Retigabine increases the conformational stability of the visual photoreceptor rhodopsin.

Rhodopsin is the key photoreceptor protein that mediates vision in low-light conditions. Mutations in rhodopsin are the cause of retinal degenerative diseases such as retinitis pigmentosa. Some of these mutations cause a decreased stability of the receptor. It is, therefore, of interest to find new approaches that can help improving rhodopsin conformational stability. In this study, we have analyzed the effect of retigabine, an anticonvulsant formerly used to treat epilepsy, on rhodopsin thermal stability, regeneration capacity, and signal transduction by means of UV-visible and fluorescence spectroscopic techniques. We find that retigabine enhances the thermal stability of dark-state rhodopsin and improves chromophore regeneration without disrupting the photobleaching process. Furthermore, retigabine does not significantly affect transducin activation. These results provide novel insights into the potential therapeutic applications of retigabine in the treatment of retinitis pigmentosa caused by rhodopsin mutations that cause a decreased stability of the mutated receptors.

Effect of Trace Metal Ions on the Conformational Stability of the Visual Photoreceptor Rhodopsin.

Trace metals are essential elements that play key roles in a number of biochemical processes governing human visual physiology in health and disease. Several trace metals, such as zinc, have been shown to play important roles in the visual phototransduction process. In spite of this, there has been little research conducted on the direct effect of trace metal elements on the visual photoreceptor rhodopsin. In the current study, we have determined the effect of several metal ions, such as iron, copper, chromium, manganese, and nickel, on the conformational stability of rhodopsin. To this aim, we analyzed, by means of UV-visible and fluorescence spectroscopic methods, the effects of these trace elements on the thermal stability of dark rhodopsin, the stability of its active Metarhodopsin II conformation, and its chromophore regeneration. Our results show that copper prevented rhodopsin regeneration and slowed down the retinal release process after illumination. In turn, Fe3+, but not Fe2+, increased the thermal stability of the dark inactive conformation of rhodopsin, whereas copper ions markedly decreased it. These findings stress the important role of trace metals in retinal physiology at the photoreceptor level and may be useful for the development of novel therapeutic strategies to treat retinal disease.

New insights into the molecular mechanism of rhodopsin retinitis pigmentosa from the biochemical and functional characterization of G90V, Y102H and I307N mutations.

Mutations in the photoreceptor protein rhodopsin are known as one of the leading causes of retinal degeneration in humans. Two rhodopsin mutations, Y102H and I307N, obtained in chemically mutagenized mice, are currently the subject of increased interest as relevant models for studying the process of retinal degeneration in humans. Here, we report on the biochemical and functional characterization of the structural and functional alterations of these two rhodopsin mutants and we compare them with the G90V mutant previously analyzed, as a basis for a better understanding of in vivo studies. This mechanistic knowledge is fundamental to use it for developing novel therapeutic approaches for the treatment of inherited retinal degeneration in retinitis pigmentosa. We find that Y102H and I307N mutations affect the inactive-active equilibrium of the receptor. In this regard, the mutations reduce the stability of the inactive conformation but increase the stability of the active conformation. Furthermore, the initial rate of the functional activation of transducin, by the I307N mutant is reduced, but its kinetic profile shows an unusual increase with time suggesting a profound effect on the signal transduction process. This latter effect can be associated with a change in the flexibility of helix 7 and an indirect effect of the mutation on helix 8 and the C-terminal tail of rhodopsin, whose potential role in the functional activation of the receptor has been usually underestimated. In the case of the Y102H mutant, the observed changes can be associated with conformational alterations affecting the folding of the rhodopsin intradiscal domain, and its presumed involvement in the retinal binding process by the receptor.

Polyphenols and Visual Health: Potential Effects on Degenerative Retinal Diseases.

