Accessibility of the iodopsin chromophore.

Iodopsin can replace its chromophore (11-cis retinal) by added 9-cis retinal, resulting in the formation of isoiodopsin. NaBH4 bleaches iodopsin in the dark. In a relatively low concentration of digitonin, the scotopsin (the protein moiety of chicken rhodopsin) removes 11-cis retinal from iodopsin in the dark. These facts suggest that the linkage of the chromophore to opsin in the iodopsin molecule (presumably a Schiff-base linkage) is accessible to these reagents, which is different from the situation in rhodopsin.     

Wavelength regulation in iodopsin, a cone pigment.

The opsin shift, the difference in wavenumber between the absorption peak of a visual pigment and the protonated Schiff base of the chromophore, represents the influence of the opsin binding site on the chromophore. The opsin shift for the chicken cone pigment iodopsin is much larger than that for rhodopsin. To understand the origin of this opsin shift and the mechanism of wavelength regulation in iodopsin, a series of synthetic 9-cis and 11-cis dehydro- and dihydro-retinals was used to regenerate iodopsin-based pigments. The opsin shifts of these pigments are quite similar to those found in bacteriorhodopsin-based artificial pigments. On the basis of these studies, a tentative model of wavelength regulation in iodopsin is proposed.     

Accessibility of the iodopsin chromophore

Iodopsin can replace its chromophore (11-cis retinal) by added 9-cis retinal, resulting in the formation of isoiodopsin. NaBH₄ bleaches iodopsin in the dark. In a relatively low concentration of digitonin, the scotopsin (the protein moiety of chicken rhodopsin) removes 11-cis retinal from isopsin in the iodopsin These facts suggests that the linkage of the chromophore to opsin in the iodopsin molecule (presumably a Schiff-base linkage) is accessible to these reagents, which is different from the situation in rhodopsin.     

Synthetic pigment analogues of the purple membrane protein.

Nonphysiological analogues of retinal have been shown to form pigments in reactions with the apoprotein of the purple membrane of Halobacterium halobium. Both the all-trans and 13-cis isomers of a retinal analogue, having an elongated chain with an extra double bond, formed pigments. Unlike the native all-trans and 13-cis retinal1-based pigments, the new pigments were not interconvertible with each other and were unstable against hydroxylamine. When incorporated into phospholipid vesicles, they showed no proton pumping activity upon illumination. The ability of the extended-length retinal to form pigments contrasts with its nonreactivity with opsin (apoprotein of rhodopsin), suggesting a less stringent binding site for the purple membrane chromophore. All-trans retinal2 also combined with bleached purple membrane to form a blue pigment absorbing at ca. 590 nm. Like the native purple membrane, the blu membrane showed proton pumping activity upon illumination in phospholipid vesicles.     

Effects of chloride on chicken iodopsin and the chromophore transfer reactions from iodopsin to scotopsin and B-photopsin

Spectroscopic properties of chicken iodopsin were investigated in correlation with the concentration of chloride in digitonin extracts. When chloride in the extract was depleted by extensive dialysis, chloride-depleted iodopsin (absorption maximum, 512 nm) was formed. It was converted to chloride-bound iodopsin (absorption maximum, 562 nm) by the addition of chloride in the extract. There existed an equilibrium between two forms of iodopsin with a dissociation constant of 0.8 mM chloride. The chromophore-transfer reaction from iodopsin to scotopsin or B-photopsin, the protein moiety of chicken rhodopsin or chicken blue-sensitive cone pigment, respectively, in digitonin extract was also investigated in correlation with the concentrations of chloride, other monovalent and divalent anions, and detergent. The chromophore of chloride-depleted iodopsin was easily transferred to scotopsin in the extract, resulting in formation of rhodopsin. On the other hand, chloride-bound iodopsin was fairly stable even in the presence of scotopsin, indicating that the reaction is inhibited by binding of chloride to iodopsin. The chromophore-transfer reaction to B-photopsin was also observed from chloride-depleted iodopsin but not from chloride-bound iodopsin. The reaction was observable in the 10% digitonin extract as well as in the 2% digitonin extract. The reaction was also observed when 25 mM Na2SO4 was present in the mixture instead of NaCl, but was not when 67 mM NaNO3 was present. All these facts suggest that the chloride binding site of iodopsin does not accept a divalent anion such as SO4(2+), but does accept a monovalent anion such as Cl- or NO3-, which causes inhibition of the chromophore transfer.     

