Recovery of visual functions in a mouse model of Leber congenital amaurosis

The visual process is initiated by the photoisomerization of 11-cis-retinal to all-trans-retinal. For sustained vision the 11-cis-chromophore must be regenerated from all-trans-retinal. This requires RPE65, a dominant retinal pigment epithelium protein. Disruption of the RPE65 gene results in massive accumulation of all-trans-retinyl esters in the retinal pigment epithelium, lack of 11-cis-retinal and therefore rhodopsin, and ultimately blindness. We reported previously (Van Hooser, J. P., Aleman, T. S., He, Y. G., Cideciyan, A. V., Kuksa, V., Pittler, S. J., Stone, E. M., Jacobson, S. G., and Palczewski, K. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8623-8628) that in Rpe65-/- mice, oral administration of 9-cis-retinal generated isorhodopsin, a rod photopigment, and restored light sensitivity to the electroretinogram. Here, we provide evidence that early intervention by 9-cis-retinal administration significantly attenuated retinal ester accumulation and supported rod retinal function for more than 6 months post-treatment. In single cell recordings rod light sensitivity was shown to be a function of the amount of regenerated isorhodopsin; high doses restored rod responses with normal sensitivity and kinetics. Highly attenuated residual rod function was observed in untreated Rpe65-/- mice. This rod function is likely a consequence of low efficiency production of 11-cis-retinal by photo-conversion of all-trans-retinal in the retina as demonstrated by retinoid analysis. These studies show that pharmacological intervention produces long lasting preservation of visual function in dark-reared Rpe65-/- mice and may be a useful therapeutic strategy in recovering vision in humans diagnosed with Leber congenital amaurosis caused by mutations in the RPE65 gene, an inherited group of early onset blinding and retinal degenerations.     

Probing mechanisms of photoreceptor degeneration in a new mouse model of the common form of autosomal dominant retinitis pigmentosa due to P23H opsin mutations

Rhodopsin, the visual pigment mediating vision under dim light, is composed of the apoprotein opsin and the chromophore ligand 11-cis-retinal. A P23H mutation in the opsin gene is one of the most prevalent causes of the human blinding disease, autosomal dominant retinitis pigmentosa. Although P23H cultured cell and transgenic animal models have been developed, there remains controversy over whether they fully mimic the human phenotype; and the exact mechanism by which this mutation leads to photoreceptor cell degeneration remains unknown. By generating P23H opsin knock-in mice, we found that the P23H protein was inadequately glycosylated with levels 1-10% that of wild type opsin. Moreover, the P23H protein failed to accumulate in rod photoreceptor cell endoplasmic reticulum but instead disrupted rod photoreceptor disks. Genetically engineered P23H mice lacking the chromophore showed accelerated photoreceptor cell degeneration. These results indicate that most synthesized P23H protein is degraded, and its retinal cytotoxicity is enhanced by lack of the 11-cis-retinal chromophore during rod outer segment development.     

Mole quantity of RPE65 and its productivity in the generation of 11-cis-retinal from retinyl esters in the living mouse eye

RPE65, a protein expressed in cells of the retinal pigment epithelium of the eye, is essential for the synthesis by isomerohydrolase of 11-cis-retinal, the chromophore of rod and cone opsins. Recent work has established that RPE65 is a retinyl ester binding protein, and as all-trans-retinyl esters are the substrate for isomerohydrolase activity, the hypothesis has emerged that RPE65 serves to deliver substrate to this enzyme or complex. We bred mice with five distinct combinations of the RPE65 Leu450/Met450 variants (Leu/Leu, Met/Met, Leu/Met, Leu/-, and Met/-), measured in mice of each genotype the mole quantity of RPE65 per eye, and measured the initial rate of rhodopsin regeneration after a nearly complete bleach of rhodopsin to estimate the maximum rate of 11-cis-retinal synthesis in vivo. The quantity of RPE65 per eye ranged from 5.7 pmol (Balb/c) to 0.32 pmol (C57BL/6N x Rpe65(-)(/)(-)); the initial rate of rhodopsin regeneration was a Michaelis function of RPE65, where V(max) = 18 pmol/min per eye and K(m) = 1.7 pmol, and not dependent on the Leu450/Met450 variant. At RPE65 levels well below the K(m), the rate of production of 11-cis-retinal per RPE65 molecule was approximately 10 min(-)(1). Thus, the results imply that as a chaperone each RPE65 molecule can deliver retinyl ester to the isomerohydrolase at a rate of 10 molecules/min; should RPE65 itself be identified as the isomerase, each copy must be able to produce at least 10 molecules of 11-cis-retinal per minute.     