Dietary polyphenols are a group of natural compounds that have been proposed to have beneficial effects on human health. They were first known for their antioxidant properties, but several studies over the years have shown that these compounds can exert protective effects against chronic diseases. Nonetheless, the mechanisms underlying these potential benefits are still uncertain and contradictory effects have been reported. In this review, we analyze the potential effects of polyphenol compounds on some visual diseases, with a special focus on retinal degenerative diseases. Current effective therapies for the treatment of such retinal diseases are lacking and new strategies need to be developed. For this reason, there is currently a renewed interest in finding novel ligands (or known ligands with previously unexpected features) that could bind to retinal photoreceptors and modulate their molecular properties. Some polyphenols, especially flavonoids (e.g., quercetin and tannic acid), could attenuate light-induced receptor damage and promote visual health benefits. Recent evidence suggests that certain flavonoids could help stabilize the correctly folded conformation of the visual photoreceptor protein rhodopsin and offset the deleterious effect of retinitis pigmentosa mutations. In this regard, certain polyphenols, like the flavonoids mentioned before, have been shown to improve the stability, expression, regeneration and folding of rhodopsin mutants in experimental in vitro studies. Moreover, these compounds appear to improve the integration of the receptor into the cell membrane while acting against oxidative stress at the same time. We anticipate that polyphenol compounds can be used to target visual photoreceptor proteins, such as rhodopsin, in a way that has only been recently proposed and that these can be used in novel approaches for the treatment of retinal degenerative diseases like retinitis pigmentosa; however, studies in this field are limited and further research is needed in order to properly characterize the effects of these compounds on retinal degenerative diseases through the proposed mechanisms.

Effect of Sodium Valproate on the Conformational Stability of the Visual G Protein-Coupled Receptor Rhodopsin.

Rhodopsin is the G protein-coupled receptor of rod photoreceptor cells that mediates vertebrate vision at low light intensities. Mutations in rhodopsin cause inherited retinal degenerative diseases such as retinitis pigmentosa. Several therapeutic strategies have attempted to address and counteract the deleterious effect of rhodopsin mutations on the conformation and function of this photoreceptor protein, but none has been successful in efficiently preventing retinal degeneration in humans. These approaches include, among others, the use of small molecules, known as pharmacological chaperones, that bind to the receptor stabilizing its proper folded conformation. Valproic acid, in its sodium valproate form, has been used as an anticonvulsant in epileptic patients and in the treatment of several psychiatric disorders. More recently, this compound has been tested as a potential therapeutic agent for the treatment of retinal degeneration associated with retinitis pigmentosa caused by rhodopsin mutations. We now report on the effect of sodium valproate on the conformational stability of heterologously expressed wild-type rhodopsin and a rhodopsin mutant, I307N, which has been shown to be an appropriate model for studying retinal degeneration in mice. We found no sign of enhanced stability for the dark inactive conformation of the I307N mutant. Furthermore, the photoactivated conformation of the mutant appears to be destabilized by sodium valproate as indicated by a faster decay of its active conformation. Therefore, our results support a destabilizing effect of sodium valproate on rhodopsin I307N mutant associated with retinal degeneration. These findings, at the molecular level, agree with recent clinical studies reporting negative effects of sodium valproate on the visual function of retinitis pigmentosa patients.

Ligand Binding Mechanisms in Human Cone Visual Pigments.

Vertebrate vision starts with light absorption by visual pigments in rod and cone photoreceptor cells of the retina. Rhodopsin, in rod cells, responds to dim light, whereas three types of cone opsins (red, green, and blue) function under bright light and mediate color vision. Cone opsins regenerate with retinal much faster than rhodopsin, but the molecular mechanism of regeneration is still unclear. Recent advances in the area pinpoint transient intermediate opsin conformations, and a possible secondary retinal-binding site, as determinant factors for regeneration. In this Review, we compile previous and recent findings to discuss possible mechanisms of ligand entry in cone opsins, involving a secondary binding site, which may have relevant functional and evolutionary implications.

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.

Flavonoid allosteric modulation of mutated visual rhodopsin associated with retinitis pigmentosa.

Dietary flavonoids exhibit many biologically-relevant functions and can potentially have beneficial effects in the treatment of pathological conditions. In spite of its well known antioxidant properties, scarce structural information is available on the interaction of flavonoids with membrane receptors. Advances in the structural biology of a specific class of membrane receptors, the G protein-coupled receptors, have significantly increased our understanding of drug action and paved the way for developing improved therapeutic approaches. We have analyzed the effect of the flavonoid quercetin on the conformation, stability and function of the G protein-coupled receptor rhodopsin, and the G90V mutant associated with the retinal degenerative disease retinitis pigmentosa. By using a combination of experimental and computational methods, we suggest that quercetin can act as an allosteric modulator of opsin regenerated with 9-cis-retinal and more importantly, that this binding has a positive effect on the stability and conformational properties of the G90V mutant associated with retinitis pigmentosa. These results open new possibilities to use quercetin and other flavonoids, in combination with specific retinoids like 9-cis-retinal, for the treatment of retinal degeneration associated with retinitis pigmentosa. Moreover, the use of flavonoids as allosteric modulators may also be applicable to other members of the G protein-coupled receptors superfamily.

