Conformational Selection and Equilibrium Governs the Ability of Retinals to Bind Opsin

Despite extensive study, how retinal enters and exits the visual G protein-coupled receptor rhodopsin remains unclear. One clue may lie in two openings between transmembrane helix 1 (TM1) and TM7 and between TM5 and TM6 in the active receptor structure. Recently, retinal has been proposed to enter the inactive apoprotein opsin (ops) through these holes when the receptor transiently adopts the active opsin conformation (ops*). Here, we directly test this “transient activation” hypothesis using a fluorescence-based approach to measure rates of retinal binding to samples containing differing relative fractions of ops and ops*. In contrast to what the transient activation hypothesis model would predict, we found that binding for the inverse agonist, 11-cis-retinal (11CR), slowed when the sample contained more ops* (produced using M257Y, a constitutively activating mutation). Interestingly, the increased presence of ops* allowed for binding of the agonist, all-trans-retinal (ATR), whereas WT opsin showed no binding. Shifting the conformational equilibrium toward even more ops* using a G protein peptide mimic (either free in solution or fused to the receptor) accelerated the rate of ATR binding and slowed 11CR binding. An arrestin peptide mimic showed little effect on 11CR binding; however, it stabilized opsin·ATR complexes. The TM5/TM6 hole is apparently not involved in this conformational selection. Increasing its size by mutagenesis did not enable ATR binding but instead slowed 11CR binding, suggesting that it may play a role in trapping 11CR. In summary, our results indicate that conformational selection dictates stable retinal binding, which we propose involves ATR and 11CR binding to different states, the latter a previously unidentified, open-but-inactive conformation.     

Diversity of Active States in TMT Opsins

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

Studies on a Missing Reaction in the Visual Cycle

DEVELOPMENT in the biochemistry of vision during the past twenty-five years can be summarized by equations (1) and (2) in Fig. 1, which envisage¹ that 11-cis-retinal combines with the visual protein opsin in a dark reaction to form the photolabile complex rhodopsin, λ_{max 497 nm}. When rhodopsin absorbs light it stimulates, through a process whose mechanism is not understood, the transmission of impulses, which are responsible for the visual sensation, although much is known about the biochemical changes accompanying the absorption of light by rhodopsin. These changes culminate in the formation of all-trans-retinal (λ_{max 385 nm}) and opsin (equation (2), Fig. 1), through a number of intermediates² and for the completion of the cycle one needs a molecular process which may regenerate 11-cis-retinal from all-trans-retinal (equation (3), Fig. 1).     

Key enzymes of the retinoid (visual) cycle in vertebrate retina

A major goal in vision research over the past few decades has been to understand the molecular details of retinoid processing within the retinoid (visual) cycle. This includes the consequences of side reactions that result from delayed all-trans-retinal clearance and condensation with phospholipids that characterize a variety of serious retinal diseases. Knowledge of the basic retinoid biochemistry involved in these diseases is essential for development of effective therapeutics. Photoisomerization of the 11-cis-retinal chromophore of rhodopsin triggers a complex set of metabolic transformations collectively termed phototransduction that ultimately lead to light perception. Continuity of vision depends on continuous conversion of all-trans-retinal back to the 11-cis-retinal isomer. This process takes place in a series of reactions known as the retinoid cycle, which occur in photoreceptor and RPE cells. All-trans-retinal, the initial substrate of this cycle, is a chemically reactive aldehyde that can form toxic conjugates with proteins and lipids. Therefore, much experimental effort has been devoted to elucidate molecular mechanisms of the retinoid cycle and all-trans-retinal-mediated retinal degeneration, resulting in delineation of many key steps involved in regenerating 11-cis-retinal. Three particularly important reactions are catalyzed by enzymes broadly classified as acyltransferases, short-chain dehydrogenases/reductases and carotenoid/retinoid isomerases/oxygenases. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.     