Lipofuscin and N-retinylidene-N-retinylethanolamine (A2E) accumulate in retinal pigment epithelium in absence of light exposure: their origin is 11-cis-retinal

The age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been associated with the development of retinal diseases, particularly age-related macular degeneration and Stargardt disease. A major component of lipofuscin is the bis-retinoid N-retinylidene-N-retinylethanolamine (A2E). The current model for the formation of A2E requires photoactivation of rhodopsin and subsequent release of all-trans-retinal. To understand the role of light exposure in the accumulation of lipofuscin and A2E, we analyzed RPEs and isolated rod photoreceptors from mice of different ages and strains, reared either in darkness or cyclic light. Lipofuscin levels were determined by fluorescence imaging, whereas A2E levels were quantified by HPLC and UV-visible absorption spectroscopy. The identity of A2E was confirmed by tandem mass spectrometry. Lipofuscin and A2E levels in the RPE increased with age and more so in the Stargardt model Abca4(-/-) than in the wild type strains 129/sv and C57Bl/6. For each strain, the levels of lipofuscin precursor fluorophores in dark-adapted rods and the levels and rates of increase of RPE lipofuscin and A2E were not different between dark-reared and cyclic light-reared animals. Both 11-cis- and all-trans-retinal generated lipofuscin-like fluorophores when added to metabolically compromised rod outer segments; however, it was only 11-cis-retinal that generated such fluorophores when added to metabolically intact rods. The results suggest that lipofuscin originates from the free 11-cis-retinal that is continuously supplied to the rod for rhodopsin regeneration and outer segment renewal. The physiological role of Abca4 may include the translocation of 11-cis-retinal complexes across the disk membrane.     

Topography of the rhodopsin molecule. Identification of the domain phosphorylated.

Studies on the light-stimulated phosphorylation of rod outer segments by [gamma-32P]ATP showed that although nearly 1 mol of [32P]phosphate was incorporated/mol of total opsin, only a small fraction of the molecules were phosphorylated, and these contained at least 2-3 mol of phosphate/mol. Rod outer segments containing the phosphorylated opsin were incubated with 11-cis-retinal to generate phosphorylated rhodopsin and then digested with papain to produce a cleaved complex comprising three fragments, heavy (H), medium (M) and light (L). It was shown that L-fragment of apparent mol.wt. 6000 contained all the phosphorylation sites. This suggests that one specific domain of rhodopsin is susceptible to multiple phosphorylation.     

Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye

Visual sensation in vertebrates is triggered when light strikes retinal photoreceptor cells causing photoisomerization of the rhodopsin chromophore 11-cis-retinal to all-trans-retinal. The regeneration of preillumination conditions of the photoreceptor cells requires formation of 11-cis-retinal in the adjacent retinal pigment epithelium (RPE). Using the intrinsic fluorescence of all-trans-retinyl esters, noninvasive two-photon microscopy revealed previously uncharacterized structures (6.9 +/- 1.1 microm in length and 0.8 +/- 0.2 microm in diameter) distinct from other cellular organelles, termed the retinyl ester storage particles (RESTs), or retinosomes. These structures form autonomous all-trans-retinyl ester-rich intracellular compartments distinct from other organelles and colocalize with adipose differentiation-related protein. As demonstrated by in vivo experiments using wild-type mice, the RESTs participate in 11-cis-retinal formation. RESTs accumulate in Rpe65-/- mice incapable of carrying out the enzymatic isomerization, and correspondingly, are absent in the eyes of Lrat-/- mice deficient in retinyl ester synthesis. These results indicate that RESTs located close to the RPE plasma membrane are essential components in 11-cis-retinal production.     