Structural and functional alterations associated with deutan N94K and R330Q mutations of green cone opsin.

Deuteranopia is an X-linked congenital dichromatic condition in which single point mutations in green cone opsin lead to defective non-functional cone photoreceptor cells. Green cone opsin belongs to the G protein-coupled receptor superfamily and consists of a seven transmembrane helical apoprotein covalently bound to 11-cis-retinal, by means of a protonated Schiff base linkage, in its inactive dark state. Several point mutations in green cone opsin have been reported to cause deuteranopia, but the structural details underlying the molecular mechanisms behind the malfunction of mutated opsins have not been clearly established. Here, deutan N94K and R330Q mutants were studied by introducing these substitutions into the native green cone opsin gene by site-directed mutagenesis. The mutant proteins were purified and analyzed using UV-vis spectroscopy and transducin activation assay. We find that the N94K mutant binds the retinal chromophore by means of an unprotonated Schiff base linkage in contrast to previous studies that reported no chromophore regeneration. The other mutant studied, R330Q, showed impaired functionality as measured by its reduced transducin activation ability when compared to wild-type green cone opsin. A double Cys mutant that could form a stabilizing disulfide bond was used in an attempt to address the instability of the green opsin mutants. Our results suggest the presence of key intramolecular networks which may be disrupted in deuteranopia, and these findings could help in finding therapeutic solutions for treating color blindness. Furthermore, our results can also have implications for the study of other visual pigments and other rhodopsin-like G protein-coupled receptors.

Docosahexaenoic acid phospholipid differentially modulates the conformation of G90V and N55K rhodopsin mutants associated with retinitis pigmentosa.

Rhodopsin is the visual photoreceptor of the retinal rod cells that mediates dim light vision and a prototypical member of the G protein-coupled receptor superfamily. The structural stability and functional performance of rhodopsin are modulated by membrane lipids. Docosahexaenoic acid has been shown to interact with native rhodopsin but no direct evidence has been established on the effect of such lipid on the stability and regeneration of rhodopsin mutants associated with retinal diseases. The stability and regeneration of two thermosensitive mutants G90V and N55K, associated with the retinal degenerative disease retinitis pigmentosa, have been analyzed in docosohexaenoic phospholipid (1,2-didocosa-hexaenoyl-sn-glycero-3-phosphocholine; DDHA-PC) liposomes. G90V mutant reconstituted in DDHA-PC liposomes significantly increased its thermal stability, but N55K mutant showed similar thermal sensitivity both in dodecyl maltoside detergent solution and in DDHA-PC liposomes. The retinal release process, measured by fluorescence spectroscopy, became faster in the lipid system for the two mutants. The opsin conformation was stabilized for the G90V mutant allowing improved retinal uptake whereas no chromophore binding could be detected for N55K opsin after photoactivation. The results emphasize the distinct role of DHA on different phenotypic rhodopsin mutations associated with classical (G90V) and sector (N55K) retinitis pigmentosa.

Functional role of positively selected amino acid substitutions in mammalian rhodopsin evolution.

Visual rhodopsins are membrane proteins that function as light photoreceptors in the vertebrate retina. Specific amino acids have been positively selected in visual pigments during mammal evolution, which, as products of adaptive selection, would be at the base of important functional innovations. We have analyzed the top candidates for positive selection at the specific amino acids and the corresponding reverse changes (F13M, Q225R and A346S) in order to unravel the structural and functional consequences of these important sites in rhodopsin evolution. We have constructed, expressed and immunopurified the corresponding mutated pigments and analyzed their molecular phenotypes. We find that position 13 is very important for the folding of the receptor and also for proper protein glycosylation. Position 225 appears to be important for the function of the protein affecting the G-protein activation process, and position 346 would also regulate functionality of the receptor by enhancing G-protein activation and presumably affecting protein phosphorylation by rhodopsin kinase. Our results represent a link between the evolutionary analysis, which pinpoints the specific amino acid positions in the adaptive process, and the structural and functional analysis, closer to the phenotype, making biochemical sense of specific selected genetic sequences in rhodopsin evolution.