Measurement of Slow Spontaneous Release of 11-cis-Retinal from Rhodopsin

The vertebrate visual photoreceptor rhodopsin (Rho) is a unique G protein-coupled receptor as it utilizes a covalently tethered inverse agonist (11-cis-retinal) as the native ligand. Previously, electrophysiological studies showed that ligand binding of 11-cis-retinal in dark-adapted Rho was essentially irreversible with a half-life estimated to be 420 years, until after thermal isomerization to all-trans-retinal, which then slowly dissociates. This long lifetime of 11-cis-retinal binding was considered to be physiologically important for minimizing background signal (dark noise) of the visual system. However, in vitro biochemical studies on the thermal stability of Rho showed that Rho decays with a half-life on the order of days. In this study, we resolve the discrepancy by measuring the chromophore exchange rate of the bound 11-cis-retinal chromophore with free 9-cis-retinal from Rho in an in vitro phospholipid/detergent bicelle system. We conclude that the thermal decay of Rho primarily proceeds through spontaneous breaking of the covalent linkage between opsin and 11-cis-retinal, which was overlooked in the electrophysiological recording. We estimate that this slow spontaneous release of 11-cis-retinal from Rho should result in 10⁴ to 10⁵ free opsin molecules in a dark-adapted rod cell—a number that is three orders of magnitude higher than previously expected. We also discuss the physiological implications of these findings on the basal activity of opsins and the associated dark noise in the visual system.     

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter,ABCA4

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport.     

Substrate Specificity and Subcellular Localization of the Aldehyde-Alcohol Redox-coupling Reaction in Carp Cones

Our previous study suggested the presence of a novel cone-specific redox reaction that generates 11-cis-retinal from 11-cis-retinol in the carp retina. This reaction is unique in that 1) both 11-cis-retinol and all-trans-retinal were required to produce 11-cis-retinal; 2) together with 11-cis-retinal, all-trans-retinol was produced at a 1:1 ratio; and 3) the addition of enzyme cofactors such as NADP(H) was not necessary. This reaction is probably part of the reactions in a cone-specific retinoid cycle required for cone visual pigment regeneration with the use of 11-cis-retinol supplied from Müller cells. In this study, using purified carp cone membrane preparations, we first confirmed that the reaction is a redox-coupling reaction between retinals and retinols. We further examined the substrate specificity, reaction mechanism, and subcellular localization of this reaction. Oxidation was specific for 11-cis-retinol and 9-cis-retinol. In contrast, reduction showed low specificity: many aldehydes, including all-trans-, 9-cis-, 11-cis-, and 13-cis-retinals and even benzaldehyde, supported the reaction. On the basis of kinetic studies of this reaction (aldehyde-alcohol redox-coupling reaction), we found that formation of a ternary complex of a retinol, an aldehyde, and a postulated enzyme seemed to be necessary, which suggested the presence of both the retinol- and aldehyde-binding sites in this enzyme. A subcellular fractionation study showed that the activity is present almost exclusively in the cone inner segment. These results suggest the presence of an effective production mechanism of 11-cis-retinal in the cone inner segment to regenerate visual pigment.     

Retinal-protein interactions in bacteriorhodopsin monomers,dispersed in the detergent L-1690

Bacteriorhodopsin monomer dispersed in a solution of the detergent L-1690 could maintain the specific interaction between retinal and protein in the pH range 9.0-0.0 at 25°C. λ_max of the absorbance spectrum was 550 nm at pH 9.0, 556 nm at pH 5.5, 609 nm at pH 2.1 and 570 nm at pH 0.0. Increasing the NaCl concentration in the solution promoted formation of the 609 nm product at pH 5.0-3.0 and also its transition to the 570 nm product at pH 2.5-1.0. Retinal isomer analysis gave a ratio of 13-cis- to all-trans-retinal of 53 : 47 at pH 5.5. When the pH of the solution was reduced, the relative content of all-trans-retinal increased and the ratio of 13-cis- to all-trans-retinal was 14 : 86 at pH 0.0. Illumination of the solution at pH 7.2 yielded a product containing 9-cis-retinal or 9-cis, 13-cis-retinal, which may be due to a reaction other than the photoreaction cycle.     

