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.     

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

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

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.     

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

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

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.     

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.     

Engineering a "steric doorstop" in rhodopsin: converting an inverse agonist to an agonist

The crystal structures of rhodopsin depict the inactive conformation of rhodopsin in the dark. The 11-cis retinoid chromophore, the inverse agonist holding rhodopsin inactive, is well-resolved. Thr118 in helix 3 is the closest amino acid residue next to the 9-methyl group of the chromophore. The 9-methyl group of retinal facilitates the transition from an inactive metarhodopsin I to the active metarhodopsin II intermediate. In this study, a site-specific mutation of Thr118 to the bulkier Trp was made with the idea to induce an active conformation of the protein. The data indicate that such a mutation does indeed result in an active protein that depends on the presence of the ligand, specifically the 9-methyl group. As a result of this mutation, 11-cis retinal has been converted to an agonist. The apoprotein form of this mutant is no more active than the wild-type apoprotein. However, unlike wild-type rhodopsin, the covalent linkage of the ligand can be attacked by hydroxylamine in the dark. The combination of the Thr118Trp mutation and the 9-methyl group of the chromophore behaves as a "steric doorstop" holding the protein in an open and active conformation.     

Synthesis of the all-trans-retinal chromophore of retinal G protein-coupled receptor opsin in cultured pigment epithelial cells.

Light-dependent production of 11-cis-retinal by the retinal pigment epithelium (RPE) and normal regeneration of rhodopsin under photic conditions involve the RPE retinal G protein-coupled receptor (RGR) opsin. This microsomal opsin is bound to all-trans-retinal which, upon illumination, isomerizes stereospecifically to the 11-cis isomer. In this paper, we investigate the synthesis of the all-trans-retinal chromophore of RGR in cultured ARPE-hRGR and freshly isolated bovine RPE cells. Exogenous all-trans-[(3)H]retinol is incorporated into intact RPE cells and converted mainly into retinyl esters and all-trans-retinal. The intracellular processing of all-trans-[(3)H]retinol results in physiological binding to RGR of a radiolabeled retinoid, identified as all-trans-[(3)H]retinal. The ARPE-hRGR cells contain a membrane-bound NADPH-dependent retinol dehydrogenase that reacts efficiently with all-trans-retinol but not the 11-cis isomer. The NADPH-dependent all-trans-retinol dehydrogenase activity in isolated RPE microsomal membranes can be linked in vitro to specific binding of the chromophore to RGR. These findings provide confirmation that RGR opsin binds the chromophore, all-trans-retinal, in the dark. A novel all-trans-retinol dehydrogenase exists in the RPE and performs a critical function in chromophore biosynthesis.     

Squid retinochrome. Configurational changes of the retinal chromophore.

The configurations of the retinal chromophore in light and dark reactions of squid retinochrome were investigated by means of high-performance liquid chromatography. Orange light isomerized the chromophore of retinochrome, all-trans-retinal, mainly to the 11-cis configuration in metaretinochrome. Irradiation with shorter-wavelength lights not only accelerates the photoreversal of metaretinochrome to retinochrome but also leads to a slight production of isoretinochrome (13-cis-retinochrome), yielding a photoequilibrium mixture of three kinds of retinochrome. 13-cis- and 9-cis-retinochromes are photosensitive, and are converted into metaretinochrome upon irradiation with orange light. When steadily exposed to orange light in the presence of a trace of retinochrome-protein, all of the all-trans-, 13-cis-, and 9-cis-retinals are catalytically isomerized only to the 11-cis form, although the reaction rate is reduced in the order of the retinals listed above. In the dark, 9-cis-retinochrome, like retinochrome, remains unchanged, but both meta- and 13-cis-retinochromes slowly change to retinochrome. The chromophore of 13-cis-retinochrome changes directly to the all-trans form, whereas the 11-cis chromophore of metaretinochrome goes to all-trans mainly through the 13-cis form. The direct isomerization from 11-cis to all-trans hardly occurs at temperatures as low as 20 degrees C, and shows high values of the activation enthalpy and entropy changes. Based upon these findings, the role of retinochrome in the photoreception of the visual cells is discussed.     