Beyond spectral tuning: human cone visual pigments adopt different transient conformations for chromophore regeneration.

Human red and green visual pigments are seven transmembrane receptors of cone photoreceptor cells of the retina that mediate color vision. These pigments share a very high degree of homology and have been assumed to feature analogous structural and functional properties. We report on a different regeneration mechanism among red and green cone opsins with retinal analogs using UV-Vis/fluorescence spectroscopic analyses, molecular modeling and site-directed mutagenesis. We find that photoactivated green cone opsin adopts a transient conformation which regenerates via an unprotonated Schiff base linkage with its natural chromophore, whereas red cone opsin forms a typical protonated Schiff base. The chromophore regeneration kinetics is consistent with a secondary retinal uptake by the cone pigments. Overall, our findings reveal, for the first time, structural differences in the photoactivated conformation between red and green cone pigments that may be linked to their molecular evolution, and support the proposal of secondary retinal binding to visual pigments, in addition to binding to the canonical primary site, which may serve as a regulatory mechanism of dark adaptation in the phototransduction process.

Zinc Is Involved in Depression by Modulating G Protein-Coupled Receptor Heterodimerization.

5-Hydroxytryptamine 1A receptor and galanin receptor 1 belong to the G protein-coupled receptors superfamily, and they have been described to heterodimerize triggering an anomalous physiological state that would underlie depression. Zinc supplementation has been widely reported to improve treatment against major depressive disorder. Our work has focused on the study and characterization of these receptors and its relationships with zinc both under purified conditions and in cell culture. To this aim, we have designed a strategy to purify the receptors in a conformationally active state. We have used receptors tagged with the monoclonal Rho-1D4 antibody and employed ligand-assisted purification in order to successfully purify both receptors in a properly folded and active state. The interaction between both purified receptors has been analyzed by surface plasmon resonance in order to determine the kinetics of dimerization. Zinc effect on heteromer has also been tested using the same methodology but exposing the 5-hydroxytryptamine 1A receptor to zinc before the binding experiment. These results, combined with Förster resonance energy transfer (FRET) measurements, in the absence and presence of zinc, suggest that this ion is capable of disrupting this interaction. Moreover, molecular modeling suggests that there is a coincidence between zinc-binding sites and heterodimerization interfaces for the serotonin receptor. Our results establish a rational explanation for the role of zinc in the molecular processes associated with receptor-receptor interactions and its relationship with depression, in agreement with previously reported evidence for the positive effects of zinc in depression treatment, and the involvement of our target dimer in the same disease.

Optogenetics Comes of Age: Novel Inhibitory Light-Gated Anionic Channels Allow Efficient Silencing of Neural Function.

Optogenetics, the developing field of research that uses light-switchable biochemical tools in a sophisticated technological approach to monitor or control neural function, is rapidly evolving with the discovery and development of novel microbial rhodopsins. Light-absorbing membrane proteins, as tools for brain research, are promoting new applications within the discipline of optogenetics. Light-gated rhodopsin ion channels with better intrinsic light sensitivity and improved resolution are needed to overcome some of the current limitations of existing molecules. The recent discovery of light-gated inhibitory anion channels opens new opportunities for studying physiological neural processes and, at the same time, represent a powerful approach for elucidating the mechanisms of neurological and mental disorders that could benefit from this approach.

The zinc binding receptor GPR39 interacts with 5-HT1A and GalR1 to form dynamic heteroreceptor complexes with signaling diversity.