Bax-Induced Apoptosis in Leber's Congenital Amaurosis: A Dual Role in Rod and Cone Degeneration

Pathogenesis in the Rpe65^−/− mouse model of Leber''s congenital amaurosis (LCA) is characterized by a slow and progressive degeneration of the rod photoreceptors. On the opposite, cones degenerate rapidly at early ages. Retinal degeneration in Rpe65 ^−/− mice, showing a null mutation in the gene encoding the retinal pigment epithelium 65-kDa protein (Rpe65), was previously reported to depend on continuous activation of a residual transduction cascade by unliganded opsin. However, the mechanisms of apoptotic signals triggered by abnormal phototransduction remain elusive. We previously reported that activation of a Bcl-2-dependent pathway was associated with apoptosis of rod photoreceptors in Rpe65^−/− mice during the course of the disease. In this study we first assessed whether activation of Bcl-2-mediated apoptotic pathway was dependent on constitutive activation of the visual cascade through opsin apoprotein. We then challenged the direct role of pro-apoptotic Bax protein in triggering apoptosis of rod and cone photoreceptors.Quantitative PCR analysis showed that increased expression of pro-apoptotic Bax and decreased level of anti-apoptotic Bcl-2 were restored in Rpe65^−/−/Gnat1^−/− mice lacking the Gnat1 gene encoding rod transducin. Moreover, photoreceptor apoptosis was prevented as assessed by TUNEL assay. These data indicate that abnormal activity of opsin apoprotein induces retinal cell apoptosis through the Bcl-2-mediated pathway. Following immunohistological and real-time PCR analyses, we further observed that decreased expression of rod genes in Rpe65-deficient mice was rescued in Rpe65^−/−/Bax^−/− mice. Histological and TUNEL studies confirmed that rod cell demise and apoptosis in diseased Rpe65^−/− mice were dependent on Bax-induced pathway. Surprisingly, early loss of cones was not prevented in Rpe65^−/−/Bax^−/− mice, indicating that pro-apoptotic Bax was not involved in the pathogenesis of cone cell death in Rpe65-deficient mice.This is the first report, to our knowledge, that a single genetic mutation can trigger two independent apoptotic pathways in rod and cone photoreceptors in Rpe65-dependent LCA disease. These results highlight the necessity to investigate and understand the specific death signaling pathways committed in rods and cones to develop effective therapeutic approaches to treat RP diseases.     

Organization of the G protein-coupled receptors rhodopsin and opsin in native membranes

G protein-coupled receptors (GPCRs), which constitute the largest and structurally best conserved family of signaling molecules, are involved in virtually all physiological processes. Crystal structures are available only for the detergent-solubilized light receptor rhodopsin. In addition, this receptor is the only GPCR for which the presumed higher order oligomeric state in native membranes has been demonstrated (Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D. A., Engel, A., and Palczewski, K. (2003) Nature 421, 127-128). Here, we have determined by atomic force microscopy the organization of rhodopsin in native membranes obtained from wild-type mouse photoreceptors and opsin isolated from photoreceptors of Rpe65-/- mutant mice, which do not produce the chromophore 11-cis-retinal. The higher order organization of rhodopsin was present irrespective of the support on which the membranes were adsorbed for imaging. Rhodopsin and opsin form structural dimers that are organized in paracrystalline arrays. The intradimeric contact is likely to involve helices IV and V, whereas contacts mainly between helices I and II and the cytoplasmic loop connecting helices V and VI facilitate the formation of rhodopsin dimer rows. Contacts between rows are on the extracellular side and involve helix I. This is the first semi-empirical model of a higher order structure of a GPCR in native membranes, and it has profound implications for the understanding of how this receptor interacts with partner proteins.     

Rpe65 is a retinyl ester binding protein that presents insoluble substrate to the isomerase in retinal pigment epithelial cells

Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the all-trans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of beta-carotene dioxygenase, appears to play a role in this pathway. Rpe65-/- knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Leber's congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds all-trans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65-/- mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65.     

The retinal G protein-coupled receptor (RGR) enhances isomerohydrolase activity independent of light

Rod and cone visual pigments use 11-cis-retinal, a vitamin A derivative, as their chromophore. Light isomerizes 11-cis- into all-trans-retinal, triggering a conformational transition of the opsin molecule that initiates phototransduction. After bleaching all-trans-retinal leaves the opsin, and light sensitivity must be restored by regeneration of 11-cis-retinal. Under bright light conditions the retinal G protein-coupled receptor (RGR) was reported to support this regeneration by acting as a photoisomerase in a proposed photic visual cycle. We analyzed the contribution of RGR to rhodopsin regeneration under different light regimes and show that regeneration, during light exposure and in darkness, is slowed about 3-fold in Rgr(-/-) mice. These findings are not in line with the proposed function of RGR as a photoisomerase. Instead, RGR, independent of light, accelerates the conversion of retinyl esters to 11-cis-retinal by positively modulating isomerohydrolase activity, a key step in the "classical" visual cycle. Furthermore, we find that light accelerates rhodopsin regeneration, independent of RGR.     

