Beta-ionone activates and bleaches visual pigment in salamander photoreceptors.

Vision begins with photoisomerization of 11-cis retinal to the all-trans conformation within the chromophore-binding pocket of opsin, leading to activation of a biochemical cascade. Release of all-trans retinal from the binding pocket curtails but does not fully quench the ability of opsin to activate transducin. All-trans retinal and some other analogs, such as beta-ionone, enhance opsin's activity, presumably on binding the empty chromophore-binding pocket. By recording from isolated salamander photoreceptors and from patches of rod outer segment membrane, we now show that high concentrations of beta-ionone suppressed circulating current in dark-adapted green-sensitive rods by inhibiting the cyclic nucleotide-gated channels. There were also decreases in circulating current and flash sensitivity, and accelerated flash response kinetics in dark-adapted blue-sensitive (BS) rods and cones, and in ultraviolet-sensitive cones, at concentrations too low to inhibit the channels. These effects persisted in BS rods even after incubation with 9-cis retinal to ensure complete regeneration of their visual pigment. After long exposures to high concentrations of beta-ionone, recovery was incomplete unless 9-cis retinal was given, indicating that visual pigment had been bleached. Therefore, we propose that beta-ionone activates and bleaches some types of visual pigments, mimicking the effects of light.

Visual cycle and its metabolic support in gecko photoreceptors.

Photoreceptors of nocturnal geckos are transmuted cones that acquired rod morphological and physiological properties but retained cone-type phototransduction proteins. We have used microspectrophotometry and microfluorometry of solitary isolated green-sensitive photoreceptors of Tokay gecko to study the initial stages of the visual cycle within these cells. These stages are the photolysis of the visual pigment, the reduction of all-trans retinal to all-trans retinol, and the clearance of all-trans retinol from the outer segment (OS) into the interphotoreceptor space. We show that the rates of decay of metaproducts (all-trans retinal release) and retinal-to-retinol reduction are intermediate between those of typical rods and cones. Clearance of retinol from the OS proceeds at a rate that is typical of rods and is greatly accelerated by exposure to interphotoreceptor retinoid-binding protein, IRBP. The rate of retinal release from metaproducts is independent of the position within the OS, while its conversion to retinol is strongly spatially non-uniform, being the fastest at the OS base and slowest at the tip. This spatial gradient of retinol production is abolished by dialysis of saponin-permeabilized OSs with exogenous NADPH or substrates for its production by the hexose monophosphate pathway (NADP+glucose-6-phosphate or 6-phosphogluconate, glucose-6-phosphate alone). Following dialysis by these agents, retinol production is accelerated by several-fold compared to the fastest rates observed in intact cells in standard Ringer solution. We propose that the speed of retinol production is set by the availability of NADPH which in turn depends on ATP supply within the outer segment. We also suggest that principal source of this ATP is from mitochondria located within the ellipsoid region of the inner segment.

Circular dichroism and cross-linking studies of bacteriorhodopsin mutants.

Circular dichroism (CD) spectroscopy was employed for native (wild type, WT) bacteriorhodopsin (bR) and several mutant derivatives: R134K, R134H, R82Q, S35C, L66C, and R134C/E194C. Comparative analysis of the CD spectra in visible range shows that only R134C/E194C exhibits biphasic CD, typical for native bR, the other mutants demonstrate CD spectra with significantly smaller or absent negative band. Since the biphasic CD is a feature of hexagonal lattice structure composed by bR trimers in the purple membrane, these mutants and WT were examined by cross-linking studies, which confirmed the same trend towards trimeric organization. Therefore, a single amino acid substitution may lead to drastically different CD spectra without disruption of bR trimeric organization. Thus, although disruption of bR trimeric crystalline lattice structure (e.g., solubilization with detergents) directly results in the disappearance of characteristic bilobe in visible CD, the lack of the bilobe in the CD alone does not predict the absence of trimers.

Differences in the pharmacological activation of visual opsins.