GPR39 is a class A G protein-coupled receptor involved in zinc binding and glucose homeostasis regulation, among other physiological processes. GPR39 was originally thought to be the receptor for obestatin peptide but this view has been challenged. However, activation of this receptor by zinc has been clearly established. Recent studies suggest that low GPR39 expression, due to deficient zinc levels, is involved in major depressive disorder. We have previously reported that zinc can alter receptor-receptor interactions and favor specific receptor interactions. In order to unravel the effect of zinc on specific G protein-coupled receptor association processes, we have performed FRET and co-immunopurification studies with GPR39 and 5-HT1A and GalR1 which have been shown to dimerize. Our results suggest that zinc can modulate the formation of specific 5-HT1A-GPR39 and GalR1-5-HT1A-GPR39 heteroreceptor complexes under our experimental conditions. We have analyzed the differences in signaling between the mono-homomeric receptors 5-HT1A, GalR1 and GPR39 and the heteroreceptor complexes between them Our results show that the GPR39-5-HT1A heterocomplex has additive functionalities when compared to the monomeric-homomeric receptors upon receptor activation. In addition, the heterocomplex including also GalR1 shows a different behavior, upon exposure to the same agonists. Furthermore, these processes appear to be regulated by zinc. These findings provide a rationale for the antidepressive effect widely described for zinc because pro-depressive heterocomplexes are predominant at low zinc concentration levels.

Phospholipid Bicelles Improve the Conformational Stability of Rhodopsin Mutants Associated with Retinitis Pigmentosa.

Mutations in the visual photoreceptor rhodopsin are the cause of the retinal degenerative disease retinitis pigmentosa. Some naturally occurring mutations can lead to protein conformational instability. Two such mutations, N55K and G90V, in the first and second transmembrane helices of the receptor, have been associated with sector and classical retinitis pigmentosa, respectively, and showed enhanced thermal sensitivity. We have carefully analyzed the effect of phospholipid bicelles on the stability and ligand binding properties of these two mutants and compared it with those of the detergent-solubilized samples. We have used a phospholipid bilayer consisting of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). We find that DMPC/DHPC bicelles dramatically increase the thermal stability of the rhodopsin mutants G90V and N55K. The chromophore stability and regeneration of the mutants were also increased in bicelles when compared to their behavior in a dodecyl maltoside detergent solution. The retinal release process was slowed in bicelles, and chromophore entry, after illumination, was improved for the G90V mutant but not for N55K. Furthermore, fluorescence spectroscopy measurements showed that bicelles allowed more exogenous retinal binding to the photoactivated G90V mutant than in a detergent solution. In contrast, N55K could not reposition any chromophore either in the detergent or in bicelles. The results demonstrate that DMPC/DHPC bicelles can counteract the destabilizing effect of the disease-causing mutations and can modulate the structural changes in the ensuing receptor photoactivation in a distinct specific manner for different retinitis pigmentosa mutant phenotypes.

Differential light-induced responses in sectorial inherited retinal degeneration.

Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous inherited degenerative retinopathies caused by abnormalities of photoreceptors or retinal pigment epithelium in the retina leading to progressive sight loss. Rhodopsin is the prototypical G-protein-coupled receptor located in the vertebrate retina and is responsible for dim light vision. Here, novel M39R and N55K variants were identified as causing an intriguing sector phenotype of RP in affected patients, with selective degeneration in the inferior retina. To gain insights into the molecular aspects associated with this sector RP phenotype, whose molecular mechanism remains elusive, the mutations were constructed by site-directed mutagenesis, expressed in heterologous systems, and studied by biochemical, spectroscopic, and functional assays. M39R and N55K opsins had variable degrees of chromophore regeneration when compared with WT opsin but showed no gross structural misfolding or altered trafficking. M39R showed a faster rate for transducin activation than WT rhodopsin with a faster metarhodopsinII decay, whereas N55K presented a reduced activation rate and an altered photobleaching pattern. N55K also showed an altered retinal release from the opsin binding pocket upon light exposure, affecting its optimal functional response. Our data suggest that these sector RP mutations cause different protein phenotypes that may be related to their different clinical progression. Overall, these findings illuminate the molecular mechanisms of sector RP associated with rhodopsin mutations.

G-protein-coupled receptors oligomerization: emerging signaling units and new opportunities for drug design.