Signaling states of rhodopsin. Formation of the storage form,metarhodopsin III,from active metarhodopsin II

Vertebrate rhodopsin consists of the apoprotein opsin and the chromophore 11-cis-retinal covalently linked via a protonated Schiff base. Upon photoisomerization of the chromophore to all-trans-retinal, the retinylidene linkage hydrolyzes, and all-trans-retinal dissociates from opsin. The pigment is eventually restored by recombining with enzymatically produced 11-cis-retinal. All-trans-retinal release occurs in parallel with decay of the active form, metarhodopsin (Meta) II, in which the original Schiff base is intact but deprotonated. The intermediates formed during Meta II decay include Meta III, with the original Schiff base reprotonated, and Meta III-like pseudo-photoproducts. Using an intrinsic fluorescence assay, Fourier transform infrared spectroscopy, and UV-visible spectroscopy, we investigated Meta II decay in native rod disk membranes. Up to 40% of Meta III is formed without changes in the intrinsic Trp fluorescence and thus without all-trans-retinal release. NADPH, a cofactor for the reduction of all-trans-retinal to all-trans-retinol, does not accelerate Meta II decay nor does it change the amount of Meta III formed. However, Meta III can be photoconverted back to the Meta II signaling state. The data are described by two quasi-irreversible pathways, leading in parallel into Meta III or into release of all-trans-retinal. Therefore, Meta III could be a form of rhodopsin that is stored away, thus regulating photoreceptor regeneration.     

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.     

G-protein-coupled receptor rhodopsin regulates the phosphorylation of retinal insulin receptor

We have shown previously that phosphoinositide 3-kinase in the retina is activated in vivo through light-induced tyrosine phosphorylation of the insulin receptor (IR). The light effect is localized to photoreceptor neurons and is independent of insulin secretion (Rajala, R. V., McClellan, M. E., Ash, J. D., and Anderson, R. E. (2002) J. Biol. Chem. 277, 43319-43326). These results suggest that there exists a cross-talk between phototransduction and other signal transduction pathways. In this study, we examined the stage of phototransduction that is coupled to the activation of the IR. We studied IR phosphorylation in mice lacking the rod-specific alpha-subunit of transducin to determine if phototransduction events are required for IR activation. To confirm that light-induced tyrosine phosphorylation of the IR is signaled through bleachable rhodopsin, we examined IR activation in retinas from RPE65(-/-) mice that are deficient in opsin chromophore. We observed that IR phosphorylation requires the photobleaching of rhodopsin but not transducin signaling. To determine whether the light-dependent activation of IR is mediated through the rod or cone transduction pathway, we studied the IR activation in mice lacking opsin, a mouse model of pure cone function. No light-dependent activation of the IR was found in the retinas of these mice. We provide evidence for the existence of a light-mediated IR pathway in the retina that is different from the known insulin-mediated pathway in nonneuronal tissues. These results suggest that IR phosphorylation in rod photoreceptors is signaled through the G-protein-coupled receptor rhodopsin. This is the first study demonstrating that rhodopsin can initiate signaling pathway(s) in addition to its classical phototransduction.     

Retinoids assist the cellular folding of the autosomal dominant retinitis pigmentosa opsin mutant P23H

The clinically common mutant opsin P23H, associated with autosomal dominant retinitis pigmentosa, yields low levels of rhodopsin when retinal is added following induction of the protein in stably transfected HEK-293 cells. We previously showed that P23H rhodopsin levels could be increased by providing a 7-membered ring, locked analog of 11-cis-retinal during expression of P23H opsin in vivo. Here we demonstrate that the mutant opsin is effectively rescued by 9- or 11-cis-retinal, the native chromophore. When retinal was added during expression, P23H rhodopsin levels were 5-fold (9-cis) and 6-fold (11-cis) higher than when retinal was added after opsin was expressed and cells were harvested. Levels of P23H opsin were increased approximately 3.5-fold with both compounds, but wild-type protein levels were only slightly increased. Addition of retinal during induction promoted the Golgi-specific glycosylation of P23H opsin and transport of the protein to the cell surface. P23H rhodopsins containing 9- or 11-cis-retinal had blue-shifted absorption maxima and altered photo-bleaching properties compared with the corresponding wild-type proteins. Significantly, P23H rhodopsins were more thermally unstable than the wild-type proteins and more rapidly bleached by hydroxylamine in the dark. We suggest that P23H opsin is similarly unstable and that retinal binds and stabilizes the protein early in its biogenesis to promote its cellular folding and trafficking. The implications of this study for treating retinitis pigmentosa and other protein conformational disorders are discussed.     