Opsins, like many other G-protein-coupled receptors, sustain constitutive activity in the absence of ligand. In partially bleached rods and cones, opsin's activity closes cGMP-gated channels and produces a state of "pigment adaptation" with reduced sensitivity to light and accelerated flash response kinetics. The truncated retinal analogue, beta-ionone, further desensitizes partially bleached green-sensitive salamander rods, but enables partially bleached red-sensitive cones to recover dark-adapted physiology. Structural differences between rod and cone opsins were proposed to explain the effect. Rods and cones, however, also contain different transducins, raising the possibility that G-protein type determines the photoreceptor-specific effects of beta-ionone. To test the two hypotheses, we applied beta-ionone to partially bleached blue-sensitive rods and cones of salamander, two cells that couple the same cone-like opsin to either rod or cone transducin, respectively. Immunocytochemistry confirmed that all salamander rods contain one form of transducin, whereas all cones contain another. beta-Ionone enhanced pigment adaptation in blue-sensitive rods, but it also did so in blue- and UV-sensitive cones. Furthermore, all recombinant salamander rod and cone opsins, with the exception of the red-sensitive cone opsin, activated rod transducin upon the addition of beta-ionone. Thus opsin structure determines the identity of beta-ionone as an agonist or an inverse agonist and in that respect distinguishes the red-sensitive cone opsin from all others.

Rhodopsin phosphorylation in rats exposed to intense light.

The damaging effects of intense light on the rat retina are known to vary depending on the time of day of exposure. The purpose of this study was to determine if rhodopsin phosphorylation patterns, a measure of the activity of the pigment, varied in a similar manner. After 10 min in strong light (1400 lux), all six threonine and serine sites in the rat rhodopsin C-terminus were phosphorylated, with mono- to tetraphosphorylation being substantially more prominent than penta- to hexaphosphorylation. The level and multiplicity of rhodopsin phosphorylations were reduced both with the duration of light exposure and the duration of subsequent darkness. Although showing vast differences in susceptibility to light damage, rats exposed at 5 P.M. or 1 A.M. showed similar rhodopsin phosphorylation levels and patterns. These data indicate that a process controlled by circadian rhythm other than rhodopsin phosphorylation is involved either in damaging or mediating the damage evoked by intense light exposure.

A visual pigment expressed in both rod and cone photoreceptors.

Rods and cones contain closely related but distinct G protein-coupled receptors, opsins, which have diverged to meet the differing requirements of night and day vision. Here, we provide evidence for an exception to that rule. Results from immunohistochemistry, spectrophotometry, and single-cell RT-PCR demonstrate that, in the tiger salamander, the green rods and blue-sensitive cones contain the same opsin. In contrast, the two cells express distinct G protein transducin alpha subunits: rod alpha transducin in green rods and cone alpha transducin in blue-sensitive cones. The different transducins do not appear to markedly affect photon sensitivity or response kinetics in the green rod and blue-sensitive cone. This suggests that neither the cell topology or the transducin is sufficient to differentiate the rod and the cone response.

Cloning and characterization of three salamander retinal G-protein beta subunits.

PURPOSE: Recent evidence has shown that the beta-gamma dimers (beta gamma) of activated heterotrimeric G-proteins are important in many cellular signaling pathways. Since two distinct transducin alpha subunits have been cloned from the salamander retina, we aimed to identify and characterize the G-protein beta (Gbeta) subunits that are involved in visual signal transduction in the salamander. METHODS: A salamander retina cDNA library was screened using degenerate oligonucleotide primers designed from a compilation of known Gbeta sequences. Tissue specific expression was determined by reverse transcriptase PCR (RT-PCR). RESULTS: The library screening resulted in the cloning of three full-length sequences, two of which encode proteins of 340 residues and the third being an iniation variant of 353 and 395 residues. No identical matches were found in GenBank but each shows highest homology to G-beta-1 (beta1), G-beta-3 (beta3), and G-beta-5 (beta5 and beta5L) subunits of other species, respectively. The beta1 and beta3 subunits are 84.7% identical to each other but both show only 52% identity to beta5 at the protein level. RT-PCR analysis showed that all the subunits are expressed in multiple tissues, including the retina. However, the beta5L splice variant was found only in the retina. CONCLUSIONS: Three distinct Gbeta subunit transcripts are expressed in the salamander retina. These subunits have proven to be important in the visual system of mammalian models.

Exploring the function of Tyr83 in bacteriorhodopsin: features of the Y83F and Y83N mutants.

