PHOTOEXCITATION OF RHODOPSIN: CONFORMATION CHANGES IN THE CHROMOPHORE, PROTEIN AND ASSOCIATED LIPIDS AS DETERMINED BY FTIR DIFFERENCE SPECTROSCOPY

Abstract— The visual pigment rhodopsin is the major membrane protein in the rod photoreceptor membrane. Rhodopsin's function is to transduce the light induced isomerization (ll-cis to all-trans) of its internally located retinylidene chromophore into transient expression of signal sites at the surface of the protein. Fourier transform infrared (FTIR) difference spectroscopy has been used to study all of the steps in the photobleaching sequence of rhodopsin. Early protein alterations involving the peptide backbone and aspartic and/or glutamic carboxyl groups were detected which increase upon lumirhodopsin formation and spread to water exposed carboxyl groups by metarhodopsin II. The intensified and frequency shifted hydrogen-out-of-plane vibrations of the chromophore that are present in bathorhodopsin are absent in lumirhodopsin. This indicates that by lumirhodopsin, the chromophore has relaxed relative to its more strained all-frans form in bathorhodopsin. Finally, the transition to metarhodopsin II is found to involve perturbation of the acyl tail region of unsaturated phospholipid molecules possibly in response to small changes in the shape of the rhodopsin.     

THE INVOLVEMENT OF WATER AT THE RETINAL BINDING SITE IN RHODOPSIN AND EARLY LIGHT-INDUCED INTRAMOLECULAR PROTON TRANSFER

Abstract Extensive dehydration of air-dried films of bovine rod outer segment membranes induces fully reversible changes in the absorption spectrum of rhodopsin, indicative of deprotonation of the retinylidene Schiff base in more than 50% of the rhodopsin molecules in the sample. This suggests that water is involved at the site of the Schiff base protonation in rhodopsin. In contrast, the spectrum of metarhodopsin I is resistant to similar dehydrating conditions, implying a significant difference in the mechanism for protonation in metarhodopsin I. The photochemistry of dehydrated membranes was also explored. Photoexcitation of deprotonated rhodopsin (λ_max 390 nm) induces a large bathochromic shift of the chromophore. The major photoproduct at room temperature was spectrally similar to metarhodopsin I (λ_max, 478 nm). These findings suggest that intramolecular proton transfer involving the Schiff base proton may occur in the earlier stages of the visual cycle, prior to or during the formation of metarhodopsin I.     

Primary events in dim light vision: a chemical and spectroscopic approach toward understanding protein/chromophore interactions in rhodopsin

The visual pigment rhodopsin (bovine) is a 40 kDa protein consisting of 348 amino acids, and is a prototypical member of the subfamily A of G protein-coupled receptors (GPCRs). This remarkably efficient light-activated protein (quantum yield = 0.67) binds the chromophore 11-cis-retinal covalently by attachment to Lys296 through a protonated Schiff base. The 11-cis geometry of the retinylidene chromophore keeps the partially active opsin protein locked in its inactive state (inverse agonist). Several retinal analogs with defined configurations and stereochemistry have been incorporated into the apoprotein to give rhodopsin analogs. These incorporation results along with the spectroscopic properties of the rhodopsin analogs clarify the mode of entry of the chromophore into the apoprotein and the biologically relevant conformation of the chromophore in the rhodopsin binding site. In addition, difference UV, CD, and photoaffinity labeling studies with a 3-diazo-4-oxo analog of 11-cis-retinal have been used to chart the movement of the retinylidene chromophore through the various intermediate stages of visual transduction.     

Iodopsin, a red-sensitive cone visual pigment in the chicken retina.

