromophore used by other opsins, and some opsins have unknown functions.     

Wavelength regulation in iodopsin, a cone pigment.

The opsin shift, the difference in wavenumber between the absorption peak of a visual pigment and the protonated Schiff base of the chromophore, represents the influence of the opsin binding site on the chromophore. The opsin shift for the chicken cone pigment iodopsin is much larger than that for rhodopsin. To understand the origin of this opsin shift and the mechanism of wavelength regulation in iodopsin, a series of synthetic 9-cis and 11-cis dehydro- and dihydro-retinals was used to regenerate iodopsin-based pigments. The opsin shifts of these pigments are quite similar to those found in bacteriorhodopsin-based artificial pigments. On the basis of these studies, a tentative model of wavelength regulation in iodopsin is proposed.     

NinaB Is Essential for Drosophila Vision but Induces Retinal Degeneration in Opsin-deficient Photoreceptors

In animals, visual pigments are essential for photoreceptor function and survival. These G-protein-coupled receptors consist of a protein moiety (opsin) and a covalently bound 11-cis-retinylidene chromophore. The chromophore is derived from dietary carotenoids by oxidative cleavage and trans-to-cis isomerization of double bonds. In vertebrates, the necessary chemical transformations are catalyzed by two distinct but structurally related enzymes, the carotenoid oxygenase β-carotenoid-15,15′-monooxygenase and the retinoid isomerase RPE65 (retinal pigment epithelium protein of 65 kDa). Recently, we provided biochemical evidence that these reactions in insects are catalyzed by a single enzyme family member named NinaB. Here we show that in the fly pathway, carotenoids are mandatory precursors of the chromophore. After chromophore formation, the retinoid-binding protein Pinta acts downstream of NinaB and is required to supply photoreceptors with chromophore. Like ninaE encoding the opsin, ninaB expression is eye-dependent and is activated as a downstream target of the eyeless/pax6 and sine oculis master control genes for eye development. The requirement for coordinated synthesis of chromophore and opsin is evidenced by analysis of ninaE mutants. Retinal degeneration in opsin-deficient photoreceptors is caused by the chromophore and can be prevented by restricting its supply as seen in an opsin and chromophore-deficient double mutant. Thus, our study identifies NinaB as a key component for visual pigment production and provides evidence that chromophore in opsin-deficient photoreceptors can elicit retinal degeneration.     

Rod and cone visual pigments and phototransduction through pharmacological, genetic, and physiological approaches

Activation of the visual pigment by light in rod and cone photoreceptors initiates our visual perception. As a result, the signaling properties of visual pigments, consisting of a protein, opsin, and a chromophore, 11-cis-retinal, play a key role in shaping the light responses of photoreceptors. The combination of pharmacological, physiological, and genetic tools has been a powerful approach advancing our understanding of the interactions between opsin and chromophore and how they affect the function of visual pigments. The signaling properties of the visual pigments modulate many aspects of the function of rods and cones, producing their unique physiological properties.     

Regeneration of bovine and octopus opsins in situ with natural and artificial retinals

We consider the problem of color regulation in visual pigments for both bovine rhodopsin (lambda max = 500 nm) and octopus rhodopsin (lambda max = 475 nm). Both pigments have 11-cis-retinal (lambda max = 379 nm, in ethanol) as their chromophore. These rhodopsins were bleached in their native membranes, and the opsins were regenerated with natural and artificial chromophores. Both bovine and octopus opsins were regenerated with the 9-cis- and 11-cis-retinal isomers, but the octopus opsin was additionally regenerated with the 13-cis and all-trans isomers. Titration of the octopus opsin with 11-cis-retinal gave an extinction coefficient for octopus rhodopsin of 27,000 +/- 3000 M-1 cm-1 at 475 nm. The absorption maxima of bovine artificial pigments formed by regenerating opsin with the 11-cis dihydro series of chromophores support a color regulation model for bovine rhodopsin in which the chromophore-binding site of the protein has two negative charges: one directly hydrogen bonded to the Schiff base nitrogen and another near carbon-13. Formation of octopus artificial pigments with both all-trans and 11-cis dihydro chromophores leads to a similar model for octopus rhodopsin and metarhodopsin: there are two negative charges in the chromophore-binding site, one directly hydrogen bonded to the Schiff base nitrogen and a second near carbon-13. The interaction of this second charge with the chromophore in octopus rhodopsin is weaker than in bovine, while in metarhodopsin it is as strong as in bovine.     

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.     