Role of LRAT on the retinoid isomerase activity and membrane association of Rpe65

Absorption of a photon by a vertebrate opsin pigment induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical re-isomerization of the chromophore via an enzyme pathway called the visual cycle. The retinoid isomerase in this pathway is Rpe65, a membrane-associated protein in the retinal pigment epithelium (RPE) with no predicted membrane-spanning segments. It has been suggested that Rpe65 is S-palmitoylated by lecithin:retinol acyl transferase (LRAT) on Cys(231), Cys(329), and Cys(330), and that this palmitoylation is required for isomerase activity and the association of Rpe65 with membranes. Here we show that the affinity of Rpe65 for membranes is similar in wild-type and lrat(-/-) mice. The isomerase activity of Rpe65 is also similar in both strains when all-trans-retinyl palmitate is used as substrate. With all-trans-retinol substrate, isomerase activity is present in wild-type but undetectable in RPE homogenates from lrat(-/-) mice. Substitution of Cys(231), Cys(329), and Cys(330) with Ser or Ala did not affect the affinity of Rpe65 for membranes. Further, these Cys residues are not palmitoylated in Rpe65 by mass spectrometric analysis. Global inhibition of protein palmitoylation by 2-bromopalmitate did not affect the solubility or isomerase activity of Rpe65. Finally, we show that soluble and membrane-associated Rpe65 possesses similar isomerase specific activities. These results indicate that LRAT is not required for isomerase activity beyond synthesis of retinyl-ester substrate, and that the association of Rpe65 with membranes is neither dependent upon LRAT nor the result of S-palmitoylation. The affinity of Rpe65 for membranes is probably an intrinsic feature of this protein.     

Rpe65 is the retinoid isomerase in bovine retinal pigment epithelium

The first event in light perception is absorption of a photon by an opsin pigment, which induces isomerization of its 11-cis-retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical regeneration of 11-cis-retinaldehyde through an enzymatic pathway called the visual cycle. The isomerase, which converts an all-trans-retinyl ester to 11-cis-retinol, has never been identified. Here, we performed an unbiased cDNA expression screen to identify this isomerase. We discovered that the isomerase is a previously characterized protein called Rpe65. We confirmed our identification of the isomerase by demonstrating catalytic activity in mammalian and insect cells that express Rpe65. Mutations in the human RPE65 gene cause a blinding disease of infancy called Leber congenital amaurosis. Rpe65 with the Leber-associated C330Y and Y368H substitutions had no isomerase activity. Identification of Rpe65 as the isomerase explains the phenotypes in rpe65-/- knockout mice and in humans with Leber congenital amaurosis.     

Ligand channeling within a G-protein-coupled receptor. The entry and exit of retinals in native opsin

Deactivation of light-activated rhodopsin (metarhodopsin II) involves, after rhodopsin kinase and arrestin interactions, the hydrolysis of the covalent bond of all-trans-retinal to the apoprotein. Although the long-lived storage form metarhodopsin III is transiently formed, all-trans-retinal is eventually released from the active site. Here we address the question of whether the release results in a retinal that is freely diffusible in the lipid phase of the photoreceptor membrane. The release reaction is accompanied by an increase in intrinsic protein fluorescence (release signal), which arises from the relief of the fluorescence quenching imposed by the retinal in the active site. An analogous fluorescence decrease (uptake signal) was evoked by exogenous retinoids when they non-covalently bound to native opsin membranes. Uptake of 11-cis-retinal was faster than formation of the retinylidene linkage to the apoprotein. Endogenous all-trans-retinal released from the active site during metarhodopsin II decay did not generate the uptake signal. The data show that in addition to the retinylidene pocket (site I) there are two other retinoidbinding sites within opsin. Site II involved in the uptake signal is an entrance site, while the exit site (site III) is occupied when retinal remains bound after its release from site I. Support for a retinal channeling mechanism comes from the rhodopsin crystal structure, which unveiled two putative hydrophobic binding sites. This mechanism enables a unidirectional process for the release of photoisomerized chromophore and the uptake of newly synthesized 11-cis-retinal for the regeneration of rhodopsin.     