The vertebrate retina contains two kinds of visual cells: rods, responsible for twilight (scotopic) vision (black and white discrimination); and cones, responsible for daylight (photopic) vision (color discrimination). Here we attempt to explain some of their functional differences and similarities in terms of their visual pigments. In the chicken retina there are four types of single cones and a double cone; each of the single cones has its own characteristic oil droplet (red, orange, blue, or colorless) and the double cone is composed of a set of principal and accessory members, the former of which has a green-colored oil droplet. Iodopsin, the chicken red-sensitive cone visual pigment, is located at outer segments of both the red single cones and the double cones, while the other single cones and the rod contain their own visual pigments with different absorption spectra. The diversity in absorption spectra among these visual pigments is caused by the difference in interaction between chromophore (11-cis retinal) and protein moiety (opsin). However, the chromophore-binding pocket in iodopsin is similar to that in rhodopsin. The difference in absorption maxima between both pigments could be explained by the difference in distances between the protonated Schiff-bases at the chromophore-binding site and their counter ions in iodopsin and rhodopsin. Furthermore, iodopsin has a unique chloride-binding site whose chloride ion serves for the red-shift of the absorption maximum of iodopsin. Visual pigment bleaches upon absorption of light through several intermediates and finally dissociates into all-trans retinal and opsin. That the sensitivity of cones is lower than rods cannot be explained by the relative photosensitivity of iodopsin to rhodopsin, but may be understood to some extent by the short lifetime of an enzymatically active intermediate (corresponding to metarhodopsin II) produced in the photobleaching process of iodopsin. The rapid formation and decay of the meta II-intermediate of iodopsin compared with metarhodopsin II are not contradictory to the rapid generation and recovery of cone receptor potential compared with rod receptor potential. The rapid recovery of the cone receptor potential may be due to a more effective shutoff mechanism of the visual excitation, including the phosphorylation of iodopsin. The rapid dark adaptation of cones compared with rods has been explained by the rapid regeneration of iodopsin from 11-cis retinal and opsin. One of the reasons for the rapid regeneration and susceptibility to chemicals of iodopsin compared with rhodopsin may be a unique structure near the chromophore-binding site of iodopsin.     

SPECTRAL PROPERTIES OF THE RHODOPSIN-SYSTEM OF THE CRAYFISH Astacus leptodactylus

Abstract— The absorption spectra of the membrane-bound and of the digitonin-solubilized visual pigment of crayfish Astacus leptodactylus were investigated by conventional spectrophotometry. A method was developed to isolate purified rhabdoms almost entirely free from screening pigments from a single retina. The quantity of isolated and purified rhabdoms from a single retina was sufficient to measure the absorption spectra of the visual pigment.
The absorption spectra of the chromoprotein system (R and M) show that both the membrane-bound and the digitonin-solubilized visual pigment isomers are stable at 0°C and pH 7.0. Rhodopsin and metarhodopsin are photoreversible under these conditions without any light-induced denaturation. The difference spectra for the chromoprotein isomers and those of different photostationary states yield maximal values for ΔE at 570 and 485 nm.
At neutral pH, 0°C, Λ_max of rhodopsin is 530 nm. Irradiation with light of Λ= 630 to 640 nm isomerizes rhodopsin nearly quantitatively to metarhodopsin with Λ_max, of 500 nm. The molar extinction coefficient of metarhodopsin is greater than that of rhodopsin by a factor of ˜ 1.41. each measured at its respective Λ_max Metarhodopsin can be isomerized to rhodopsin by irradiating at Λ > 630 nm. As the absorption spectra of the two chromoprotein isomers overlap, only part of the metarhodopsin can be reversed to rhodopsin. The maximal photoreversion can be achieved by irradiating at 460 nm. The stability of the digitonin-solubilized chromoprotein is remarkably dependent on temperature. Warming the digitonin extract of rhabdoms from 0 to 20 or 30°C caused a shift of the rhodopsin spectrum to shorter wavelengths (Λ_max= 485 nm) accompanied by a decrease of E_Λmax by about 30%.     

Steric Hindrance between Chromophore Substituents as the Driving Force of Rhodopsin Photoisomerization: 10-Methyl-13-Demethyl Retinal Containing Rhodopsin

Abstract— A visual chromophore analogue, 10-methyl-13-demethyl (dm) retinal, was synthesized and reconstituted with bleached bovine rhodopsin to form a visual pigment derivative with absorbance maximum at 505 nm. The investigations with this new compound were stimulated from recent results using 13-dm retinal as a chromophore that revealed a remarkable loss in quantum efficiency (φ of 13-dm retinal-containing rhodopsin: 0.30, Ternieden and Gartner, J. Photochem. Photobiol. B Biol. 33, 83–86, 1996). The quantum efficiency of the new pigment was determined as 0.59 by quantitative bleaching using reconstituted rhodopsin as a reference. The very similar quantum efficiencies of rhodopsin and the new pigment give experimental support for the recently presented hypothesis that a steric hindrance between the substituents at positions 10 and 13 in 11- cis -retinal is elevated during the photoisomerization and thus facilitates the rapid photoisomerization of the visual chromophore (Peteanu et al., Proc. Natl. Acad. Sci. USA 90, 11762–11766, 1993). Such steric hindrance is removed from the molecule by the elimination of the methyl group from position 13 and can be re-established via a rearrangement of the substitution pattern by introducing a methyl group at position 10 of 13-dm retinal.     

