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.     

Turning cones off: the role of the 9-methyl group of retinal in red cones

Our ability to see in bright light depends critically on the rapid rate at which cone photoreceptors detect and adapt to changes in illumination. This is achieved, in part, by their rapid response termination. In this study, we investigate the hypothesis that this rapid termination of the response in red cones is dependent on interactions between the 9-methyl group of retinal and red cone opsin, which are required for timely metarhodopsin (Meta) II decay. We used single-cell electrical recordings of flash responses to assess the kinetics of response termination and to calculate guanylyl cyclase (GC) rates in salamander red cones containing native visual pigment as well as visual pigment regenerated with 11-cis 9-demethyl retinal, an analogue of retinal in which the 9-methyl group is missing. After exposure to bright light that photoactivated more than approximately 0.2% of the pigment, red cones containing the analogue pigment had a slower recovery of both flash response amplitudes and GC rates (up to 10 times slower at high bleaches) than red cones containing 11-cis retinal. This finding is consistent with previously published biochemical data demonstrating that red cone opsin regenerated in vitro with 11-cis 9-demethyl retinal exhibited prolonged activation as a result of slowed Meta II decay. Our results suggest that two different mechanisms regulate the recovery of responsiveness in red cones after exposure to light. We propose a model in which the response recovery in red cones can be regulated (particularly at high light intensities) by the Meta II decay rate if that rate has been inhibited. In red cones, the interaction of the 9-methyl group of retinal with opsin promotes efficient Meta II decay and, thus, the rapid rate of recovery.     

Effect of 11-cis 13-demethylretinal on phototransduction in bleach-adapted rod and cone photoreceptors

We used 11-cis 13-demethylretinal to examine the physiological consequences of retinal's noncovalent interaction with opsin in intact rod and cone photoreceptors during visual pigment regeneration. 11-Cis 13-demethylretinal is an analog of 11-cis retinal in which the 13 position methyl group has been removed. Biochemical experiments have shown that it is capable of binding in the chromophore pocket of opsin, forming a Schiff-base linkage with the protein to produce a pigment, but at a much slower rate than the native 11-cis retinal (Nelson, R., J. Kim deReil, and A. Kropf. 1970. Proc. Nat. Acad. Sci. USA. 66:531-538). Experimentally, this slow rate of pigment formation should allow separate physiological examination of the effects of the initial binding of retinal in the pocket and the subsequent formation of the protonated Schiff-base linkage. Currents from solitary rods and cones from the tiger salamander were recorded in darkness before and after bleaching and then after exposure to 11-cis 13-demethylretinal. In bleach-adapted rods, 11-cis 13-demethylretinal caused transient activation of phototransduction, as evidenced by a decrease of the dark current and sensitivity, acceleration of the dim flash responses, and activation of cGMP phosphodiesterase and guanylyl cyclase. The steady state of phototransduction activity was still higher than that of the bleach-adapted rod. In contrast, exposure of bleach-adapted cones to 11-cis 13-demethylretinal resulted in an immediate deactivation of transduction as measured by the same parameters. These results extend the validity of a model for the effects of the noncovalent binding of a retinoid in the chromophore pockets of rod and cone opsins to analogs capable of forming a Schiff-base and imply that the noncovalent binding by itself may play a role for the dark adaptation of photoreceptors.     

Occupancy of the chromophore binding site of opsin activates visual transduction in rod photoreceptors

The retinal analogue beta-ionone was used to investigate possible physiological effects of the noncovalent interaction between rod opsin and its chromophore 11-cis retinal. Isolated salamander rod photoreceptors were exposed to bright light that bleached a significant fraction of their pigment, were allowed to recover to a steady state, and then were exposed to beta-ionone. Our experiments show that in bleach-adapted rods beta-ionone causes a decrease in light sensitivity and dark current and an acceleration of the dim flash photoresponse and the rate constants of guanylyl cyclase and cGMP phosphodiesterase. Together, these observations indicate that in bleach-adapted rods beta-ionone activates phototransduction in the dark. Control experiments showed no effect of beta-ionone in either fully dark-adapted or background light-adapted cells, indicating direct interaction of beta-ionone with the free opsin produced by bleaching. We speculate that beta-ionone binds specifically in the chromophore pocket of opsin to produce a complex that is more catalytically potent than free opsin alone. We hypothesize that a similar reaction may occur in the intact retina during pigment regeneration. We propose a model of rod pigment regeneration in which binding of 11-cis retinal to opsin leads to activation of the complex accompanied by a decrease in light sensitivity. The subsequent covalent attachment of retinal to opsin completely inactivates opsin and leads to the recovery of sensitivity. Our findings resolve the conflict between biochemical and physiological data concerning the effect of the occupancy of the chromophore binding site on the catalytic potency of opsin. We show that binding of beta-ionone to rod opsin produces effects opposite to its previously described effects on cone opsin. We propose that this distinction is due to a fundamental difference in the interaction of rod and cone opsins with retinal, which may have implications for the different physiology of the two types of photoreceptors.     