Orientation of retinal in bovine rhodopsin determined by cross-linking using a photoactivatable analog of 11-cis-retinal

A photoactivatable analog of 11-cis-retinal has been used to probe the orientation of retinal in bovine rhodopsin. The analog binds to the opsin to regenerate a chromophore with lambda max at 458 nm. The linkage site of the analog to the opsin was confirmed to be Lys-296 as in 11-cis-retinal rhodopsin. The analog-reconstituted rhodopsin activated transducin and was phosphorylated by rhodopsin kinase on illumination. On photolysis of rhodopsin containing the radioactively labeled analog at 365 nm at -15 degrees C, 20-25% of the analog was covalently linked to the protein. Proteolysis of the labeled protein and characterization of the appropriate peptides showed that cross-linking of the analog was predominantly to helices C or F. When analog reconstituted rhodopsin in rod outer segments was photolyzed, cross-linking was predominantly to helix C. However, when analog-reconstituted rhodopsin, purified in lauryl maltoside, was photolyzed, labeling occurred mainly in helix F. Sequence analysis showed major sites of cross-linking to be Phe-115, Ala-117, Glu-122, Trp-126, and Ser-127 in helix C while Trp-265 was the major site in helix F. The results suggest that the beta-ionone ring of retinal orients toward helices C and F.     

Characterization of the recombination reaction of rhodopsin.

The kinetics of recombination of 11-cis-retinal with bleached rod outer segments and sodium cholate solubilized rhodopsin have been investigated. At neutral pH, it was found that bleached rod outer segments in the presence of an excess of 11-cis-retinal follow pseudo-first-order kinetics. The results suggest the second-order formation of an intermediate addition compound followed by a first-order dehydration step to form a protonated aldimine linkage. In addition, at pH values above 7.5 or below 6.5 the kinetics of recombination are complex, indicating the formation of a molecular species inactive in recombination which is in equilibrium with the active form of opsin. Based upon the observed rate constants as a function of pH, a scheme is presented to describe the recombination reaction in bleached rod outer segments. The kinetics of recombination of sodium cholate solubilized opsin were also analyzed. In terms of formation of an intermediate addition compound and subsequent dehydration, the values for the individual rate constants for both bleached rod outer segments and cholate-solubilized opsin were found to compare very favorably. These results demonstrate that the sodium cholate (2 mg/ml) maintains opsin in a conformation very similar to that in the rod outer segment membrane and suggest that the cholate-opsin complex is an excellent model system for studies on opsin-membrane interactions.     

Deactivation and proton transfer in light-induced metarhodopsin II/metarhodopsin III conversion: a time-resolved fourier transform infrared spectroscopic study

Vertebrate rhodopsin shares with other retinal proteins the 11-cis-retinal chromophore and the light-induced 11-cis/trans isomerization triggering its activation pathway. However, only in rhodopsin the retinylidene Schiff base bond to the apoprotein is eventually hydrolyzed, making a complex regeneration pathway necessary. Metabolic regeneration cannot be short-cut, and light absorption in the active metarhodopsin (Meta) II intermediate causes anti/syn isomerization around the retinylidene linkage rather than reversed trans/cis isomerization. A new deactivating pathway is thereby triggered, which ends in the Meta III "retinal storage" product. Using time-resolved Fourier transform infrared spectroscopy, we show that the identified steps of receptor activation, including Schiff base deprotonation, protein structural changes, and proton uptake by the apoprotein, are all reversed. However, Schiff base reprotonation is much faster than the activating deprotonation, whereas the protein structural changes are slower. The final proton release occurs with pK approximately 4.5, similar to the pK of a free Glu residue and to the pK at which the isolated opsin apoprotein becomes active. A forced deprotonation, equivalent to the forced protonation in the activating pathway, which occurs against the unfavorable pH of the medium, is not observed. This explains properties of the final Meta III product, which displays much higher residual activity and is less stable than rhodopsin arising from regeneration with 11-cis-retinal. We propose that the anti/syn conversion can only induce a fast reorientation and distance change of the Schiff base but fails to build up the full set of dark ground state constraints, presumably involving the Glu(134)/Arg(135) cluster.