QUANTUM EFFICIENCY AND PHOTOSENSITIVITY OF THE RHODOPSIN ⇌ METARHODOPSIN CONVERSION IN CRAYFISH PHOTORECEPTORS

Photosensitivity (K_λ) of a visual pigment is the product of the molecular absorption coefficient (α_λ) and the quantum efficiency for photoconversion (γ). Among the invertebrates, many visual pigments are stable not only in the rhodopsin (R) conformation but also as the photoproduct, metarhodopsin (M), We here employ a method for determining the photosensitivities of the two stable pigments of a rhodopsin-metarhodopsin pair, using kinetic analysis of fluorescence from metarhodopsin combined with measurements of spectral absorption made before and after saturation at the isosbestic wavelength of the pigment pair. A curve fitting technique, in which a theoretical function is scaled for best fit to the measured absorption spectrum of the photosteady-state mixture, yields values for the photosensitivity of rhodopsin at λ._max, the ratio of quantum efficiencies for rhodopsin—metarhodopsin interconversion, and the fractional composition of the steady-state mixture. With knowledge of the molecular extinction coefficient, the absolute values of quantum efficiency can be calculated. For crayfish ( Orconectes, Procambarus ) rhodopsin, measured in isolated rhabdoms, K_max= 1.05 x 10^-16 cm² at 535 nm with >7λR→M0.69. These values are similar to the photosensitivity and quantum efficiency of bleaching of vertebrate rhodopsins in digitonin solution (Dartnall, 1972). For the metarhodopsin, K_max= 1.02 x 10^-16 cm² at 510 nm, and λ_M-R= 0.49.     

FLUORESCENCE OF HOUSEFLY VISUAL PIGMENT

Abstract —The fluorescence of housefly photoreceptors was studied in vivo by using the deep pseudopupil technique. Whereas the rhodopsin R490 of the peripheral retinular cells fluoresces negligibly the metarhodopsin M580 fluoresces distinctly in the red. The newly discovered metarhodopsin M'is produced by intense blue light and can be reconverted into rhodopsin by intense long wavelength light. M'also fluoresces in the red; its excitation spectrum and emission spectrum peak at _max= 570 and 660 nm respectively.
Intense ultraviolet light irreversibly reduces the visual pigment fluorescence as well as the broad band autofluorescence (kmnx 470 nm) originating from non-visual pigments in the fly's eye.     

Retinal photoisomerase: role in invertebrate visual cells.

In invertebrate visual cells, the rhodopsin content is maintained at a high level by the fast process of photoregeneration during daylight. Rhodopsin is converted by photoabsorption to metarhodopsin, which is reconverted to rhodopsin by light. In addition, rhodopsin is regenerated by a slow process of renewal which takes days to complete and involves the biosynthesis of opsin. It is well known that rhodopsin can be formed from opsin only when 11-cis-retinal is present; this requires the existence of an isomerizing enzyme which is capable of transforming all-trans-retinal, released from the degradation of metarhodopsin, into the 11-cis-retinal isomer. In some invertebrate visual systems, experiments on rhodopsin regeneration have been interpreted by assuming that the isomerization reaction is a light-dependent process involving a retinal-protein complex. Two retinal photoisomerases which have been well characterized, i.e. bee photoisomerase and cephalopod retinochrome, are reviewed here. Their properties are compared in order to determine their physiological role, which is likely to be in the renewal of visual pigment rhodopsin. To conclude, a visual pigment cycle is proposed in which rhodopsin regeneration follows two light-dependent pathways. This greatly simplifies the rhodopsin regeneration scheme for invertebrate visual systems.     

ON THE CHROMOPHORE CONFIGURATION OF METARHODOPSIN II

Abstract The nature of the chromophore configuration of metarhodopsin II (meta II) has been a subject of recent controversy. Earlier resonance Raman results from this laboratory had indicated that meta II has an unprotonated Schiff base linkage between the chromophore and apoprotein. It has been argued, on the other hand, that the Raman evidence does not exclude a non-covalently bound retinal chromophore. In this communication we present additional evidence and further arguments in favor of our earlier conclusion regarding the state of the chromophore in meta II.     