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.     

Low aqueous solubility of 11-cis-retinal limits the rate of pigment formation and dark adaptation in salamander rods

We report experiments designed to test the hypothesis that the aqueous solubility of 11-cis-retinoids plays a significant role in the rate of visual pigment regeneration. Therefore, we have compared the aqueous solubility and the partition coefficients in photoreceptor membranes of native 11-cis-retinal and an analogue retinoid, 11-cis 4-OH retinal, which has a significantly higher solubility in aqueous medium. We have then correlated these parameters with the rates of pigment regeneration and sensitivity recovery that are observed when bleached intact salamander rod photoreceptors are treated with physiological solutions containing these retinoids. We report the following results: (a) 11-cis 4-OH retinal is more soluble in aqueous buffer than 11-cis-retinal. (b) Both 11-cis-retinal and 11-cis 4-OH retinal have extremely high partition coefficients in photoreceptor membranes, though the partition coefficient of 11-cis-retinal is roughly 50-fold greater than that of 11-cis 4-OH retinal. (c) Intact bleached isolated rods treated with solutions containing equimolar amounts of 11-cis-retinal or 11-cis 4-OH retinal form functional visual pigments that promote full recovery of dark current, sensitivity, and response kinetics. However, rods treated with 11-cis 4-OH retinal regenerated on average fivefold faster than rods treated with 11-cis-retinal. (d) Pigment regeneration from recombinant and wild-type opsin in solution is slower when treated with 11-cis 4-OH retinal than with 11-cis-retinal. Based on these observations, we propose a model in which aqueous solubility of cis-retinoids within the photoreceptor cytosol can place a limit on the rate of visual pigment regeneration in vertebrate photoreceptors. We conclude that the cytosolic gap between the plasma membrane and the disk membranes presents a bottleneck for retinoid flux that results in slowed pigment regeneration and dark adaptation in rod photoreceptors.     

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.     

The 9-methyl group of retinal is essential for rapid Meta II decay and phototransduction quenching in red cones

Cone photoreceptors of the vertebrate retina terminate their response to light much faster than rod photoreceptors. However, the molecular mechanisms underlying this rapid response termination in cones are poorly understood. The experiments presented here tested two related hypotheses: first, that the rapid decay rate of metarhodopsin (Meta) II in red-sensitive cones depends on interactions between the 9-methyl group of retinal and the opsin part of the pigment molecule, and second, that rapid Meta II decay is critical for rapid recovery from saturation of red-sensitive cones after exposure to bright light. Microspectrophotometric measurements of pigment photolysis, microfluorometric measurements of retinol production, and single-cell electrophysiological recordings of flash responses of salamander cones were performed to test these hypotheses. In all cases, cones were bleached and their visual pigment was regenerated with either 11-cis retinal or with 11-cis 9-demethyl retinal, an analogue of retinal lacking the 9-methyl group. Meta II decay was four to five times slower and subsequent retinol production was three to four times slower in red-sensitive cones lacking the 9-methyl group of retinal. This was accompanied by a significant slowing of the recovery from saturation in cones lacking the 9-methyl group after exposure to bright (>0.1% visual pigment photoactivated) but not dim light. A mathematical model of the turn-off process of phototransduction revealed that the slower recovery of photoresponse can be explained by slower Meta decay of 9-demethyl visual pigment. These results demonstrate that the 9-methyl group of retinal is required for steric chromophore–opsin interactions that favor both the rapid decay of Meta II and the rapid response recovery after exposure to bright light in red-sensitive cones.     