Squid retinochrome

Retinochrome is a photosensitive pigment located primarily in the inner portions of the visual cells of cephalopods. Its absorption spectrum resembles that of rhodopsin, but its chromophore is all-trans retinal, which light isomerizes to 11-cis, the reverse of the situation in rhodopsin. The 11-cis photoproduct of retinochrome slowly reverts to retinochrome in the dark. The chromophoric site of retinochrome is more reactive than that of most visual pigments: (a) Hydroxylamine converts retinochrome in the dark to all-trans retinal oxime + retinochrome opsin. (by Sodium borohydride reduces it to N-retinyl opsin. (c) Lambda max of retinochrome shifts from 500 to 515 nm as the pH is raised from 6 to 10, with a loss of absorption above pH 8; meanwhile above this PH a second band appears at shorter wavelengths with lambda max 375 nm. These changes are reversible. (d) If retinochrome is incubated with all-trans 3-dehydroretinal (retinal2) in the dark, some 3-dehydroretinochrome (retinochrome2, lambda max about 515 nm) is formed. Conversely, when retinochrome2, made by adding all-trans retinal2 to bleached retinochrome or retinochrome opsin, is incubated in the dark with all-trans retinal some of it is converted to retinochrome. Retinal and 3-dehydroretinal therefore can replace each other as chromophores in the dark.     

Spectral tuning in salamander visual pigments studied with dihydroretinal chromophores.

In visual pigments, opsin proteins regulate the spectral absorption of a retinal chromophore by mechanisms that change the energy level of the excited electronic state relative to the ground state. We have studied these mechanisms by using photocurrent recording to measure the spectral sensitivities of individual red rods and red (long-wavelength-sensitive) and blue (short-wavelength-sensitive) cones of salamander before and after replacing the native 3-dehydro 11-cis retinal chromophore with retinal analogs: 11-cis retinal, 3-dehydro 9-cis retinal, 9-cis retinal, and 5,6-dihydro 9-cis retinal. The protonated Schiff's bases of analogs with unsaturated bonds in the ring had broader spectra than the same chromophores bound to opsins. Saturation of the bonds in the ring reduced the spectral bandwidths of the protonated Schiff's bases and the opsin-bound chromophores and made them similar to each other. This indicates that torsion of the ring produces spectral broadening and that torsion is limited by opsin. Saturating the 5,6 double bond in retinal reduced the perturbation of the chromophore by opsin in red and in blue cones but not in red rods. Thus an interaction between opsin and the chromophoric ring shifts the spectral maxima of the red and blue cone pigments, but not that of the red rod pigment.     

Photoreactions of metarhodopsin III

Meta III is an inactive intermediate thermally formed following light activation of the visual pigment rhodopsin. It is produced from the Meta I/Meta II photoproduct equilibrium of rhodopsin by a thermal isomerization of the protonated Schiff base C=N bond of Meta I, and its chromophore configuration is therefore all-trans 15-syn. In contrast to the dark state of rhodopsin, which catalyzes exclusively the cis to trans isomerization of the C11=C12 bond of its 11-cis 15-anti chromophore, Meta III does not acquire this photoreaction specificity. Instead, it allows for light-dependent syn to anti isomerization of the C15=N bond of the protonated Schiff base, yielding Meta II, and for trans to cis isomerizations of C11=C12 and C9=C10 of the retinal polyene, as shown by FTIR spectroscopy. The 11-cis and 9-cis 15-syn isomers produced by the latter two reactions are not stable, decaying on the time scale of few seconds to dark state rhodopsin and isorhodopsin by thermal C15=N isomerization, as indicated by time-resolved FTIR methods. Flash photolysis of Meta III produces therefore Meta II, dark state rhodopsin, and isorhodopsin. Under continuous illumination, the latter two (or its unstable precursors) are converted as well to Meta II by presumably two different mechanisms.     

The endogenous chromophore of retinal G protein-coupled receptor opsin from the pigment epithelium

The recent identification of nonvisual opsins has revealed an expanding family of vertebrate opsin genes. The retinal pigment epithelium (RPE) and Müller cells contain a blue and UV light-absorbing opsin, the RPE retinal G protein-coupled receptor (RGR, or RGR opsin). The spectral properties of RGR purified from bovine RPE suggest that RGR is conjugated in vivo to a retinal chromophore through a covalent Schiff base bond. In this study, the isomeric structure of the endogenous chromophore of RGR was identified by the hydroxylamine derivatization method. The retinaloximes derived from RGR in the dark consisted predominantly of the all-trans isomer. Irradiation of RGR with 470-nm monochromatic or near-UV light resulted in stereospecific isomerization of the bound all-trans-retinal to an 11-cis configuration. The stereospecificity of photoisomerization of the all-trans-retinal chromophore of RGR was lost by denaturation of the protein in SDS. Under the in vitro conditions, the photosensitivity of RGR is at least 34% that of bovine rhodopsin. These results provide evidence that RGR is bound in vivo primarily to all-trans-retinal and is capable of operating as a stereospecific photoisomerase that generates 11-cis-retinal in the pigment epithelium.     