Effects of water re-location and cavity trimming on the CASPT2//CASSCF/AMBER excitation energy of Rhodopsin

We have used quantum-mechanics/molecular-mechanics computations based on ab initio multiconfigurational perturbation theory to determine and rationalize the effect of the re-location of one crystallographic water molecule on the vertical excitation energy of the visual pigment rhodopsin. It is found that the re-location of one water molecule to the opposite side of the 11-cis retinal chromophore leads to a large 0.7–0.8 Å contraction in the chromophore—counterion salt-bridge distance. In spite of this structural effect, the change in excitation energy is found to be limited (< 1.5 kcal mol^?1). Through an analysis of different rhodopsin models in terms of “components” (isolated chromophore, isolated chromophore—counterion ion-pair and models deprived of the counterion charges) we show that the limited change of the excitation energy can be related to a displacement of the retinal chromophore to a different spot of the protein cavity.     

Structure, spectroscopy, and spectral tuning of the gas-phase retinal chromophore: the beta-ionone "handle" and alkyl group effect

The low-lying singlet states (i.e. S0, S1, and S2) of the chromophore of rhodopsin, the protonated Schiff base of 11-cis-retinal (PSB11), and of its all-trans photoproduct have been studied in isolated conditions by using ab initio multiconfigurational second-order perturbation theory. The computed spectroscopic features include the vertical excitation, the band origin, and the fluorescence maximum of both isomers. On the basis of the S0-->S1 vertical excitation, the gas-phase absorption maximum of PSB11 is predicted to be 545 nm (2.28 eV). Thus, the predicted absorption maximum appears to be closer to that of the rhodopsin pigment (2.48 eV) and considerably red-shifted with respect to that measured in solution (2.82 eV in methanol). In addition, the absorption maxima associated with the blue, green, and red cone visual pigments are tentatively rationalized in terms of the spectral changes computed for PSB11 structures featuring differently twisted beta-ionone rings. More specifically, a blue-shifted absorption maximum is explained in terms of a large twisting of the beta-ionone ring (with respect to the main conjugated chain) in the visual S-cone (blue) pigment chromophore. In contrast, the chromophore of the visual L-cone (red) pigment is expected to have a nearly coplanar beta-ionone ring yielding a six double bond fully conjugated framework. Finally, the M-cone (green) chromophore is expected to feature a twisting angle between 10 and 60 degrees. The spectroscopic effects of the alkyl substituents on the PSB11 spectroscopic properties have also been investigated. It is found that they have a not negligible stabilizing effect on the S1-S0 energy gap (and, thus, cause a red shift of the absorption maximum) only when the double bond of the beta-ionone ring conjugates significantly with the rest of the conjugated chain.     

PHOTOSENSITIVITIES OF IODOPSIN AND RHODOPSINS

The relative photosensitivity and the molar extinction coefficient of a highly purified iodopsin (chicken red sensitive cone visual pigment) solubilized in a mixture of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and phosphatidylcholine (CHAPS-PC) were measured using bovine rhodopsin solubilized in 2% digitonin as a standard and compared with those of chicken and bovine rhodopsins. The photosensitivity obtained (1.08) was close to those of rhodopsins (chicken, 1.04; bovine, 0.99) in CHAPS-PC. The molar extinction coefficient of iodopsin (47,200) was 1.15-1.17 times higher than those of rhodopsins (chicken, 40,500; bovine, 41,200). The oscillator strength of iodopsin (0.60) calculated from the extinction coefficient was nearly identical to that of chicken rhodopsin (0.61), suggesting that the chromophore of iodopsin is similar in configuration to rhodopsin. In contrast, the difference in quantum yield between iodopsin (0.62) and chicken rhodopsin (0.70) suggests that the chromophore-opsin interaction after absorption of a photon by the chromophore may be different.     