Tautomeric Forms of Metarhodopsin

Light isomerizes the chromophore of rhodopsin, 11-cis retinal (formerly retinene), to the all-trans configuration. This introduces a succession of unstable intermediates—pre-lumirhodopsin, lumirhodopsin, metarhodopsin —in which all-trans retinal is still attached to the chromophoric site on opsin. Finally, retinal is hydrolyzed from opsin. The present experiments show that metarhodopsin exists in two tautomeric forms, metarhodopsins I and II, with λ_max 478 and 380 mµ. Metarhodopsin I appears first, then enters into equilibrium with metarhodopsin II. In this equilibrium, the proportion of metarhodopsin II is favored by higher temperature or pH, neutral salts, and glycerol. The change from metarhodopsin I to II involves the binding of a proton by a group with pK 6.4 (imidazole?), and a large increase of entropy. Metarhodopsin II has been confused earlier with the final mixture of all-trans retinal and opsin (λ_max 387 mµ), which it resembles in spectrum. These two products are, however, readily distinguished experimentally.     

Biosynthesis of 9-cis-Retinoic Acid from 9-cis-β-Carotene in Human Intestinal Mucosa in Vitro

The role of 9-cis-β-carotene (9-cis-β-C) as a potential precursor of 9-cis-retinoic acid (9-cis-RA) has been examined in human intestinal microcosa in vitro. By using HPLC, uv spectra, and chemical derivatization analysis, both 9-cis-RA and all-trans-retinoic acid (all-trans-RA) have been identified in the postnuclear fraction of human intestinal microcosa after incubation with 9-cis-β-C at 37°C. The biosynthesis of both 9-cis-RA and all-trans-RA from 9-cis-β-C was linear with increasing concentrations of 9-cis-β-C (2-30 μM) and was linear with respect to tissue protein concentration up to 0.75 mg/ml. Retinoic acid was not detected when a boiled incubation mixture was incubated in the presence of 9-cis-β-C. The rate of synthesis of 9-cis- and all-trans-RA from 4 μM 9-cis-β-C were 16 ± 1 and 18 ± 2 pmol/hr/mg of protein, respectively. However, when 2 μM all-trans-β-C was added to the 4 μM 9-cis-β-C, the rate of all-trans-RA synthesis was increased to 38 ± 6 pmol/hr/mg of protein, whereas the rate of 9-cis-RA synthesis remained the same. These results suggest that 9-cis-RA is produced directly from 9-cis-β-C. Furthermore, incubations of either 0.1 μM 9-cis- or all-trans-retinal under the same incubation conditions showed that 9-cis-RA could also arise through oxidative conversion of 9-cis-retinal. Although only 9-cis-RA was detected when 9-cis-RA was used as the substrate, the isomerization of the all-trans-RA to 9-cis-RA cannot be ruled out, since both all-trans-RA and trace amounts of 9-cis-RA were detected when all-trans-retinal was incubated as the substrate. These data indicate that 9-cis-β-C can be a source of 9-cis-RA in the human. This conversion may have a significance in the anticarcinogenic action of β-C.     