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.     

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.     

Cone visual pigments in two marsupial species: the fat-tailed dunnart (Sminthopsis crassicaudata) and the honey possum (Tarsipes rostratus)

Uniquely for non-primate mammals, three classes of cone photoreceptors have been previously identified by microspectrophotometry in two marsupial species: the polyprotodont fat-tailed dunnart (Sminthopsis crassicaudata) and the diprotodont honey possum (Tarsipes rostratus). This report focuses on the genetic basis for these three pigments. Two cone pigments were amplified from retinal cDNA of both species and identified by phylogenetics as members of the short wavelength-sensitive 1 (SWS1) and long wavelength-sensitive (LWS) opsin classes. In vitro expression of the two sequences from the fat-tailed dunnart confirmed the peak absorbances at 363 nm in the UV for the SWS1 pigment and 533 nm for the LWS pigment. No additional expressed cone opsin sequences that could account for the middle wavelength cones could be amplified. However, amplification from the fat-tailed dunnart genomic DNA with RH1 (rod) opsin primer pairs identified two genes with identical coding regions but sequence differences in introns 2 and 3. Uniquely therefore for a mammal, the fat-tailed dunnart has two copies of an RH1 opsin gene. This raises the possibility that the middle wavelength cones express a rod rather than a cone 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.     

Bleached pigment activates transduction in salamander cones

We have used suction electrode recording together with rapid steps into 0.5 mM IBMX solution to investigate changes in guanylyl cyclase velocity produced by pigment bleaching in isolated cones of the salamander Ambystoma tigrinum. Both backgrounds and bleaches accelerate the time course of current increase during steps into IBMX. We interpret this as evidence that the velocity of the guanylyl cyclase is increased in background light or after bleaching. Our results indicate that cyclase velocity increases nearly linearly with increasing percent pigment bleached but nonlinearly (and may saturate) with increasing back-ground intensity. In cones (as previously demonstrated for rods), light-activated pigment and bleached pigment appear to have somewhat different effects on the transduction cascade. The effect of bleaching on cyclase rate is maintained for at least 15-20 min after the light is removed, much longer than is required after a bleach for circulating current and sensitivity to stabilize in an isolated cone. The effect on the cyclase rate can be completely reversed by treatment with liposomes containing 11-cis retinal. The effects of bleaching can also be partially reversed by beta-ionone, an analogue of the chromophore 11- cis-retinal which does not form a covalent attachment to opsin. Perfusion of a bleached cone with beta-ionone produces a rapid increase in circulating current and sensitivity, which rapidly reverses when the beta-ionone is removed. Perfusion with beta-ionone also causes a partial reversal of the bleach-induced acceleration of cyclase velocity. We conclude that bleaching produces an "equivalent background" excitation of the transduction cascade in cones, perhaps by a mechanism similar to that in rods.     

Comparative study on the chromophore binding sites of rod and red-sensitive cone visual pigments by use of synthetic retinal isomers and analogues

A comparative study on the chromophore (retinal) binding sites of the opsin (R-photopsin) from chicken red-sensitive cone visual pigment (iodopsin) and that scotopsin) from bovine rod pigment (rhodopsin) was made by the aid of geometric isomers of retinal (all-trans, 13-cis, 11-cis, 9-cis, and 7-cis) and retinal analogues including fluorinated (14-F, 12-F, 10-F, and 8-F) and methylated (12-methyl) 11-cis-retinals. The stereoselectivity of R-photopsin for the retinal isomers and analogues was almost identical with that of scotopsin, indicating that the shapes of the chromophore binding sites of both opsins are similar, although the former appears to be somewhat more restricted than the latter. The rates of pigment formation from R-photopsin were considerably greater than those from scotopsin. In addition, all the iodopsin isomers and analogues were more susceptible to hydroxylamine than were the rhodopsin ones. These observations suggest that the retinal binding site of iodopsin is located near the protein surface. On the basis of the spectral properties of fluorinated analogues, a polar group in the chromophore binding site of iodopsin as well as rhodopsin was estimated to be located near the hydrogen atom at the C10 position of the retinylidene chromophore. A large difference in wavelength between the absorption maxima of iodopsin and rhodopsin was significantly reduced in the 9-cis and 7-cis pigments. On the assumption that the retinylidene chromophore is anchored rigidly at the alpha-carbon of the lysine residue and loosely at the cyclohexenyl ring, each of the two isomers would have the Schiff-base nitrogen at a position altered from that of the 11-cis pigments.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of retinal photoisomerase in the visual cycle of the honeybee