Cyclic nucleotide-gated ion channels in rod photoreceptors are protected from retinoid inhibition

In vertebrate rods, photoisomerization of the 11-cis retinal chromophore of rhodopsin to the all-trans conformation initiates a biochemical cascade that closes cGMP-gated channels and hyperpolarizes the cell. All-trans retinal is reduced to retinol and then removed to the pigment epithelium. The pigment epithelium supplies fresh 11-cis retinal to regenerate rhodopsin. The recent discovery that tens of nanomolar retinal inhibits cloned cGMP-gated channels at low [cGMP] raised the question of whether retinoid traffic across the plasma membrane of the rod might participate in the signaling of light. Native channels in excised patches from rods were very sensitive to retinoid inhibition. Perfusion of intact rods with exogenous 9- or 11-cis retinal closed cGMP-gated channels but required higher than expected concentrations. Channels reopened after perfusing the rod with cellular retinoid binding protein II. PDE activity, flash response kinetics, and relative sensitivity were unchanged, ruling out pharmacological activation of the phototransduction cascade. Bleaching of rhodopsin to create all-trans retinal and retinol inside the rod did not produce any measurable channel inhibition. Exposure of a bleached rod to 9- or 11-cis retinal did not elicit channel inhibition during the period of rhodopsin regeneration. Microspectrophotometric measurements showed that exogenous 9- or 11-cis retinal rapidly cross the plasma membrane of bleached rods and regenerate their rhodopsin. Although dark-adapted rods could also take up large quantities of 9-cis retinal, which they converted to retinol, the time course was slow. Apparently cGMP-gated channels in intact rods are protected from the inhibitory effects of retinoids that cross the plasma membrane by a large-capacity buffer. Opsin, with its chromophore binding pocket occupied (rhodopsin) or vacant, may be an important component. Exceptionally high retinoid levels, e.g., associated with some retinal degenerations, could overcome the buffer, however, and impair sensitivity or delay the recovery after exposure to bright light.     

Photosensitivity of 10-substituted visual pigment analogues: detection of a specific secondary opsin-retinal interaction

The photosensitivities of the bovine rhodopsin and gecko pigment 521 analogues regenerated from C-10-substituted analogues of 11-cis- and 9-cis-retinals were determined by two different methods. A similar reactivity trend was noted for both pigment systems as revealed in the photosensitivity of the gecko pigments and relative quantum yields of the bovine analogues. The 10-fluoro-11-cis photopigments had a photosensitivity less than, but approaching, that of the native (11-cis) visual pigment while the 10-fluoro-9-cis photopigments had a much lower photosensitivity than the parent 9-cis regenerated pigment. The results are interpreted in terms of recently described models of rhodopsin architecture and of the primary molecular reaction of visual pigments to light. The unusually low photoreactivity of the 10-fluoro-9-cis pigment molecule is viewed as the result of a regiospecific hydrogen-bonding interaction of the electronegative fluorine atom to the opsin.     

Bathoproducts of rhodopsin, isorhodopsin I, and isorhodopsin II.

Bathorhodopsins were prepared by partially (10--15%) photoconverting bovine rhodopsin (11-cis chromophore) or isorhodopsin I (9-cis chromophore) at 77 degrees K; care was taken to avoid establishing photostationary states. The absorption spectra calculated for the bathorhodopsins derived from the two parent pigments are identical in their lambda max 'S, bandwidths, and extinction coefficients. This result provides further support for the hypothesis that bathorhodopsin is a common intermediate between an 11-cis pigment (rhodopsin) and a 9-cis one (isorhodopsin I) and thus probably has an all-trans chromophore. This in turn is strong evidence for the cis-trans isomerization model of the primary event in vision. The spectrum of the bathoproduct of isorhodopsin II (9,13-dicis chromophore) is different from the other pigments' bathoproducts.     

Characterization of the chromophore of the third rhodopsin-like pigment of Halobacterium halobium and its photoproduct.