Involvement of All-trans-retinal in Acute Light-induced Retinopathy of Mice

Exposure to bright light can cause visual dysfunction and retinal photoreceptor damage in humans and experimental animals, but the mechanism(s) remain unclear. We investigated whether the retinoid cycle (i.e. the series of biochemical reactions required for vision through continuous generation of 11-cis-retinal and clearance of all-trans-retinal, respectively) might be involved. Previously, we reported that mice lacking two enzymes responsible for clearing all-trans-retinal, namely photoreceptor-specific ABCA4 (ATP-binding cassette transporter 4) and RDH8 (retinol dehydrogenase 8), manifested retinal abnormalities exacerbated by light and associated with accumulation of diretinoid-pyridinium-ethanolamine (A2E), a condensation product of all-trans-retinal and a surrogate marker for toxic retinoids. Now we show that these mice develop an acute, light-induced retinopathy. However, cross-breeding these animals with lecithin:retinol acyltransferase knock-out mice lacking retinoids within the eye produced progeny that did not exhibit such light-induced retinopathy until gavaged with the artificial chromophore, 9-cis-retinal. No significant ocular accumulation of A2E occurred under these conditions. These results indicate that this acute light-induced retinopathy requires the presence of free all-trans-retinal and not, as generally believed, A2E or other retinoid condensation products. Evidence is presented that the mechanism of toxicity may include plasma membrane permeability and mitochondrial poisoning that lead to caspase activation and mitochondria-associated cell death. These findings further understanding of the mechanisms involved in light-induced retinal degeneration.The retinoid cycle is a fundamental metabolic process in the vertebrate retina responsible for continuous generation of 11-cis-retinal from its all-trans-isomer (1-3). Because 11-cis-retinal is the chromophore of rhodopsin and cone visual pigments (4), disabling mutations in genes encoding proteins of the retinoid cycle can cause a spectrum of retinal diseases affecting sight (3). Moreover, the efficiency of the mammalian visual system and health of photoreceptors and retinal pigment epithelium (RPE)² decrease significantly with age. Even in the presence of a functional retinoid cycle, A2E, retinal dimer (RALdi), and other toxic all-trans-retinal condensation products (5-7) can accumulate as a consequence of aging (8). Under experimental conditions, these compounds can produce toxic effects on RPE cells (9-11). Patients affected by age-related macular degeneration, Stargardt disease, or other retinal diseases associated with accumulation of surrogate markers, such as A2E, all develop retinal degeneration (12). Thus, elucidating the fundamental causes of these age-dependent changes is of increasing importance. Encouragingly, our understanding of both retinoid metabolism outside the eye and production of 11-cis-retinal unique to the eye has accelerated recently (Scheme 1) (1-3), and genetic mouse models are readily available to study these processes and their potential aberrations in vivo (13). Thus, a central question can be addressed, namely what initiates the death of photoreceptor cells and the underlining RPE?Open in a separate windowSCHEME 1.Retinoid flow and all-trans-retinal clearance in the visual cycle. After diffusion from the RPE, the visual chromophore, 11-cis-retinal, combines with rhodopsin and then is photoisomerized to all-trans-retinal. Most of the all-trans-retinal dissociates from opsin into the cytoplasm, where it is reduced to all-trans-retinol by RDHs, including RDH8. The fraction of all-trans-retinal that dissociates into the disc lumen is transported by ABCA4 into the cytoplasm (23) before it is reduced. All-trans-retinol then is translocated to the RPE, esterified by LRAT, and recycled back to 11-cis-retinal. Mutations of ABCA4 are associated with human macular degeneration, Stargardt disease, and age-related macular degeneration (55, 56).Several mechanisms associated with retinoid metabolism may contribute to different retinopathies (1). For example, lack of retinoids in LRAT (lecithin:retinol acyltransferase) or chromophore in retinoid isomerase knock-out (Rpe65^-/-) mice leads to rapid degeneration of cone photoreceptors and slowly progressive death of rods (14). Such mice do not produce toxic condensation products from all-trans-retinal. Instead, their retinopathies have been attributed to continuous activation of visual phototransduction (15) due to either the basal activity of opsin (16-18) or disordered vectorial transport of cone visual pigments without bound chromophore (19). Paradoxically, an abnormally high flux of retinoids through the retinoid cycle can also lead to retinopathy in other mouse models (20, 21). Animal models featuring anomalies in the retinoid cycle illustrate the importance of chromophore regeneration and provide an approach to elucidating mechanisms involved in human retinal dysfunction and disease.Recently, we showed that mice carrying a double knock-out of Rdh8 (retinol dehydrogenase 8), one of the main enzymes that reduces all-trans-retinal in rod and cone outer segments (22), and Abca4 (ATP-binding cassette transporter 4), which transports all-trans-retinal from the inside to the outside of disc membranes (23), rapidly accumulate all-trans-retinal condensation products and exhibit accentuated RPE/photoreceptor dystrophy at an early age (24). Although these studies suggest retinoid toxicity, it is still unclear if the elevated levels of retinal and/or its condensation products, such as A2E, are the cause of this retinopathy or merely a nonspecific reflection of impaired retinoid metabolism. Here, we report that spent chromophore, all-trans-retinal, is most likely responsible for photoreceptor degeneration in Rdh8^-/-Abca4^-/- mice. Toxic effects of all-trans-retinal include caspase activation and mitochondria-associated cell death.     

