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PubMedDate not supplied

Receptor demise from alteration of glycosylation site in Drosophila opsin: electrophysiology, microspectrophotometry, and electron microscopy.

In the delta Asn20 Drosophila stock, the N-linked glycosylation site of opsin in R1-6 receptors (Rh1) is absent. We used electroretinography (ERG), microspectrophotometry (MSP), and electron microscopy (EM) to quantify visual cell defects. Positive controls, w9, had wild type Rh1. MSP revealed minimal photopigment in delta Asn20 for 6 days posteclosion; w9 had near normal visual pigment. ERG sensitivity and prolonged depolarizing afterpotential (PDA) were compared for delta Asn20 and w9. Delta Asn20's R1-6 function is decreased 100-fold at eclosion and diminishes until only R7/8 functions at 11 days. What little rhodopsin is routed to the rhabdomere functions. Morphometry showed smaller R1-6 rhabdomeres in delta Asn20 for 8 days posteclosion. Rhabdomeres in w9 were normal. A negative control, ninaE(ol17), a deletion of the Rh1 gene, also has small rhabdomeres. Delta Asn20 and ninaE(ol17) lack the extreme rhabdomere elimination of ora (outer rhabdomeres absent), a nonsense mutant interrupting Rh1's coding sequence. Delta Asn20 and ora have surplus membrane while ninaE(ol17) does not. Freeze fracture reveals that delta Asn20's rhabdomeric P-face particle count is as low as for vitamin A deprivation, consistent with an opsin defect. High particle density, organized into rows, is present in adjacent plasmalemma where surplus membrane accumulates. In summary, delta Asn20 interferes with either synthesis, deployment, or maintenance of opsin.

Animals↗
PubMed1996

Mechanisms of opsin activation.

Rhodopsin is constrained in an inactive conformation by interactions with 11-cis-retinal including formation of a protonated Schiff base with Lys296. Upon photoisomerization, major structural rearrangements that involve protonation of the active site Glu113 and cytoplasmic acidic residues, including Glu134, lead to the formation of the active form of the receptor, metarhodopsin II b, which decays to opsin. However, an activated receptor may be generated without illumination by addition of all-trans-retinal or its analogues to opsin, as measured in this study by the increased phosphorylation of opsin by rhodopsin kinase. The potency of stimulation depended on the chemical and isomeric nature of the analogues and the length of the polyene chain with all-trans-C17 aldehyde and all-trans-retinal being the most active and trans-C12 aldehyde being the least active. Certain cis-isomers, 11-cis-13-demethyl-retinal and 9-cis-C17 aldehyde, were also active. Most of the retinal analogues tested did not regenerate a spectrally identifiable pigment, and many were incapable of Schiff base formation (ketone, stable oximes, and Schiff base-derivatives of retinal). Thus, receptor activation resulted from formation of non-covalent complexes with opsin. pH titrations suggested that an equilibrium exists between partially active (protonated) and inactive (deprotonated) forms of opsin. These findings are consistent with a model in which protonation of one or more cytoplasmic carboxyl groups of opsin is essential for activity. Upon addition of retinoids, the partially active conformation of opsin is converted to a more active intermediate similar to metarhodopsin II b. The model provides an understanding of the structural requirements for opsin activation and an interpretation of the observed activities of natural and experimental opsin mutants.

Animals↗
PubMed1998

Histopathologic and immunohistochemical study of dominant cone degeneration.

PURPOSE: To report the histopathologic and immunohistochemical findings in autosomal dominant cone degeneration. METHODS: The autopsy eyes of a 75-year-old man with autosomal dominant cone degeneration were studied with both light and electron microscopy. In addition, immunofluorescent studies using antibodies to cone opsins and cone alpha transducin were performed. RESULTS: Histopathologic examination of the fovea disclosed loss of photoreceptors and attenuated retinal pigment epithelium. Reduced numbers of cones could be seen in the parafovea, and only occasional cones were visible in the periphery; rods were preserved in the periphery. CONCLUSION: The histopathologic and immunohistochemical findings in this case of dominantly inherited cone degeneration correlate well with the loss of cone function and preservation of normal or nearly normal rod function upon clinical examination. This condition affects red, green, and blue cones.

Aged↗
PubMed1991

Turnover of membrane and opsin in visual receptors of normal and mutant Drosophila.