Halobacterium halobium contains at least three retinal-containing pigments: bacteriorhodopsin, halorhodopsin, and a third rhodopsin-like pigment (tR) absorbing at approximately 590 nm, tR590. Illumination of tR590 gives rise to a very long-lived blue absorbing photoproduct, tR370. Using high-performance liquid chromatography we show that the chromophore of tR590 is primarily all-trans retinal and its conversion by light to tR370 causes the chromophore to isomerize primarily to the 13-cis conformation. Irradiation of the tR370 gives rise to a transient photoproduct absorbing at approximately 520 nm that decays back to the initial pigment tR590. In addition to all-trans retinal, the apomembrane of tR can also combine with 13-cis retinal but not with the 9- or 11-cis isomers.     

Methyl substituents at the 11 or 12 position of retinal profoundly and differentially affect photochemistry and signalling activity of rhodopsin

The C-11=C-12 double bond of the retinylidene chromophore of rhodopsin holds a central position in its light-induced photoisomerization and hence the photosensory function of this visual pigment. To probe the local environment of the HC-11=C-12H element we have prepared the 11-methyl and 12-methyl derivatives of 11-Z retinal and incorporated these into opsin to generate the rhodopsin analogs 11-methyl and 12-methyl rhodopsin. These analog pigments form with much slower kinetics and lower efficiency than the native pigment. The initial photochemistry and the signaling activity of the analog pigments were investigated by UV-vis and FTIR spectroscopy, and by a G protein activation assay. Our data indicate that the ultrafast formation of the first photointermediate is strongly perturbed by the presence of an 11-methyl substituent, but much less by a 12-methyl substituent. These results support the current concept of the mechanism of the primary photoisomerization event in rhodopsin. An important stronghold of this concept is an out-of-plane movement of the C-12H element, which is facilitated by torsion as well as extended positive charge delocalization into the C-10-C-13 segment of the chromophore. We argue that this mechanism is maintained principally with a methyl substituent at C-12. In addition, we show that both an 11-methyl and a 12-methyl substitutent perturb the photointermediate cascade and finally yield a low-activity state of the receptor. The 11-methyl pigment retains about 30% of the G protein activation rate of native rhodopsin, while the 12-methyl chromophore behaves like an inverse agonist up to at least 20 degrees C, trapping the protein in a perturbed Meta-I-like conformation. We conclude that the isomerization region of the chromophore and the spatial structure of the binding site are finely tuned, in order to achieve a high photosensory potential with an efficient pathway to a high-activity state.     

Effect of anion binding on the thermal reverse reaction of bathoiodopsin: anion stabilizes two forms of iodopsin

The thermal reactions of the bathoproduct of the long wavelength sensitive visual pigment iodopsin were investigated under various anionic and environmental conditions, to get an insight into the mechanism leading to the unusual thermal isomerization of the retinal chromophore from the trans to the 11-cis form at very low temperatures (-160 degrees C). The all-trans chromophore of the bathoiodopsin produced from iodopsin in the presence of chloride thermally reverted to the 11-cis form, while in the presence of nitrate it kept its all-trans configuration upon warming. Different protein environments, either in a detergent or in phosphatidylcholine (PC) liposomes, did not change the reaction characteristics of the bathoiodopsins under the two anionic conditions. However, reaction characteristics of bathoiodopsins produced in the absence of small anions were dependent on the environment. The trans-to-cis isomerization occurred upon warming of bathoiodopsin in the presence of detergent but not in liposomes. Spectral measurements revealed that iodopsin in the absence of small anions is a mixture of two spectrally distinct forms that exhibit absorption maxima and reaction characteristics similar to those of chloride-bound and nitrate-bound iodopsins, respectively. Thus, iodopsin exhibits two conformational states, each of which is stabilized by the binding of chloride and nitrate, respectively.     

Spectroscopic study of the batho-to-lumi transition during the photobleaching of rhodopsin using ring-modified retinal analogues