Coupling of retinal, protein, and water dynamics in squid rhodopsin

The light-induced isomerization of the retinal from 11-cis to all-trans triggers changes in the conformation of visual rhodopsins that lead to the formation of the activated state, which is ready to interact with the G protein. To begin to understand how changes in the structure and dynamics of the retinal are transmitted to the protein, we performed molecular dynamics simulations of squid rhodopsin with 11-cis and all-trans retinal, and with two different force fields for describing the retinal molecule. The results indicate that structural rearrangements in the binding pocket, albeit small, propagate toward the cytoplasmic side of the protein, and affect the dynamics of internal water molecules. The sensitivity of the active-site interactions on the retinal force-field parameters highlights the coupling between the retinal molecule and its immediate protein environment.     

Mouse alcohol dehydrogenase 4: kinetic mechanism,substrate specificity and simulation of effects of ethanol on retinoid metabolism

Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37°C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD⁺ is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the K_m for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.     

The Action of 11-cis-Retinol on Cone Opsins and Intact Cone Photoreceptors

11-cis-Retinol has previously been shown in physiological experiments to promote dark adaptation and recovery of photoresponsiveness of bleached salamander red cones but not of bleached salamander red rods. The purpose of this study was to evaluate the direct interaction of 11-cis-retinol with expressed human and salamander cone opsins, and to determine by microspectrophotometry pigment formation in isolated salamander photoreceptors. We show here in a cell-free system using incorporation of radioactive guanosine 5′-3-O-(thio)triphosphate into transducin as an index of activity, that 11-cis-retinol inactivates expressed salamander cone opsins, acting an inverse agonist. Similar results were obtained with expressed human red and green opsins. 11-cis-Retinol had no significant effect on the activity of human blue cone opsin. In contrast, 11-cis-retinol activates the expressed salamander and human red rod opsins, acting as an agonist. Using microspectrophotometry of salamander cone photoreceptors before and after bleaching and following subsequent treatment with 11-cis-retinol, we show that 11-cis-retinol promotes pigment formation. Pigment was not formed in salamander red rods or green rods (containing the same opsin as blue cones) treated under the same conditions. These results demonstrate that 11-cis-retinol is not a useful substrate for rod photoreceptors although it is for cone photoreceptors. These data support the premise that rods and cones have mechanisms for handling retinoids and regenerating visual pigment that are specific to photoreceptor type. These mechanisms are critical to providing regenerated pigments in a time scale required for the function of these two types of photoreceptors.11-cis-Retinol is the precursor to 11-cis-retinal, the 11-cis-aldehyde form of vitamin A and the chromophore that combines covalently with rod and cone opsin proteins to form visual pigments. 11-cis-Retinal is consumed during visual signaling, and its continual synthesis is required. Photon absorption by the visual pigments causes the isomerization of its chromophore to the all-trans configuration. This initiates two processes critical for vision: activation of the photoreceptor cell and the eventual recovery of the original photosensitivity of the cells, requiring regeneration of the visual pigments. As cones are used for bright light vision, these two processes must work more rapidly in cones than in rods and thus cones have a higher requirement of 11-cis-retinoids as suggested by Rushton (1, 2).Photoreceptor activation begins with photoisomerization of the chromophore within the visual pigment. This results in a subsequent conformational change of the protein part of the visual pigment that is able to activate its G protein transducin, which in turn activates a PDE that lowers the concentration of cGMP and closes cGMP-gated ion channels. These steps comprise the visual signal transduction cascade (see Ref. 3 for review).The visual cycle involves regeneration of the visual pigment, which ultimately deactivates the protein and accomplishes the recovery of the photosensitivity of the photoreceptor cell. Classically, this process involves both the photoreceptor cell and the retinal pigment epithelium (RPE).⁴ After photoisomerization of the chromophore and formation of the active visual pigment, all-trans-retinal is released from the opsin and reduced to all-trans-retinol, which is then transported to the RPE where it is isomerized to 11-cis-retinol through a number of steps. In the RPE, 11-cis-retinol is oxidized to the aldehyde form, which is transported back to the photoreceptor cell and can be directly used by all of the opsins to regenerate an inactive pigment ready for photoactivation. The details of this model have been extensively reviewed (4, 5). Alternatively, recent work suggests that cones have an additional source of 11-cis-retinoids from Müller cells (6–8). Like the RPE cells, Müller cells have been shown to be able to convert all-trans-retinol to 11-cis-retinol (6). Unlike in the RPE cells, 11-cis-retinol is not oxidized to 11-cis-retinal in Müller cells.Jones et al. (9) demonstrated that administration of 11-cis-retinol to bleached salamander red cones could restore photosensitivity. A logical conclusion was that red cones were able to oxidize 11-cis-retinol to the aldehyde and regenerate visual pigments although noncovalent binding of 11-cis-retinol to red cone opsins generating a light-sensitive complex could not be excluded. On the other hand, 11-cis-retinol does not restore photosensitivity to bleached salamander rod cells but appears to directly activate the cells (9, 10). The data suggested that the rods were not able to oxidize 11-cis-retinol, but that the retinol itself could activate the signal transduction cascade, and indeed we recently demonstrated that 11-cis-retinol acts as an agonist to expressed bovine rod opsin (11). Our aim here was to study the action of 11-cis-retinol on cone opsins and cone photoreceptor cells to determine the efficacy of an alternate visual cycle for cones.The photoreceptor cells used in this study are from tiger salamander, and the expressed opsins used for biochemical experiments are those from salamander and human. Photoreceptor cells are generally identified by cell morphology and the type of opsin it contains that can be further complicated by the findings that some cone cells have multiple opsins (12, 13). Recently genetic analysis has determined that opsins fall into five classes (reviewed in Refs. 14 and 15). We have studied opsins falling into four of these classes and use common color-derived names for the opsins and photoreceptor cells. The classic rod cells used for scotopic vision contain rhodopsin, the visual pigment for the rod opsin (RH1 opsin) and appeared red and thus have been designated as red rods. Some species such as salamanders have an additional rod cell whose photosensitivity is blue-shifted from that of the red rod and thus designated as green rods. In the tiger salamander, the green rods contain the identical opsin (SWS2 opsin) found in blue cones (16). The human blue cones contain an opsin from a different class (SWS1 opsin), which is homologous to the salamander UV cone opsin. The human red and green and salamander red cone opsins all belong to the same class of opsins (M/LWS opsins). Absorption properties of visual pigments are further modulated in some animals including the tiger salamander by use of 11-cis-retinal with an additional double bond (3,4-dehydro or A2 11-cis-retinal) resulting in red-shifted absorbance from pigments containing 11-cis-retinal (A1 11-cis-retinal).We show here that 11-cis-retinol is not an agonist to cone opsins and does not itself generate a light-sensitive opsin. We further show using microspectrophotometry that both red and blue salamander cone cells regenerate visual pigments from 11-cis-retinol, whereas pigments could not be regenerated with 11-cis-retinol in bleached salamander red and green rods even though the latter contains the same opsin as the salamander blue cone. Thus, rods and cones have mechanisms for handling retinoids and regenerating visual pigment that are specific to photoreceptor type, and these mechanisms are critical to providing regenerated pigments in a time scale required for the function of these two types of photoreceptors.     