Tyrosine-83, a residue which is conserved in all halobacterial retinal proteins, is located at the extracellular side in helix C of bacteriorhodopsin. Structural studies indicate that its hydroxyl group is hydrogen bonded to Trp189 and possibly to Glu194, a residue which is part of the proton release complex (PRC) in bacteriorhodopsin. To elucidate the role of Tyr83 in proton transport, we studied the Y83F and Y83N mutants. The Y83F mutation causes an 11 nm blue shift of the absorption spectrum and decreases the size of the absorption changes seen upon dark adaptation. The light-induced fast proton release, which accompanies formation of the M intermediate, is observed only at pH above 7 in Y83F. The pK(a) of the PRC in M is elevated in Y83F to about 7.3 (compared to 5.8 in WT). The rate of the recovery of the initial state (the rate of the O --> BR transition) and light-induced proton release at pH below 7 is very slow in Y83F (ca. 30 ms at pH 6). The amount of the O intermediate is decreased in Y83F despite the longer lifetime of O. The Y83N mutant shows a similar phenotype in respect to proton release. As in Y83F, the recovery of the initial state is slowed several fold in Y83N. The O intermediate is not seen in this mutant. The data indicate that the PRC is functional in Y83F and Y83N but its pK(a) in M is increased by about 1.5 pK units compared to the WT. This suggests that Tyr83 is not the main source for the proton released upon M formation in the WT; however, Tyr83 is involved in the proton release affecting the pK(a) of the PRC in M and the rate of proton transport from Asp85 to PRC during the O --> bR transition. Both the Y83F and the Y83N mutations lead to a greatly decreased functionality of the pigment at high pH because most of the pigment is converted into the inactive P480 species, with a pK(a) 8-9.

Mass spectrometric analysis of integral membrane proteins at the subnanomolar level: application to recombinant photopigments.

Integral membrane proteins produced by eukaryotic expression systems are a subject of much current interest in biomedical investigation. Due to the low efficiency of their expression and the limited quantity of the expressed to the total amount of the membrane proteins, they have evaded mass spectrometric analysis. The methodology previously developed for mass spectrometric analysis of integral membrane proteins required proteins that were obtained relatively pure from their native membranes. The previously developed methodology has been modified and applied to the analysis of subnanomolar samples of rhodopsin. Bovine rhodopsin purified by affinity chromatography, from native membranes and from a eukaryotic expression system, was successfully analyzed, obtaining complete sequence coverage for the detection and localization of posttranslational modifications. The methodology presented here will enable mass spectrometric analysis of subnanomolar levels of photopigments or other integral membrane proteins either from their native membranes or as products of expression systems.

Prolongation of actions of Ca2+ early in phototransduction by 9-demethylretinal.

During adaptation Ca2+ acts on a step early in phototransduction, which is normally available for only a brief period after excitation. To investigate the identity of this step, we studied the effect of the light-induced decline in intracellular Ca2+ concentration on the response to a bright flash in normal rods, and in rods bleached and regenerated with 11-cis 9-demethylretinal, which forms a photopigment with a prolonged photoactivated lifetime. Changes in cytoplasmic Ca2+ were opposed by rapid superfusion of the outer segment with a 0Na+/0Ca2+ solution designed to minimize Ca2+ fluxes across the surface membrane. After regeneration of a bleached rod with 9-demethlyretinal, the response in Ringer's to a 440-nm bright flash was prolonged in comparison with the unbleached control, and the response remained in saturation for 10-15s. If the dynamic fall in Ca2+i induced by the flash was delayed by stepping the outer segment to 0Na+/0Ca2+ solution just before the flash and returning it to Ringer's shortly before recovery, then the response saturation was prolonged further, increasing linearly by 0.41 +/- 0.01 of the time spent in this solution. In contrast, even long exposures to 0Na+/0Ca2+ solution of rods containing native photopigment evoked only a modest response prolongation on the return to Ringer's. Furthermore, if the rod was preexposed to steady subsaturating light, thereby reducing the cytoplasmic calcium concentration, then the prolongation of the bright flash response evoked by 0Na+/0Ca2+ solution was reduced in a graded manner with increasing background intensity. These results indicate that altering the chromophore of rhodopsin prolongs the time course of the Ca2+-dependent step early in the transduction cascade so that it dominates response recovery, and suggest that it is associated with photopigment quenching by phosphorylation.

Salamander UV cone pigment: sequence, expression, and spectral properties.