CHROMOPHORE OF A LONG-LIVED PHOTOPRODUCT FORMED WITH METARHODOPSIN III IN THE ISOLATED FROG RETINA

Abstract Long-lived photoproducts of frog rhodopsin in isolated retina and digitonin solution have been investigated by spectrophotometry and their chromophores have been analyzed by high-pressure liquid chromatography (HPLC). By irradiation (> 560 nm) at 3°C and pH 8.6, a product analogous to metarhodopsin III (MIII) is formed, whose absorption maximum is at about 450 nm. This product decays more slowly than MIII does. The results of HPLC analysis indicate that the chromophore of this photoproduct is 7- cis retinal and that of MIII is all-trans retinal. The product possessing 7- cis retinal is called 7- cis photoproduct. The amount of 7- cis isomer in rhodopsin solution irradiated at various temperatures between 15°C and –82°C, has been determined. The results suggest that the 7- cis photoproduct can be formed by the photoconversion of lumirhodopsin and metarhodopsin I.     

Time-resolved resonance Raman analysis of chromophore structural changes in the formation and decay of rhodopsin's BSI intermediate

Time-resolved resonance Raman microchip flow experiments are performed to obtain the vibrational spectrum of the chromophore in rhodopsin's BSI intermediate and to probe structural changes in the bathorhodopsin-to-BSI and BSI-to-lumirhodopsin transitions. Kinetic Raman spectra from 250 ns to 3 micros identify the key vibrational features of BSI. BSI exhibits relatively intense HOOP modes at 886 and 945 cm(-1) that are assigned to C(14)H and C(11)H=C(12)H A(u) wags, respectively. This result suggests that in the bathorhodopsin-to-BSI transition the highly strained all-trans chromophore has relaxed in the C(10)-C(11)=C(12)-C(13) region, but is still distorted near C(14). The low frequency of the 11,12 A(u) HOOP mode in BSI compared with that of lumirhodopsin and metarhodopsin I indicates weaker coupling between the 11H and 12H wags due to residual distortion of the BSI chromophore near C(11)=C(12). The C=NH(+) stretching mode in BSI at 1653 cm(-1) exhibits a normal deuteriation induced downshift of 23 cm(-1), implying that there is no significant structural rearrangement of the Schiff base counterion region in the transition of bathorhodopsin to BSI. However, a dramatic Schiff base environment change occurs in the BSI-to-lumirhodopsin transition, because the 1638 cm(-1) C=NH(+) stretching mode in lumirhodopsin is unusually low and shifts only 7 cm(-1) in D(2)O, suggesting that it has essentially no H-bonding acceptor. With these data we can for the first time compare and discuss the room temperature resonance Raman vibrational structure of all the key intermediates in visual excitation.     

Activity switches of rhodopsin

Rhodopsin, the visual pigment of the rod photoreceptor cell contains as its light-sensitive cofactor 11-cis retinal, which is bound by a protonated Schiff base between its aldehyde group and the Lys296 side chain of the apoprotein. Light activation is achieved by 11-cis to all-trans isomerization and subsequent thermal relaxation into the active, G protein-binding metarhodopsin II state. Metarhodopsin II decays via two parallel pathways, which both involve hydrolysis of the Schiff base eventually to opsin and released all-trans retinal. Subsequently, rhodopsin's dark state is regenerated by a complicated retinal metabolism, termed the retinoid cycle. Unlike other retinal proteins, such as bacteriorhodopsin, this regeneration cycle cannot be short cut by light, because blue illumination of active metarhodopsin II does not lead back to the ground state but to the formation of largely inactive metarhodopsin III. In this review, mechanistic details of activating and deactivating pathways of rhodopsin, particularly concerning the roles of the retinal, are compared. Based on static and time-resolved UV/Vis and FTIR spectroscopic data, we discuss a model of the light-induced deactivation. We describe properties and photoreactions of metarhodopsin III and suggest potential roles of this intermediate for vision.     

Modulating rhodopsin receptor activation by altering the pKa of the retinal Schiff base