Binding affinities of CRBPI and CRBPII for 9-cis-retinoids

Background

Cellular retinol binding-protein I (CRBPI) and cellular retinol binding-protein II (CRBPII) serve as intracellular retinoid chaperones that bind retinol and retinal with high affinity and facilitate substrate delivery to select enzymes that catalyze retinoic acid (RA) and retinyl ester biosynthesis. Recently, 9-cis-RA has been identified in vivo in the pancreas, where it contributes to regulating glucose-stimulated insulin secretion. In vitro, 9-cis-RA activates RXR (retinoid × receptors), which serve as therapeutic targets for treating cancer and metabolic diseases. Binding affinities and structure–function relationships have been well characterized for CRBPI and CRBPII with all-trans-retinoids, but not for 9-cis-retinoids. This study extended current knowledge by establishing binding affinities for CRBPI and CRBPII with 9-cis-retinoids.

Methods

We have determined apparent dissociation constants, K′_d, through monitoring binding of 9-cis-retinol, 9-cis-retinal, and 9-cis-RA with CRBPI and CRBPII by fluorescence spectroscopy, and analyzing the data with non-linear regression. We compared these data to the data we obtained for all-trans- and 13-cis-retinoids under identical conditions.

Results

CRBPI and CRBPII, respectively, bind 9-cis-retinol (K′_d, 11 nM and 68 nM) and 9-cis-retinal (K′_d, 8 nM and 5 nM) with high affinity. No significant 9-cis-RA binding was observed with CRBPI or CRBPII.