Photochemical and subsequent thermal reactions of rhodopsin containing 9-cis-retinal [Rh(9)] or one of four analogues with 9-cis geometries formed from ring-modified retinals, alpha-retinal [alpha Rh(9)], acyclic retinal [AcRh(9)], acyclic alpha-retinal [Ac alpha Rh(9)], and 5-isopropyl-alpha-retinal [P alpha Rh(9)] were investigated by low-temperature spectrophotometry and nanosecond laser photolysis. Irradiation of each pigment at -180 degrees C produced a photosteady-state mixture containing the original 9-cis pigment, its 11-cis pigment, and a photoproduct, indicating that the primary process of each pigment is a photoisomerization of its chromophore. The photoproduct produced by the irradiation of AcRh(9) had an absorption spectrum red shifted from the original AcRh(9) and was identified as the batho intermediate of AcRh(9). It was converted to the lumi intermediate through a metastable species, the BL intermediate, which has never been detected in Rh(9) at low temperature and whose absorption maximum was at shorter wavelengths than that of the batho intermediate. In contrast, the absorption maxima of the photoproducts produced from the other analogue pigments were at shorter wavelengths than those of the original pigments. They were identified as BL intermediates on the basis of their absorption maxima and thermal stabilities. The formation time constant of the lumi intermediate at room temperature was found to be dependent on the extent of modification of the ring portion of the chromophore, decreasing with the complete truncation of the cyclohexenyl ring [Ac alpha Rh(9)] and increasing with the attachment of the isopropyl group to the ring [P alpha Rh(9)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Regeneration of bovine and octopus opsins in situ with natural and artificial retinals

We consider the problem of color regulation in visual pigments for both bovine rhodopsin (lambda max = 500 nm) and octopus rhodopsin (lambda max = 475 nm). Both pigments have 11-cis-retinal (lambda max = 379 nm, in ethanol) as their chromophore. These rhodopsins were bleached in their native membranes, and the opsins were regenerated with natural and artificial chromophores. Both bovine and octopus opsins were regenerated with the 9-cis- and 11-cis-retinal isomers, but the octopus opsin was additionally regenerated with the 13-cis and all-trans isomers. Titration of the octopus opsin with 11-cis-retinal gave an extinction coefficient for octopus rhodopsin of 27,000 +/- 3000 M-1 cm-1 at 475 nm. The absorption maxima of bovine artificial pigments formed by regenerating opsin with the 11-cis dihydro series of chromophores support a color regulation model for bovine rhodopsin in which the chromophore-binding site of the protein has two negative charges: one directly hydrogen bonded to the Schiff base nitrogen and another near carbon-13. Formation of octopus artificial pigments with both all-trans and 11-cis dihydro chromophores leads to a similar model for octopus rhodopsin and metarhodopsin: there are two negative charges in the chromophore-binding site, one directly hydrogen bonded to the Schiff base nitrogen and a second near carbon-13. The interaction of this second charge with the chromophore in octopus rhodopsin is weaker than in bovine, while in metarhodopsin it is as strong as in bovine.     

Photochromicity of Anabaena sensory rhodopsin, an atypical microbial receptor with a cis-retinal light-adapted form

We characterize changes in isomeric states of the retinylidene chromophore during light-dark adaptation and photochemical reactions of Anabaena (Nostoc) sp. PCC7120 sensory rhodopsin (ASR). The results show that ASR represents a new type of microbial rhodopsin with a number of unusual characteristics. The three most striking are: (i) a primarily all-trans configuration of retinal in the dark-adapted state and (ii) a primarily 13-cis light-adapted state with a blue-shifted and lower extinction absorption spectrum, opposite of the case of bacteriorhodopsin; and (iii) efficient reversible light-induced interconversion between the 13-cis and all-trans unphotolyzed states of the pigment. The relative amount of ASR with cis and trans chromophore forms depends on the wavelength of illumination, providing a mechanism for single-pigment color sensing analogous to that of phytochrome pigments. In addition ASR exhibits unusually slow formation of L-like and M-like intermediates, with a dominant accumulation of M during the photocycle. Co-expression of ASR with its putative cytoplasmic transducer protein shifts the absorption maximum and strongly decreases the rate of dark adaptation of ASR, confirming interaction between the two proteins. Thus ASR, the first non-haloarchaeal sensory rhodopsin characterized, demonstrates the diversity of photochemistry of microbial rhodopsins. Its photochromic properties and the position of its two ground state absorption maxima suggest it as a candidate for controlling differential photosynthetic light-harvesting pigment synthesis (chromatic adaptation) or other color-sensitive physiological responses in Anabaena cells.     