G-protein-coupled receptors (GPCRs) are a widespread family of transmembrane receptors with different physiologically relevant functions. Alterations in the structure and function of these receptors at different levels (ligand binding, signaling and trafficking) may result in a number of pathological conditions which represent a major health problem. Mutations in these receptors are also linked to different inherited diseases for which there is no cure to date. Rationale design, based on receptor structural knowledge, is needed for the discovery of novel drugs with higher selectivity and less side effects. In fact, about 50% of the drugs currently under development target this kind of receptors. Oligomerization among GPCRs has been clearly established from experimental, particularly in vitro, studies. Moreover, homo and heterodimerization provide new unexpected clues for explaining the molecular mechanisms underlying some diseases in which GPCRs signaling might be affected. In this review we will analyze GPCRs structure and function for a better understanding of the dimerization process and the experimental approaches currently used to detect such interactions. Furthermore, how drugs targeting heteromers can represent new opportunities to tackle novel and safer treatments of some pathologies will be described. Recent results, in this regard, will be reported as encouraging examples in the field. Finally, the newest technologies available for developing drugs targeting heteromers will also be reviewed highlighting the importance of bivalent ligands that emerge as very powerful molecules interacting with heteromers.

Preferential activation by galanin 1-15 fragment of the GalR1 protomer of a GalR1-GalR2 heteroreceptor complex.

The three cloned galanin receptors show a higher affinity for galanin than for galanin N-terminal fragments. Galanin fragment (1-15) binding sites were discovered in the rat Central Nervous System, especially in dorsal hippocampus, indicating a relevant role of galanin fragments in central galanin communication. The hypothesis was introduced that these N-terminal galanin fragment preferring sites are formed through the formation of GalR1-GalR2 heteromers which may play a significant role in mediating galanin fragment (1-15) signaling. In HEK293T cells evidence for the existence of GalR1-GalR2 heteroreceptor complexes were obtained with proximity ligation and BRET(2) assays. PLA positive blobs representing GalR1-GalR2 heteroreceptor complexes were also observed in the raphe-hippocampal system. In CRE luciferase reporter gene assays, galanin (1-15) was more potent than galanin (1-29) in inhibiting the forskolin-induced increase of luciferase activity in GalR1-GalR2 transfected cells. The inhibition of CREB by 50nM of galanin (1-15) and of galanin (1-29) was fully counteracted by the non-selective galanin antagonist M35 and the selective GalR2 antagonist M871. These results suggested that the orthosteric agonist binding site of GalR1 protomer may have an increased affinity for the galanin (1-15) vs galanin (1-29) which can lead to its demonstrated increase in potency to inhibit CREB vs galanin (1-29). In contrast, in NFAT reporter gene assays galanin (1-29) shows a higher efficacy than galanin (1-15) in increasing Gq/11 mediated signaling over the GalR2 of these heteroreceptor complexes. This disbalance in the signaling of the GalR1-GalR2 heteroreceptor complexes induced by galanin (1-15) may contribute to depression-like actions since GalR1 agonists produce such effects.

Mercury-induced dark-state instability and photobleaching alterations of the visual g-protein coupled receptor rhodopsin.

Mercuric compounds were previously shown to affect the visual phototransduction cascade, and this could result in vision impairment. We have analyzed the effect of mercuric chloride on the structure and stability of the dim light vision photoreceptor rhodopsin. For this purpose, we have used both native rhodopsin immunopurified from bovine retinas and a recombinant mutant rhodopsin carrying several Cys to Ser substitutions in order to investigate the potential binding site of mercury on the receptor. Our results show that mercuric chloride dramatically reduces the stability of dark-state rhodopsin and alters the molecular features of the photoactived conformation obtained upon illumination by eliciting the formation of an altered photointermediate. The thermal bleaching kinetics of native and mutant rhodopsin is markedly accelerated by mercury in a concentration-dependent manner, and its chromophore regeneration ability is severely reduced without significantly affecting its G-protein activation capacity. Furthermore, fluorescence spectroscopic measurements on the retinal release process, ensuing illumination, suggest that mercury impairs complete retinal release from the receptor binding pocket. Our results provide further support for the capacity of mercury as a hazardous metal ion with reported deleterious effect on vision and provide a molecular explanation for such an effect at the rhodopsin photoreceptor level. We suggest that mercury could alter vision by acting in a specific manner on the molecular components of the retinoid cycle, particularly by modifying the ability of the visual photoreceptor protein rhodopsin to be regenerated and to be normally photoactivated by light.