Conclusions

CRBPI and CRBPII bind 9-cis-retinol and 9-cis-retinal with high affinities, albeit with affinities somewhat lower than for all-trans-retinol and all-trans-retinal.

General significance

These data provide further insight into structure–binding relationships of cellular retinol binding-proteins and are consistent with a model of 9-cis-RA biosynthesis that involves chaperoned delivery of 9-cis-retinoids to enzymes that recognize retinoid binding-proteins.     

Enzymatic reduction of 11-cis-retinal bound to cellular retinal-binding protein

Cellular retinal-binding protein from bovine retina purifies with bound 11-cis-retinal and 11-cis-retinol as endogenous ligands. Inasmuch as these retinoids are interconvertible by a dehydrogenase reaction the accessibility of the aldehyde function of bound 11-cis-retinal to chemical and enzymatic reducing agents was determined. An 11-cis-retinol dehydrogenase from retinal pigment epithelial microsomes, first described by Lion, F., Rotmans, J.P., Daemen, F.J.M. and Bonting, S.L. (Biochim. Biophys. Acta 384, 283–292, 1975) was found to reduce complexed 11-cis-retinal at pH 5.5 and 37°C rapidaly and nearly quantitatively. The product of the reduction, 11-cis-retinol, remained complexed with the binding protein following the reaction. Reduction proceeded 3-times more rapidly with NADH than with NADPH. No change in geometrical isomeric configuration occurred during the reaction. The dehydrogenase from retinal pigment epithelium oxidized 11-cis-retinol complexed with cellular retinal-binding protein at pH 8.5 in the presence of NAD. In spite of the ready enzymatic reduction of 11-cis-retinal complexed with cellular retinal-binding protein, the aldehyde function was inaccessible to several chemical reducing agents. Incubation of the complex with NaBH₄ at pH 7.5 and NaCNBH₃ or borane dimethylamine at pH 5.5 did not result in reduction of 11-cis-retinal unless the complex had been exposed to white light, a treatment known to produce all-rans-retinal which has little affinity for the binding protein. Liver alcohol dehydrogenase produced only 10% reduction of 11-cis-retinal complexed with cellular retinal-binding protein in 15 min at 37°C when added in amounts which produced about 60% reduction of the uncomplexed retinoid. The results suggest that the interaction between the 11-cis-retinol dehydrogenase and the 11-cis-retinal complexed to cellular retinal-binding protein is a specific one of that the binding protein may function as a substrate carrier for a dehydrogenase.     

The Visual Cycle in the Inner Retina of Chicken and the Involvement of Retinal G-Protein-Coupled Receptor (RGR)

The vertebrate retina contains typical photoreceptor (PR) cones and rods responsible for day/night vision, respectively, and intrinsically photosensitive retinal ganglion cells (ipRGCs) involved in the regulation of non-image-forming tasks. Rhodopsin/cone opsin photopigments in visual PRs or melanopsin (Opn4) in ipRGCs utilizes retinaldehyde as a chromophore. The retinoid regeneration process denominated as “visual cycle” involves the retinal pigment epithelium (RPE) or Müller glial cells. Opn4, on the contrary, has been characterized as a bi/tristable photopigment, in which a photon of one wavelength isomerizes 11-cis to all-trans retinal (Ral), with a second photon re-isomerizing it back. However, it is unknown how the chromophore is further metabolized in the inner retina. Nor is it yet clear whether an alternative secondary cycle occurs involving players such as the retinal G-protein-coupled receptor (RGR), a putative photoisomerase of unidentified inner retinal activity. Here, we investigated the role of RGR in retinoid photoisomerization in Opn4x (Xenopus ortholog) (+) RGC primary cultures free of RPE and other cells from chicken embryonic retinas. Opn4x (+) RGCs display significant photic responses by calcium fluorescent imaging and photoisomerize exogenous all-trans to 11-cis Ral and other retinoids. RGR was found to be expressed in developing retina and in primary cultures; when its expression was knocked down, the levels of 11-cis, all-trans Ral, and all-trans retinol in cultures exposed to light were significantly higher and those in all-trans retinyl esters lower than in dark controls. The results support a novel role for RGR in ipRGCs to modulate retinaldehyde levels in light, keeping the balance of inner retinal retinoid pools.     