Role of the 9-methyl group of retinal in cone visual pigments

In rhodopsin, the 9-methyl group of retinal has previously been identified as being critical in linking the ligand isomerization with the subsequent protein conformational changes that result in the activation of its G protein, transducin. Here, we report studies on the role of this methyl group in the salamander rod and cone pigments. Pigments were generated by combining proteins expressed in COS cells with 11-cis 9-demethyl retinal, where the 9-methyl group on the polyene chain has been deleted. The absorption spectra of all pigments were blue-shifted. The red cone and blue cone/green rod pigments were unstable to hydroxylamine; whereas, the rhodopsin and UV cone pigments were stable. The lack of the 9-methyl group of the chromophore did not affect the ability of the red cone and blue cone/green rod pigments to activate transducin. On the other hand, with the rhodopsin and UV cone pigments, activation was diminished. Interestingly, the red cone pigment containing the retinal analogue remained active longer than the native pigment. Thus, the 9-methyl group of retinal is not important in the activation pathway of the red cone and blue cone/green rod pigments. However, for the red cone pigment, the 9-methyl group of retinal appears to be critical in the deactivation pathway.     

Protein-bound water molecules in primate red- and green-sensitive visual pigments

Protein-bound water molecules play crucial roles in the structure and function of proteins. The functional role of water molecules has been discussed for rhodopsin, the light sensor for twilight vision, on the basis of X-ray crystallography, Fourier transform infrared (FTIR) spectroscopy, and a radiolytic labeling method, but nothing is known about the protein-bound waters in our color visual pigments. Here we apply low-temperature FTIR spectroscopy to monkey red (MR)- and green (MG)-sensitive color pigments at 77 K and successfully identify water vibrations using D(2)O and D(2)(18)O in the whole midinfrared region. The observed water vibrations are 6-8 for MR and MG, indicating that several water molecules are present near the retinal chromophore and change their hydrogen bonds upon retinal photoisomerization. In this sense, color visual pigments possess protein-bound water molecules essentially similar to those of rhodopsin. The absence of strongly hydrogen-bonded water molecules (O-D stretch at <2400 cm(-1)) is common between rhodopsin and color pigments, which greatly contrasts with the case of proton-pumping microbial rhodopsins. On the other hand, two important differences are observed in water signal between rhodopsin and color pigments. First, the water vibrations are identical between the 11-cis and 9-cis forms of rhodopsin, but different vibrational bands are observed at >2550 cm(-1) for both MR and MG. Second, strongly hydrogen-bonded water molecules (2303 cm(-1) for MR and 2308 cm(-1) for MG) are observed for the all-trans form after retinal photoisomerization, which is not the case for rhodopsin. These specific features of MR and MG can be explained by the presence of water molecules in the Cl(-)-biding site, which are located near positions C11 and C9 of the retinal chromophore. The averaged frequencies of the observed water O-D stretching vibrations for MR and MG are lower as the λ(max) is red-shifted, suggesting that water molecules are involved in the color tuning of our vision.     

The pKa of the protonated Schiff bases of gecko cone and octopus visual pigments.

A visual pigment is composed of retinal bound to its apoprotein by a protonated Schiff base linkage. Light isomerizes the chromophore and eventually causes the deprotonation of this Schiff base linkage at the meta II stage of the bleaching cycle. The meta II intermediate of the visual pigment is the active form of the pigment that binds to and activates the G protein transducin, starting the visual cascade. The deprotonation of the Schiff base is mandatory for the formation of meta II intermediate. We studied the proton binding affinity, pKa, of the Schiff base of both octopus rhodopsin and the gecko cone pigment P521 by spectral titration. Several fluorinated retinal analogs have strong electron withdrawing character around the Schiff base region and lower the Schiff base pKa in model compounds. We regenerated octopus and gecko visual pigments with these fluorinated and other retinal analogs. Experiments on these artificial pigments showed that the spectral changes seen upon raising the pH indeed reflected the pKa of the Schiff base and not the denaturation of the pigment or the deprotonation of some other group in the pigment. The Schiff base pKa is 10.4 for octopus rhodopsin and 9.9 for the gecko cone pigment. We also showed that although the removal of Cl- ions causes considerable blue-shift in the gecko cone pigment P521, it affects the Schiff base pKa very little, indicating that the lambda max of visual pigment and its Schiff base pKa are not tightly coupled.     