The compound eye of the honeybee has previously been shown to contain a soluble retinal photoisomerase which, in vitro, is able to catalyze stereospecifically the photoconversion of all-trans retinal to 11-cis retinal. In this study we combine in vivo and in vitro techniques to demonstrate how the retinal photoisomerase is involved in the visual cycle, creating 11-cis retinal for the generation of visual pigment. Honeybees have approximately 2.5 pmol/eye of retinal associated with visual pigments, but larger amounts (4-12 pmol/eye) of both retinal and retinol bound to soluble proteins. When bees are dark adapted for 24 h or longer, greater than 80% of the endogenous retinal, mostly in the all-trans configuration, is associated with the retinal photoisomerase. On exposure to blue light the retinal is isomerized to 11-cis, which makes it available to an alcohol dehydrogenase. Most of it is then reduced to 11-cis retinol. The retinol is not esterified and remains associated with a soluble protein, serving as a reservoir of 11-cis retinoid available for renewal of visual pigment. Alternatively, 11-cis retinal can be transferred directly to opsin to regenerate rhodopsin, as shown by synthesis of rhodopsin in bleached frog rod outer segments. This retinaldehyde cycle from the honeybee is the third to be described. It appears very similar to the system in another group of arthropods, flies, and differs from the isomerization processes in vertebrates and cephalopod mollusks.     

An alternative isomerohydrolase in the retinal Müller cells of a cone-dominant species

Cone photoreceptors have faster light responses than rods and a higher demand for 11-cis retinal (11cRAL), the chromophore of visual pigments. RPE65 is the isomerohydrolase in the retinal pigment epithelium (RPE) that converts all-trans retinyl ester to 11-cis retinol, a key step in the visual cycle for regenerating 11cRAL. Accumulating evidence suggests that cone-dominant species express an alternative isomerase, likely in retinal Müller cells, to meet the high demand for the chromophore by cones. In the present study, we describe the identification and characterization of a novel isomerohydrolase, RPE65c, from the cone-dominant zebrafish retina. RPE65c shares 78% amino acid sequence identity with RPE-specific zebrafish RPE65a (orthologue of human RPE65) and retains all of the known key residues for the enzymatic activity of RPE65. Similar to the other RPE-specific RPE65, RPE65c was present in both the membrane and cytosolic fractions, used all-trans retinyl ester as its substrate and required iron for its enzymatic activity. However, immunohistochemistry detected RPE65c in the inner retina, including Müller cells, but not in the RPE. Furthermore, double-immunostaining of dissociated retinal cells using antibodies for RPE65c and glutamine synthetase (a Müller cell marker), showed that RPE65c co-localized with the Müller cell marker. These results suggest that RPE65c is the alternative isomerohydrolase in the intra-retinal visual cycle, providing 11cRAL to cone photoreceptors in cone-dominant species. Identification of an alternative visual cycle will contribute to the understanding of the functional differences of rod and cone photoreceptors.     

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.     

M opsin phosphorylation in intact mammalian retinas

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

Role of noncovalent binding of 11-cis-retinal to opsin in dark adaptation of rod and cone photoreceptors

Regeneration of visual pigments of vertebrate rod and cone photoreceptors occurs by the initial noncovalent binding of 11-cis-retinal to opsin, followed by the formation of a covalent bond between the ligand and the protein. Here, we show that the noncovalent interaction between 11-cis-retinal and opsin affects the rate of dark adaptation. In rods, 11-cis-retinal produces a transient activation of the phototransduction cascade that precedes sensitivity recovery, thus slowing dark adaptation. In cones, 11-cis-retinal immediately deactivates phototransduction. Thus, the initial binding of the same ligand to two very similar G protein receptors, the rod and cone opsins, activates one and deactivates the other, contributing to the remarkable difference in the rates of rod and cone dark adaptation.