Novel fluorescent GPCR biosensor detects retinal equilibrium binding to opsin and active G protein and arrestin signaling conformations

Rhodopsin is a canonical class A photosensitive G protein–coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new approach for both identifying compounds that can activate and attenuate rhodopsin and testing the hypothesis that opsin binds retinal in equilibrium. Our method uses opsin-based fluorescent sensors, which directly report the formation of active receptor conformations by detecting the binding of G protein or arrestin fragments that have been fused onto the receptor''s C terminus. We show that these biosensors can be used to monitor equilibrium binding of the agonist, ATR, as well as the noncovalent binding of β-ionone, an antagonist for G protein activation. Finally, we use these novel biosensors to observe ATR release from an activated, unlabeled receptor and its subsequent transfer to the sensor in real time. Taken together, these data support the retinal equilibrium binding hypothesis. The approach we describe should prove directly translatable to other GPCRs, providing a new tool for ligand discovery and mutant characterization.     

All-trans retinal constitutes the functional chromophore in Chlamydomonas rhodopsin

Orientation of the green alga Chlamydomonas in light (phototaxis and stop responses) is controlled by a visual system with a rhodopsin as the functional photoreceptor. Here, we present evidence that in Chlamydomonas wild-type cells all-trans retinal is the predominant isomer and that it is present in amounts similar to that of the rhodopsin itself.

The ability of different retinal isomers and analog compounds to restore photosensitivity in blind Chlamydomonas cells (strain CC2359) was tested by means of flash-induced light scattering transients or by measuring phototaxis in a taxigraph. All-trans retinal reconstitutes behavioral light responses within one minute, whereas cis-isomers require at least 50 × longer incubation times, suggesting that the retinal binding site is specific for all-trans retinal. Experiments with 13-demethyl(dm)-retinal and short-chained analogs reveal that only chromophores with a β-methyl group and at least three double bonds in conjugation with the aldehyde mediate function. Because neither 13-dm-retinal, nor 9,12-phenylretinal restores a functional rhodopsin, a trans/13-cis isomerisation seems to take place in the course of the activation mechanism. We conclude that with respect to its chromophore, Chlamydomonas rhodopsin bears a closer resemblence to bacterial rhodopsins than to visual rhodopsins of higher animals.

     

Retinal-Protein Interactions in Halorhodopsin from Natronomonas pharaonis: Binding and Retinal Thermal Isomerization Catalysis

Halorhodopsin from Natronomonas pharaonis (NpHR) is a member of the retinal protein group and serves as a light-driven chloride pump in which chloride ions are transported through the membrane following light absorption by the retinal chromophore. In this study, we examined two main issues: (1) factors controlling the binding of the retinal chromophore to the NpHR opsin and (2) the ability of the NpHR opsin to catalyze the thermal isomerization of retinal isomers. We have revealed that the reconstitution process of pharaonis HR (NpHR) pigment from its apoprotein and all-trans retinal depends on the pH, and the process has a pK_a of 5.8 ± 0.1. It was proposed that this pK_a is associated with the pK_a of the lysine residue that binds the retinal chromophore (Lys256). The pigment formation is regulated by the concentration of sodium chloride, and the maximum yield was observed at 3.7 M NaCl. The low yield of pigment in a lower concentration of NaCl (< 3 M) may be due to an altered conformation adopted by the apomembrane, which is not capable of forming the pigment. Unexpectedly and unlike the apomembrane of bacteriorhodopsin, NpHR opsin produces pigments with 11-cis retinal and 9-cis retinal owing to the thermal isomerization of these retinal isomers to all-trans retinal. The isomerization rate depends on the pH, and it is faster at a higher pH. The pK_a value of the isomerization process is similar to the pK_a of the binding process of these retinals, which suggests that Lys256 is also involved in the isomerization process. The isomerization is independent of the sodium chloride concentration. However, in the absence of sodium chloride, the apoprotein adopts such a conformation, which does not prevent the isomerization of retinal, but it prevents a covalent bond formation with the lysine residue. The rate and the thermodynamic parameter analysis of the retinal isomerization by NpHR apoprotein led to the conclusion that the apomembrane catalyzes the isomerization via a triplet mechanism.