The visual pigment rhodopsin is a seven-transmembrane (7-TM) G protein-coupled receptor (GPCR). Activation of rhodopsin involves two pH-dependent steps: proton uptake at a conserved cytoplasmic motif between TM helices 3 and 6, and disruption of a salt bridge between a protonated Schiff base (PSB) and its carboxylate counterion in the transmembrane core of the receptor. Formation of an artificial pigment with a retinal chromophore fluorinated at C14 decreases the intrinsic pKa of the PSB and thereby destabilizes this salt bridge. Using Fourier transform infrared difference and UV-visible spectroscopy, we characterized the pH-dependent equilibrium between the active photoproduct Meta II and its inactive precursor, Meta I, in the 14-fluoro (14-F) analogue pigment. The 14-F chromophore decreases the enthalpy change of the Meta I-to-Meta II transition and shifts the Meta I/Meta II equilibrium toward Meta II. Combining C14 fluorination with deletion of the retinal beta-ionone ring to form a 14-F acyclic artificial pigment uncouples disruption of the Schiff base salt bridge from transition to Meta II and in particular from the cytoplasmic proton uptake reaction, as confirmed by combining the 14-F acyclic chromophore with the E134Q mutant. The 14-F acyclic analogue formed a stable Meta I state with a deprotonated Schiff base and an at least partially protonated protein counterion. The combination of retinal modification and site-directed mutagenesis reveals that disruption of the protonated Schiff base salt bridge is the most important step thermodynamically in the transition from Meta I to Meta II. This finding is particularly important since deprotonation of the retinal PSB is known to precede the transition to the active state in rhodopsin activation and is consistent with models of agonist-dependent activation of other GPCRs.     

(9Z)- and (11Z)-8-methylretinals for artificial visual pigment studies: stereoselective synthesis, structure, and binding models

Artificial visual pigment formation was studied by using 8-methyl-substituted retinals in an effort to understand the effect that alkyl substitution of the chromophore side chain has on the visual cycle. The stereoselective synthesis of the 9-cis and 11-cis isomers of 8-methylretinal, as well as the 5-demethylated analogues is also described. The key bond formations consist of a thallium-accelerated Suzuki cross-coupling reaction between cyclohexenylboronic acids and dienyliodides (C6-C7), and a highly stereocontrolled Horner-Wadsworth-Emmons or Wittig condensation (C11-C12). The cyclohexenylboronic acid was prepared by trapping the precursor cyclohexenyllithium species with B(OiPr)(3) or B(OMe)(3). The cyclohexenyllithium species is itself obtained by nBuLi-induced elimination of a trisylhydrazone (Shapiro reaction), or depending upon the steric hindrance of the ring, by iodine-metal exchange. In binding experiments with the apoprotein opsin, only 9-cis-5-demethyl-8-methylretinal yielded an artificial pigment; 9-cis-8-methylretinal simply provided residual binding, while evidence of artificial pigment formation was not found for the 11-cis analogues. Molecular-mechanics-based docking simulations with the crystal structure of rhodopsin have allowed us to rationalize the lack of binding displayed by the 11-cis analogues. Our results indicate that these isomers are highly strained, especially when bound, due to steric clashes with the receptor, and that these interactions are undoubtedly alleviated when 9-cis-5-demethyl-8-methylretinal binds opsin.     

EXTRACTION OF RETINAL FROM Paramecium bursaria

Abstract— The ciliated protozoan Paramecium bursaria is photosensitive. Retinal, which is the chromophore of the visual pigments of many multicellular animals, was extracted from P. bursaria and identified by HPLC. The procedure of identification was based on the change in polarity achieved by reducing retinal with sodium borohydrate to form retinol. This method is useful for the detection of small amounts of retinal in cells containing large amounts of other substances with similar polarity. The presence of retinal in P. bursaria suggests that this ciliate may contain a photoreceptor pigment with retinal as the chromophore.     

9-cis Retinal increased in retina of RPE65 knockout mice with decrease in coat pigmentation

The protein RPE65 is essential for the generation of the native chromophore, 11-cis retinal, of visual pigments. However, the Rpe65 knockout (Rpe65-/-) mouse shows a minimal visual response due to the presence of a pigment, isorhodopsin, formed with 9-cis retinal. Isorhodopsin accumulates linearly with prolonged dark-rearing of the animals. The majority of Rpe65-/- mice have an agouti coat color. A tan coat color subset of Rpe65-/- mice was found to have an enhanced visual response as measured by electroretinograms. The enhanced response was found to be due to increased levels of 9-cis retinal and isorhodopsin pigment levels. Animals of both coat colors reared in cyclic light have minimal levels of regenerated pigment and show photoreceptor degeneration. On dark-rearing, pigment accumulates and photoreceptor degeneration is decreased. In the tan Rpe65-/- mice, the level of photoreceptor degeneration is less than in the agouti animals, which have an increased pigment and decreased free opsin level. Therefore, photoreceptor damage correlates with the amount of the apoprotein present, supporting findings that the activity from unregenerated opsin can lead to photoreceptor degeneration.