The visual pigment from the ultraviolet (UV) cone photoreceptor of the tiger salamander has been cloned, expressed, and characterized. The cDNA contains a full-length open reading frame encoding 347 amino acids. The phylogenetic analysis indicates that the highest sequence homology is to the visual pigments in the S group. The UV opsin was tagged at the carboxy-terminus with the sequence for the 1D4 epitope. This fusion opsin was expressed in COS-1 cells, regenerated with 11-cis retinal (A1) and immuno-purified, yielding a pigment with an absorbance maximum (lambdamax) of 356 nm which is blue shifted from the absorption of retinal itself. The transducin activation assay demonstrated that this pigment is able to activate rod transducin in a light-dependent manner. Regeneration with 11-cis 3,4-dehydroretinal (A2) yielded a pigment with a lambdamax of 360 nm, only 4 nm red shifted from that of the A1 pigment, while bovine rhodopsin generated with A2 showed a 16-nm red shift from the corresponding A1 pigment. These results demonstrate that the trend for a shorter wavelength pigment to have a smaller shift of lambdamax between the A1 and A2 pigments also fits UV pigments. We hypothesize that the small red shift with A2 could be due to a twist in the chromophore that essentially isolates the ring double bond(s) from conjugation with the rest of the polyene chain.

Expression, purification, and MALDI analysis of RPE65.

PURPOSE: RPE65 is preferentially expressed in the retinal pigment epithelium (RPE) and is essential for retinal function. The purpose of the study was to develop methods for the expression of the protein, determine the accurate molecular weight of this expressed protein, and quantitate the amount of RPE65 in the bovine RPE. METHODS: Human RPE65 was expressed in Sf9 cells using the baculovirus system. The subcellular localization was determined by Western blot analysis and immunocytochemistry. An ELISA was developed for RPE65 and used to measure levels in bovine RPE. Recombinant and native RPE65 were purified by affinity chromatography. Molecular mass was determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. RESULTS: Recombinant human (rH)RPE65 was expressed as a major protein associated with cell membrane in Sf9 cells. The recombinant protein was purified to apparent homogeneity from both the membrane and nonmembrane fractions. The identity of the purified protein was confirmed by Western blot analysis and by partial peptide sequencing. rHRPE65 from the nonmembrane fraction has a mass of 64,867 +/- 80 which is close to the calculated molecular weight from the amino acid sequence including the His-tag (64,663), whereas the membrane-associated rHRPE65 has a molecular mass of 65,380 +/- 150, which is significantly higher than that of the non-membrane-associated form and the calculated molecular weight, suggesting posttranslational modifications. Similarly, native RPE65 was detected in the cytosolic and microsomal fractions of the bovine RPE, with an average level of 3.8 +/- 1.3 and 7.2 +/- 0.4 microg RPE65 per eye, respectively. The cytosolic form had a molecular mass of 61,161 +/- 60, which is close to the calculated value (60,944), whereas that of the microsomal form was 61,961 +/- 170. CONCLUSIONS: RPE65 is expressed in two forms, one of which is membrane associated and contains significant posttranslational modifications, similar to the native membrane-associated form.

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.

Fluorescence properties of pyrylretinol.

A fluorescent analog of retinol, 3,7-dimethyl-9-(1-pyryl)-2E,4E,6E,8E-nonatetr aene-1-ol (referred to as pyrylretinol, or 1) has been synthesized. The fluorescence properties (e.g. quantum yield, lifetime, steady-state anisotropy, and excitation/emission spectra) of this compound in various organic solvents and in dimyristoylphosphatidylcholine (DMPC) liposomes have been studied, and the results are compared with those obtained from 3-methyl-5-(1-pyryl)-2E,4E-pentadiene-1-ol (2), which has the same fused aromatic ring system but a much shorter acyclic chain. 1 and 2 form excimer in aqueous media and fluorescence anisotropies of both 1 and 2 in DMPC liposomes exhibit an abrupt decrease at approximately 21-23 degrees C, which coincides with the main phase transition temperature of DMPC liposomes, indicating that both compounds may be a useful membrane probe. In addition, the binding and quenching capability of pyrylretinol (1) to bovine serum albumin has been investigated. Pyrylretinol (1) binds with BSA with a binding constant of 3.6 x 10(4) M-1, although the value is somewhat lower than that obtained for retinol (3.06 x 10(5) M-1). Pyrylretinol (1) also quenches the BSA intrinsic fluorescence with the quenching rate constant of 1.67 x 10(13) M-1 s-1 and the value is lower than that obtained for retinol (4.06 x 10(13) M-1 s-1). The binding and quenching studies suggest that pyrylretinol (1) may serve as a useful fluorescence probe for structure/function studies of different retinoid binding proteins.

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.

Mass spectrometric analysis of rhodopsin from light damaged rats.