Electron microscopy was used to investigate membrane turnover in the photoreceptors of Drosophila. Coated pits and vesicles, multivesicular bodies, primary lysosomes, multilamellate bodies, residual bodies and Golgi complexes are present throughout a light/dark cycle. Serial sections reveal that the membrane bounding of multivesicular bodies is only seen at an optimal plane of section. The temperature-sensitive shibire (shi(ts)) mutant has a defect in conversion of coated pits into vesicles which may also affect visual receptors. We used monoclonal antibodies to Rh1 in R1-6 receptors in the compound eye (also to Rh2 in ocellar receptors in the simple eyes) ro relate turnover processes at the visual pigment compared with membrane levels. Compound eye rhabdomeres but not rhabdomere caps stained selectively. Immunogold labelling was equivocal in multivesicular bodies. Further, early in the process of carotenoid replacement therapy, labelling is high in the rough endoplasmic reticulum, demonstrating de novo opsin synthesis.

Animals↗
PubMed2005

A dark and constitutively active mutant of the tiger salamander UV pigment.

A triple mutant (F86L/T93P/S118T; bovine rhodopsin numbering) of the tiger salamander UV cone pigment appears to be trapped in an open conformation that is metarhodopsin-II-like. The pigment is able to activate transducin in the dark, and the ligand-free apoprotein is also able to activate transducin constitutively. The pigment permits protons and chloride ions from solution access to the active site as it displays a pH- and NaCl-dependent absorption spectrum not observed with the wild-type pigment. However, the wild-type properties of light-dependent activity and a pH-independent absorption spectrum are recovered upon reconstitution of the triple mutant with 11-cis-9-demethyl retinal. These results suggest that binding the native chromophore cannot deactivate the protein because of steric interactions between the protein, possibly residue 118, and the 9-methyl group of the chromophore. Furthermore, the absorption spectrum of the 9-demethyl retinal regenerated pigment exhibits a band broader and with lower extinction at the absorption maximum than either the human blue or salamander UV wild-type pigments generated with the same retinal analogue. The broad spectrum appears to be comprised of two or more species and can be well-fit by a sum of scaled spectra of the two wild-type pigments. Binding the chromophore appears to trap the pigment in two or more conformations. The triple mutant reported here represents the first example of a dark-active cone pigment and constitutively active cone opsin.

Ambystoma↗
PubMedDate not supplied

[Inherited colour vision deficiencies--from Dalton to molecular genetics].

In recent years, great advances have been made in our understanding of the molecular basis of colour vision defects, as well as of the patterns of genetic variation in individuals with normal colour vision. Molecular genetic analyses have explained the diversity of types and degrees of severity in colour vision anomalies, their frequencies, pronounced individual variations in test results, etc. New techniques have even enabled the determination of John Dalton's real colour vision defect, 150 years after his death. Inherited colour vision deficiencies most often result from the mutations of genes that encode cone opsins. Cone opsin genes are linked to chromosomes 7 (the S or "blue" gene) and X (the L or "red" gene and the M or "green" gene). The L and M genes are located on the q arm of the X chromosome in a head-to-tail array, composed of 2 to 6 (typically 3) genes--a single L is followed by one or more M genes. Only the first two genes of the array are expressed and contribute to the colour vision phenotype. The high degree of homology (96%) between the L and M genes predisposes them to unequal recombination, leading to gene deletion or the formation of hybrid genes (comprising portions of both the L and M genes), explaining the majority of the common red-green colour vision deficiencies. The severity of any deficiency is influenced by the difference in spectral sensitivity between the opsins encoded by the first two genes of the array. A rare defect, S monochromacy, is caused either by the deletion of the regulatory region of the array or by mutations that inactivate the L and M genes. Most recent research concerns the molecular basis of complete achromatopsia, a rare disorder that involves the complete loss of all cone function. This is not caused by mutations in opsin genes, but in other genes that encode cone-specific proteins, e.g. channel proteins and transducin.

Color Vision Defects↗
PubMed1992

In vitro galactosylation of rhodopsin and opsin: kinetics, properties and characterization.

At best, only trace amounts of galactose have been detected as constituents of rhodopsin as analysed by several laboratories. Nevertheless, the enzymatic galactosylation of rhodopsin proceeds readily in vitro, a process which can be catalysed by galactosyltransferases from several sources. Little information is available, however, concerning the properties of the in vitro reaction. We have examined characteristics of the latter process with the hope of shedding light on the capacity of the retina to carry out this reaction. Kinetic properties of the galactosyltransferases of bovine and embryonic chick retinas, bovine milk and rat liver-Golgi were examined using rhodopsin, opsin, N-acetylglucosamine and ovalbumin as exogenous acceptors. All of these studies demonstrated the very limited activity of the galactosyltransferases of the retina as compared to the milk and rat liver systems. The subcellular distribution of the galactosyltransferases of bovine retina was examined. The influence of compounds that might modulate the reaction was also examined. alpha-Lactalbumin, a modifier of the galactosyltransferase in milk, acted as a competitive inhibitor of the galactosylation of opsin. Analogs of vitamin A, shown to inhibit galactosyltransferases in other systems, did not have this effect on the galactosylation of opsin. Similarly, mixing experiments could not demonstrate the presence of endogenous material that inhibited the reaction in the retina. The conformation of the visual pigment was shown to influence the reaction. After bleaching by visible light, opsin was preferred over rhodopsin as an acceptor of galactose by the galactosyltransferases of bovine and embryonic chick retinas and by rat liver. This distinction was only minimally demonstrated by the milk enzyme. The galactosylation of ovalbumin was unaffected by conditions of light or dark by any of the enzymes. While the mode ratio of galactose to rhodopsin after catalysis by the milk enzyme was about 1.6, this ratio was only about 0.01 after reaction with the enzyme from bovine retina. The linkage of galactose in enzymatically galactosylated rhodopsin and opsin was beta(1-4).