Squid retinochrome

Retinochrome is a photosensitive pigment located primarily in the inner portions of the visual cells of cephalopods. Its absorption spectrum resembles that of rhodopsin, but its chromophore is all-trans retinal, which light isomerizes to 11-cis, the reverse of the situation in rhodopsin. The 11-cis photoproduct of retinochrome slowly reverts to retinochrome in the dark. The chromophoric site of retinochrome is more reactive than that of most visual pigments: (a) Hydroxylamine converts retinochrome in the dark to all-trans retinal oxime + retinochrome opsin. (by Sodium borohydride reduces it to N-retinyl opsin. (c) Lambda max of retinochrome shifts from 500 to 515 nm as the pH is raised from 6 to 10, with a loss of absorption above pH 8; meanwhile above this PH a second band appears at shorter wavelengths with lambda max 375 nm. These changes are reversible. (d) If retinochrome is incubated with all-trans 3-dehydroretinal (retinal2) in the dark, some 3-dehydroretinochrome (retinochrome2, lambda max about 515 nm) is formed. Conversely, when retinochrome2, made by adding all-trans retinal2 to bleached retinochrome or retinochrome opsin, is incubated in the dark with all-trans retinal some of it is converted to retinochrome. Retinal and 3-dehydroretinal therefore can replace each other as chromophores in the dark.     

Interaction of 11-cis-retinol dehydrogenase with the chromophore of retinal g protein-coupled receptor opsin

Vertebrate opsins in both photoreceptors and the retinal pigment epithelium (RPE) have fundamental roles in the visual process. The visual pigments in photoreceptors are bound to 11-cis-retinal and are responsible for the initiation of visual excitation. Retinochrome-like opsins in the RPE are bound to all-trans-retinal and play an important role in chromophore metabolism. The retinal G protein-coupled receptor (RGR) of the RPE and Müller cells is an abundant opsin that generates 11-cis-retinal by stereospecific photoisomerization of its bound all-trans-retinal chromophore. We have analyzed a 32-kDa protein (p32) that co-purifies with bovine RGR from RPE microsomes. The co-purified p32 was identified by mass spectrometric analysis as 11-cis-retinol dehydrogenase (cRDH), and enzymatic assays have confirmed the isolation of an active cRDH. The co-purified cRDH showed marked substrate preference to 11-cis-retinal and preferred NADH rather than NADPH as the cofactor in reduction reactions. cRDH did not react with endogenous all-trans-retinal bound to RGR but reacted specifically with 11-cis-retinal that was generated by photoisomerization after irradiation of RGR. The reduction of 11-cis-retinal to 11-cis-retinol by cRDH enhanced the net photoisomerization of all-trans-retinal bound to RGR. These results indicate that cRDH is involved in the processing of 11-cis-retinal after irradiation of RGR opsin and suggest that cRDH has a novel role in the visual cycle.     

Convergent Phenotypic Evolution of Rhodopsin for Dim-Light Sensing across Deep-Diving Vertebrates

Rhodopsin comprises an opsin attached to a retinal chromophore and is the only visual pigment conferring dim-light vision in vertebrates. On activation by photons, the retinal group becomes detached from the opsin, which is then inactive until it is recharged. Of all vertebrate species, those that dive face unique visual challenges, experiencing rapid decreases in light level and hunting in near darkness. Here, we combine sequence analyses with functional assays to show that the rhodopsin pigments of four divergent lineages of deep-diving vertebrates have undergone convergent increases in their retinal release rate. We compare gene sequences and detect parallel amino acids between penguins and diving mammals and perform mutagenesis to show that a single critical residue fully explains the observed increases in retinal release rate in both the emperor penguin and beaked whale. At the same time, we find that other shared sites have no significant effect on retinal release, implying that convergence does not always signify adaptive significance. We propose that accelerated retinal release confers rapid rhodopsin recharging, enabling the visual systems of diving species to adjust quickly to changing light levels as they descend through the water column. This contrasts with nocturnal species, where adaptation to darkness has been attributed to slower retinal release rates.     

Photoisomerization efficiency in UV-absorbing visual pigments: protein-directed isomerization of an unprotonated retinal Schiff base

A visual pigment consists of an opsin protein and a chromophore, 11-cis-retinal, which binds to a specific lysine residue of opsin via a Schiff base linkage. The Schiff base chromophore is protonated in pigments that absorb visible light, whereas it is unprotonated in ultraviolet-absorbing visual pigments (UV pigments). To investigate whether an unprotonated Schiff base can undergo photoisomerization as efficiently as a protonated Schiff base in the opsin environment, we measured the quantum yields of the bovine rhodopsin E113Q mutant, in which the Schiff base is unprotonated at alkaline pH, and the mouse UV pigment (mouse UV). Photosensitivities of UV pigments were measured by irradiation of the pigments followed by chromophore extraction and HPLC analysis. Extinction coefficients were estimated by comparing the maximum absorbances of the original pigments and their acid-denatured states. The quantum yield of the bovine rhodopsin E113Q mutant at pH 8.2, where the Schiff base is unprotonated, was significantly lower than that of wild-type rhodopsin, whereas the mutant gave a quantum yield almost identical to that of the wild type at pH 5.5, where the Schiff base is protonated. These results suggest that Schiff base protonation plays a role in increasing quantum yield. The quantum yield of mouse UV, which has an unprotonated Schiff base chromophore, was significantly higher than that of the unprotonated form of the rhodopsin E113Q mutant, although it was still lower than the visible-absorbing pigments. These results suggest that the mouse UV pigment has a specific mechanism for the efficient photoisomerization of its unprotonated Schiff base chromophore.     