PURPOSE: It is well established that the retina is damaged by intense visible light. Rhodopsin has been proposed to be involved in this process. We therefore undertook to examine whether rhodopsin isolated from light damaged animals is structurally altered at the molecular level. METHODS: Dark reared and dim cyclic light reared 8 week old Sprague-Dawley rats were exposed to intense visible light and sacrificed immediately or 24 h after exposure together with unexposed control animals reared under the same conditions. Rod outer segments were isolated by sucrose gradient ultracentrifugation, their membranes treated with urea, then washed with Tris buffer. The rhodopsin preparations were then reduced, pyridylethylated, delipidated, and cleaved with CNBr. Reversed phase HPLC was used to separate the fragments, and the effluent was analyzed online with a Finnigan LCQ ion trap mass spectrometer. C-terminal phosphorylation was investigated following Asp-N cleavage. MALDI-TOF mass spectrometry was used for the identification of glycosylation. RESULTS: The rat rhodopsin protein was mapped with the exception of two single amino acid fragments. The reported sequence was confirmed with the exception of the controversial T/S320 residue, which was found to be a threonine. Mono-, di-, tri-, and tetraphosphorylated forms of rhodopsin were found in the light damaged animals. Three sites of phosphorylation were confirmed with MS/MS (tandem mass spectral) data. Single or double phosphorylations were found among these three sites, in various combinations. Dark adaptation completely reversed the phosphorylation in all light damaged animals. Other posttranslational modifications were as previously reported. CONCLUSIONS: Our results indicate that intense visible light exposure of rats does not lead to oxidative or other primary structural alterations in the rhodopsin protein of rod outer segments. We also report that the mutated rhodopsin (P23H) is present in rat rod outer segments from heterozygous animals and that residue 320 in both normal and mutated rhodopsins is threonine, not serine.

Intrahelical arrangement in the integral membrane protein rhodopsin investigated by site-specific chemical cleavage and mass spectrometry.

Site-specific cleavage on the interhelical loop I on the cytoplasmic face of rhodopsin has been observed after activation of a Cu-phenanthroline tethered cleavage reagent attached on the cytoplasmic loop IV. The characterization of the reaction products by mass spectrometry, both of the membrane-bound protein and of the CNBr-cleaved peptides, allows the site of cleavage to be determined precisely. The specific cleavage of the peptide bond between Q64 and H65 on loop I leaves the N-terminal peptide (M1-Q64) intact, confirmed by MALDI-MS detection of the two N-linked glycosyl groups near the N-terminus of rhodopsin. The limited extension of the tether side chain requires a interresidue distance between the cleavage site, Q64, and the site of ligand attachment, C316, of less than 12 A. Upon photoactivation of the receptor, no change in the cleavage pattern is observed; however, a simulated Meta II intermediate activation state indicates a much more complex cleavage pattern. The development of this cleavage method, previously used primarily as a "chemical nuclease", in combination with mass spectrometry, may provide a powerful method on membrane protein conformation studies that can be used to complement other biophysical characterizations.

Evidence for the rate of the final step in the bacteriorhodopsin photocycle being controlled by the proton release group: R134H mutant.

Light absorbed by bacteriorhodopsin (bR) leads to a proton being released at the extracellular surface of the purple membrane. Structural studies as well as studies of mutants of bR indicate that several groups form a pathway for proton transfer from the Schiff base to the extracellular surface. These groups include D85, R82, E204, E194, and water molecules. Other residues may be important in tuning the initial state pK(a) values of these groups and in mediating light-induced changes of the pK(a) values. A potentially important residue is R134: it is located close to E194 and might interact electrostatically to affect the pK(a) of E194 and light-induced proton release. In this study we investigated effects of the substitution of R134 with a histidine on light-induced proton release and on the photocycle transitions associated with proton transfer. By measuring the light-induced absorption changes versus pH, we found that the R134H mutation results in an increase in the pK(a) of the proton release group in both the M (0.6 pK unit) and O (0.7 pK unit) intermediate states. This indicates the importance of R134 in tuning the pK(a) of the group that, at neutral and high pH, releases the proton upon M formation (fast proton release) and that, at low pH, releases the proton simultaneously with O decay (slow proton release). The higher pK(a) of the proton release group found in R134H correlates with the slowing of the rate of the O --> bR transition at low pH and probably is the cause of this slowing. The pH dependence of the fraction of the O intermediate is altered in R134H compared to the WT but is similar to that in the E194D mutant: a very small amount of O is present at neutral pH, but the fraction of O increases greatly upon decreasing the pH. These results provide further support for the hypothesis that the O --> bR transition is controlled by the rate of deprotonation of the proton release group. These data also provide further evidence for the importance of the R134-E194 interaction in modulating proton release from D85 after light has led to its being protonated.