Animals↗
PubMed1998

Role of the C9 methyl group in rhodopsin activation: characterization of mutant opsins with the artificial chromophore 11-cis-9-demethylretinal.

Activation of the visual pigment rhodopsin involves both steric and electrostatic interactions between the chromophore and opsin within the retinal-binding site. Removal of the C9 methyl group of 11-cis-retinal inhibits light-dependent activation of the G protein, transducin, suggesting a direct steric contact. More recently, we have shown that steric interactions lead to receptor activation when Gly121 in the middle of transmembrane helix 3 is replaced by larger hydrophobic residues. In order to understand in more detail the role of the C9 methyl group of retinal in the structure and function of rhodopsin, we first studied the properties of recombinant 9-dm-Rho (opsin reconstituted with 11-cis-9-demethylretinal). The 9-dm-Rho pigment displayed a blue-shifted lambdamax, increased hydroxylamine reactivity, and decreased ability to activate transducin. These properties are consistent with the hypothesis that the C9 methyl group is a crucial structural anchor for the correct docking of the chromophore in its binding site. Next, we investigated the possible interaction between Gly121 of opsin and the C9 methyl group of retinal by characterizing recombinant pigments produced by combining mutant opsins (G121A, -V, -I, -L, and -W) with 11-cis-9-demethylretinal. Mutant opsins G121I, -L, and -W failed to bind the chromophore. However, the double mutant G121L/F261A bound 11-cis-9-demethylretinal to form a stable pigment with a lambdamax of 451 nm. When activity was assayed in membranes, the reduction in transducin activation by 9-dm-Rho caused by the lack of a C9 methyl group on the chromophore could be partially restored by replacing Gly121 with a bulky residue (leucine, isoleucine, or tryptophan). These results support a model of receptor activation that involves steric interaction between the C9 methyl group of the chromophore and the opsin in the vicinity of Gly121 on transmembrane helix 3.

Isomerism↗
PubMed2001

Conformations of the active and inactive states of opsin.

The signaling state metarhodopsin II of the visual pigment rhodopsin decays to the apoprotein opsin and all-trans retinal, which are then regenerated to rhodopsin by the visual cycle. Opsin is known to have at neutral pH only a small residual constitutive activity toward its G protein transducin, which is thought to play a considerable role in light adaptation (bleaching desensitization). In this study we show with Fourier-transform infrared spectroscopy that after metarhodopsin II decay, opsin exists in two conformational states that are in a pH-dependent equilibrium at 30 degrees C with a pK of 4.1 in the presence of hydroxylamine scavenging the endogenous all-trans retinal. Despite the lack of the native agonist in its binding pocket, the low pH opsin conformation is very similar to that of metarhodopsin II and is likewise stabilized by peptides derived from rhodopsin's cognate G protein, transducin. The high pH form, on the other hand, has some conformational similarity to the inactive metarhodopsin I state. We therefore conclude that the opsin apoprotein displays intrinsic conformational states that are merely modulated by bound all-trans retinal.

Animals↗
PubMed1992

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.

Animals↗
PubMed2002

Convergent evolution on the molecular level.

Divergence and convergence are two evolutionary processes by which organisms become adapted to their environments. With the advent of molecular biological techniques it is possible to ask if these processes are observed at the molecular level. There are many examples of molecular divergence in which molecular sequence or function change over evolutionary time. There are fewer reports of convergent evolution on the molecular level, and these claims are sometimes controversial. In this paper I discuss the types of convergent molecular evolution, describe the criteria for accepting or rejecting convergence, and give some examples relevant to neurobiology where convergence has been claimed. These include convergent evolution of opsins, gap junction proteins, neurotransmitter receptors, ion channels, and venoms directed against ion channels.

Animals↗
PubMed1997

Immunoreactive pinopsin in pineal and retinal photoreceptors of various vertebrates.