Photosensitivities of rhodopsin mutants with a displaced counterion

Visual pigments consist of a protein moiety opsin and an 11-cis-retinal chromophore that is covalently bound to the opsin via a Schiff base linkage. They have a high photosensitivity, which can be attributed to the high probability of photon absorption and the high photoisomerization quantum yield of the retinal chromophore. Both of these parameters are regulated by the opsin, though the precise mechanism is unknown. We previously found that counterion residue E113, which stabilizes the proton on the Schiff base, is involved in the efficient photoisomerization in vertebrate visual pigments. To test the positional effect of the counterion on the photon absorption and the photoisomerization, we measured the photosensitivities of a set of mutants of bovine rhodopsin in which the counterion was displaced to position 90, 94, 117, or 292. The molar extinction coefficient was reduced in many of the mutants, leading to reductions in the photosensitivity for monochromatic lights. However, the oscillator strength, the probability of photon absorption integrated over the entire wavenumber range of the absorption band, was relatively similar among the mutants and the wild type. In addition, the quantum yields of the mutants were not markedly different from that of the wild type. These results indicate that the counterion does not need to be located at position 113 for a high photosensitivity for natural light. Interestingly, all of the mutants exhibited greatly increased hydroxylamine sensitivity. This result suggests that the counterion in vertebrate visual pigments is optimally located for the stability of the Schiff base linkage.     

Vertebrate ancient-long opsin has molecular properties intermediate between those of vertebrate and invertebrate visual pigments

VA/VAL opsin is one of the four kinds of nonvisual opsins that are closely related to vertebrate visual pigments in the phylogenetic tree of opsins. Previous studies indicated that among these opsins, parapinopsin and pinopsin exhibit molecular properties similar to those of invertebrate bistable visual pigments and vertebrate visual pigments, respectively. Here we show that VA/VAL opsin exhibits molecular properties intermediate between those of parapinopsin and pinopsin. VAL opsin from Xenopus tropicalis was expressed in cultured cells, and the pigment with an absorption maximum at 501 nm was reconstituted by incubation with 11-cis-retinal. Light irradiation of this pigment caused cis-to-trans isomerization of the chromophore to form a state having an absorption maximum in the visible region. This state has the ability to activate Gi and Gt types of G proteins. Therefore, the active state of VAL opsin is a visible light-absorbing intermediate, which probably has a protonated retinylidene Schiff base as its chromophore, like the active state of parapinopsin. However, this state was apparently photoinsensitive and did not show reverse reaction to the original pigment, unlike the active state of parapinopsin, and instead similar to that of pinopsin. Furthermore, the Gi activation efficiency of VAL opsin was between those of pinopsin and parapinopsin. Thus, the molecular properties of VA/VAL opsin give insights into the mechanism of conversion of the molecular properties from invertebrate to vertebrate visual pigments.     

Breaking the covalent bond--a pigment property that contributes to desensitization in cones

Retinal rod and cone pigments consist of an apoprotein, opsin, covalently linked to a chromophore, 11-cis retinal. Here we demonstrate that the formation of the covalent bond between opsin and 11-cis retinal is reversible in darkness in amphibian red cones, but essentially irreversible in red rods. This dissociation, apparently a general property of cone pigments, results in a surprisingly large amount of free opsin--about 10% of total opsin--in dark-adapted red cones. We attribute this significant level of free opsin to the low concentration of intracellular free 11-cis retinal, estimated to be only a tiny fraction (approximately 0.1 %) of the pigment content in red cones. With its constitutive transducin-stimulating activity, the free cone opsin produces an approximately 2-fold desensitization in red cones, equivalent to that produced by a steady light causing 500 photoisomerizations s-1. Cone pigment dissociation therefore contributes to the sensitivity difference between rods and cones.     

Synthetic pigment analogues of the purple membrane protein.

Nonphysiological analogues of retinal have been shown to form pigments in reactions with the apoprotein of the purple membrane of Halobacterium halobium. Both the all-trans and 13-cis isomers of a retinal analogue, having an elongated chain with an extra double bond, formed pigments. Unlike the native all-trans and 13-cis retinal1-based pigments, the new pigments were not interconvertible with each other and were unstable against hydroxylamine. When incorporated into phospholipid vesicles, they showed no proton pumping activity upon illumination. The ability of the extended-length retinal to form pigments contrasts with its nonreactivity with opsin (apoprotein of rhodopsin), suggesting a less stringent binding site for the purple membrane chromophore. All-trans retinal2 also combined with bleached purple membrane to form a blue pigment absorbing at ca. 590 nm. Like the native purple membrane, the blu membrane showed proton pumping activity upon illumination in phospholipid vesicles.