Studies on structure and function of rhodopsin by use of cyclopentatrienylidene 11-cis-locked-rhodopsin

The photochemical reaction of cyclopentatrienylidene 11-cis-locked-rhodopsin derived from cyclopentatrienylidene 11-cis-locked-retinal and cattle opsin was spectrophotometrically studied. The difference absorption spectrum between the cyclopentatrienylidene 11-cis-locked-rhodopsin and its retinal oxime had its maximum at 495 nm (P-495). Irradiation of P-495 at -196 degrees C with either blue light or orange light caused no spectral change, supporting the cis-trans isomerization hypothesis for formation of bathorhodopsin. Upon irradiation of P-495 at 0 degree C with orange light, however, its absorption spectrum shifted to a shorter wavelength owing to formation of a hypsochromic product. The difference absorption spectrum between this product (P-466) and its retinal oxime showed its maximum at 466 nm. Analysis of retinal isomers by high-performance liquid chromatography showed that this spectral shift was not accompanied by photoisomerization of the chromophore. P-466 could almost completely be photoconverted to the original pigment (P-495) by irradiation at 0 degree C with blue light with little formation of the other isomeric form of its chromophore. The alpha-band of the circular dichroism spectrum of P-495 was very small in comparison with that of rhodopsin, while that of P-466 was comparable to it. These facts suggest that P-495 has a planar conformation in the side chain of the chromophore and that P-466 has a twisted one, probably at the C8-C9 single bond. Cyclic-GMP phosphodiesterase in frog rod outer segment was activated by neither P-495 nor P-466. This result suggests that the isomerization of the retinylidene chromophore of rhodopsin is indispensable in the phototransduction process.     

A nonbleachable rhodopsin analogue with a slow photocycle

Archaeal rhodopsins, e.g. bacteriorhodopsin, all have cyclic photoreactions. Such cycles are achieved by a light-induced isomerization step of their retinal chromophores, which thermally re-isomerize in the dark. Visual pigment rhodopsins, which contain in the dark state an 11-cis retinal Schiff base, do not share such a mechanism, and following light absorption, they experience a bleaching process and a subsequent release of the photo-isomerized all-trans chromophore from the binding pocket. The pigment is eventually regenerated by the rebinding of a new 11-cis retinal. In the artificial visual pigment, Rh(6.10), in which the retinal chromophore is locked in an 11-cis geometry by the introduction of a six-member ring structure, an activated receptor may be formed by light-induced isomerization around other double bonds. We have examined this activation of Rh(6.10) by UV-visible and FTIR spectroscopy and have revealed that Rh(6.10) is a nonbleachable pigment. We could further show that the activated receptor consists of two different subspecies corresponding to 9-trans and 9-cis isomers of the chromophore. Both subspecies relax in the dark via separate pathways back to their respective inactive states by thermal isomerization presumably around the C(13)=C(14) double bond. This nonbleachable pigment can be repeatedly photolyzed to undergo identical activation-relaxation cycles. The rate constants of these photocycles are pH-dependent, and the half-times vary between several hours at acidic pH and about 1.5 min at neutral to alkaline pH, which is several orders of magnitude longer than for bacteriorhodopsin.     

Utilization of retinoids in the bullfrog retina

The capacity to generate 11-cis retinal from retinoids arising naturally in the eye was examined in the retina of the bullfrog, Rana catesbeiana. Retinoids, co-suspended with phosphatidylcholine, were applied topically to the photoreceptor surface of the isolated retina after substantial bleaching of the native visual pigment. The increase in photoreceptor sensitivity associated with the formation of rhodopsin, used as an assay for the appearance of 11-cis retinal in the receptors, was analyzed by extracellular measurement of the photoreceptor potential; in separate experiments using the isolated retina or receptor outer segment preparations, the formation of rhodopsin was measured spectrophotometrically. Treatments with the 11- cis isomers of retinal and retinol induced significant increases in both the rhodopsin content and photic sensitivity of previously bleached receptors. The all-trans isomers of retinyl palmitate, retinol, and retinal, as well as the 11-cis isomer of retinyl palmitate, were inactive by both the electrophysiological and spectrophotometric criteria for the generation of rhodopsin. Treatment with any one of the "inactive" retinoids did not abolish the capacity of subsequently applied 11-cis retinal or 11-cis retinol to promote the formation of rhodopsin. The data are discussed in relation to the interconversions of retinoids ("visual cycle of vitamin A") thought to mediate the regeneration of rhodopsin in vivo after extensive bleaching.