Molecular mechanism of spontaneous pigment activation in retinal cones

Spontaneous current and voltage fluctuations (dark noise) in the photoreceptor cells of the retina limit the ability of the visual system to detect dim light. We recorded the dark current noise of individual salamander L cones. Previous work showed that the dark noise in these cells arises from thermal activation of the visual pigment. From the temperature dependence of the rate of occurrence of elementary noise events, we found an Arrhenius activation energy E(a) of 25 +/- 7 kcal/mol (mean +/- SD). This E(a) is similar to that reported for the thermal isomerization of 11-cis retinal in solution, suggesting that the cone pigment noise results from isomerization of the retinal chromophore. E(a) for the cone noise is similar to that previously reported for the "photon-like" noise of rods, but the preexponential factor is five orders of magnitude higher. To test the hypothesis that thermal isomerization can only occur in molecules whose Schiff base linkage is unprotonated, we changed the pH of the solution bathing the cone outer segment. This had little effect on the rate of occurrence of elementary noise events. The rate was also unchanged when the cone was exposed to Ringer solution made up from heavy water, whose solvent isotope effect should reduce the probability, that the Schiff base nitrogen is naked.     

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.     

Assignment of the vibrational modes of the chromophores of iodopsin and bathoiodopsin: low-temperature fourier transform infrared spectroscopy of 13C- and 2H-labeled iodopsins

To investigate the chromophore structures of iodopsin and its low-temperature photoproducts, we have assigned their vibrational bands in the Fourier transform infrared (FTIR) spectra using iodopsin samples that were reconstituted with a series of (13)C- and deuterium-labeled retinals. The analyses of the vibrational bands in the fingerprint and hydrogen-out-of-plane (HOOP) regions indicated that the structure of the chromophores in the iodopsin system differs near their centers from those in the rhodopsin system. Compared to rhodopsin, the chromophore of the batho intermediate of iodopsin is twisted in the C(12) to C(14) regions but is more planar around C(11) region. The large amount of twisting was reduced by removing the chloride ion from the iodopsin, suggesting that this twisting hinders the relaxation of the torsion near C(11) necessary for the transition to the lumi intermediate and thus results in the thermal reversion of the batho intermediate back to the iodopsin. From the analyses of the C=NH and C=ND stretching bands, we conclude that the displacement of the Schiff base region upon photoisomerization of the chromophore is restricted, as is the case for rhodopsin. These results indicated that iodopsin's chromophore has a unique structure near its center and that this difference is enhanced by the binding of chloride nearby.     

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

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

Why 11-cis-Retinal?

The C₂₀ diterpenoid compound retinal is the chromophore of thevisual pigments the rhodopsins, and the pigments present inHalobacterium halobium, namely, bacteriorhodopsin (proton pump),halorhodopsin (chloride pump), and the sensory rhodopsins (phototaxisreceptor). In all cases, they are bound covalently to the receptorprotein by a protonated Schiff base. However, in rhodopsins,the retinal is the 11-cis isomer, whereas in H. halobium pigmentsit is the all-trans isomer. Why did Nature choose retinal asthe chromophore, and why 11-cis in some cases and all-transin other cases? Also why is the chromophore a protonated Schiffbase? These points are addressed after giving an outline ofthe current status of the various photoreceptor pigments     

Retinal analog restoration of photophobic responses in a blind Chlamydomonas reinhardtii mutant. Evidence for an archaebacterial like chromophore in a eukaryotic rhodopsin.

The strain CC-2359 of the unicellular eukaryotic alga Chlamydomonas reinhardtii originally described as a low pigmentation mutant is found to be devoid of photophobic stop responses to photostimuli over a wide range of light intensities. Photophobic responses of the mutant are restored by exogenous addition of all-trans retinal. We have combined computer-based cell-tracking and motion analysis with retinal isomer and retinal analog reconstitution of CC-2359 to investigate properties of the photophobic response receptor. Most rapid and most complete reconstitution is obtained with all-trans retinal compared to 13-cis, 11-cis, and 9-cis retinal. An analog locked by a carbon bridge in a 6-s-trans conformation reconstitutes whereas the corresponding 6-s-cis locked analog does not. Retinal analogs prevented from isomerization around the 13-14 double bond by a five-membered ring in the polyene chain (locked in either the 13-trans or 13-cis configuration) do not restore the response, but enter the chromophore binding pocket as evidenced by their inhibition of all-trans retinal regeneration of the response. Results of competition experiments between all-trans and each of the 13-locked analogs fit a model in which each chromophore exhibits reversible binding to the photoreceptor apoprotein. A competitive inhibition scheme closely fits the data and permits calculation of apparent dissociation constants for the in vivo reconstitution process of 2.5 x 10(-11) M, 5.2 x 10(-10) M, and 5.4 x 10(-9) M, for all-trans, 13-trans-locked and 13-cis-locked analogs, respectively. The chromophore requirement for the trans configuration and 6-s-trans conformation, and the lack of signaling function from analogs locked at the 13 position, are characteristic of archaebacterial rhodopsins, rather than the previously studied eukaryotic rhodopsins (i.e., visual pigments).     

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

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