Pinopsin is a pineal specific opsin newly identified in the pineal of birds which has an absorption maximum at 470 nm. As the opsin content of photoreceptors in the pineal complex of several species is not yet known, in the present work, we studied their pinopsin immunoreactivity in various vertebrates from cyclostomes to mammals. We also compared the immunoreactivity of pineal photoreceptors to that of retinal cones and rods of each animal. For the immunocytochemistry, we raised antibodies in rabbits against a 14 amino acids containing part of the chicken pinopsin molecule. The immunoreaction was performed at the electron microscopic level. The pineal organs show a great diversity in vertebrates: there is a pineal organ present from cyclostomes to mammals, in addition, there is a parapineal organ in cyclostomes and fishes, a frontal organ in frogs and a parietal eye in several reptiles. We detected a strong pinopsin immunoreaction on most of the pinealocytes of birds and on the large photoreceptor-type of the pineal of reptiles. Rod-type photoreceptors of the avian retina and a cone of the reptile retina was immunoreactive as well. According to the known absorption maximum of pinopsin, the immunoreactivity may indicate a green-blue light-sensitivity for these photoreceptors. The immunoreactivity was less pronounced or absent in mammals as well as in less differentiated species. The pineal organ of snakes and the parietal eye of reptiles equally failed to exhibit pinopsin immunoreactive photoreceptors, presumably, due to the absence of green-blue light-sensitive photoreceptors of pinopsin-type in these species.

Animals↗
PubMed2000

Diversity of opsin immunoreactivities in the extraretinal tissues of four anuran amphibians.

The pineal complex, deep brain, and skin have been known to function as extraretinal photoreceptors in non-mammalian vertebrates. To see the diversity of localization of extraretinal photoreceptors in lower vertebrates having different habitats, we analyzed the opsin-like immunoreactivities in anuran amphibians, Xenopus laevis, Rana catesbeiana, Rana nigromaculata, and Bufo japonicus. An antiserum (toad Rh-AS) was raised against rhodopsin purified from the retinas of Japanese toad, B. japonicus. In the retina of all the anurans examined, the outer segments of rods were immunopositive to toad Rh-AS. The outer segments of most pinealocytes were immunopositive in R. catesbeiana, R. nigromaculata, and B. japonicus. The outer segments of photoreceptor-like cells within the frontal organ of R. nigromaculata were immunostained. Interestingly, toad Rh-AS immunostained many secretory cells of mucous glands in the head skin of B. japonicus, implying the presence of a novel photoreceptive molecule. Within the hypothalamus, toad Rh-AS immunostained many cells in the magnocellular preoptic nucleus of R. catesbeiana and B. japonicus. Toad Rh-AS also labeled cerebrospinal fluid (CSF)-contacting cells in the anterior preoptic nucleus of R. nigromaculata and those adjacent to the lateral ventricle within the septum of R. catesbeiana. Thus the distribution patterns of the rhodopsin-like immunoreactivities among the anurans were highly diverged, and there was no relationship between the distribution patterns and their habitats. J. Exp. Zool. 286:136-142, 2000.

Animals↗
PubMed1988

A specific ELISA using purified opsin, for studying autoimmunity in retinal diseases.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) was developed to measure nanogram quantities of rhodopsin or its apoprotein, opsin, in bovine retinal rod outer segment (ROS) preparations. Anti-opsin anti-sera could detect as little as 4 ng of purified opsin or of opsin in ROS preparations. The purified opsin was prepared by quantitative elution from a preparative polyacrylamide gel, and showed higher immunoreactivity with anti-opsin than did ROS when the same amount (per weight) of protein was allowed to bind in the wells of the ELISA plates. The effect of the ionic detergent SDS (sodium dodecyl sulphate) on the immunoreactivity and antigen binding to the ELISA wells was studied. Concentrations of 0.1% SDS and above reduced the apparent binding of opsin with anti-opsin when examined by ELISA. This may have been because the negatively charged SDS reduced the efficiency of the antigen coating process, or because changes in the epitopes' conformations made them less recognisable by the corresponding antibodies. A similar ELISA system using a specific anti-S-antigen anti-serum allowed the detection of even very small amounts (nanograms) of S-antigen in ROS preparations. The presence of S-antigen in ROS preparations was confirmed by immunoblotting. Thus purified opsin is preferable to ROS for ELISA tests of autoimmunity to rhodopsin in retinal diseases. These sensitive ELISA techniques could be used to examine the presence of minute amounts of rhodopsin, opsin or S-antigen in different retinal preparations.

Animals↗
PubMed1996

Positional information and opsin identity in retinal cones.

To test the hypothesis that local environmental cues regulate the expression of middle wavelength-sensitive (MWS) and short wavelength-sensitive (SWS) opsins in cone photoreceptors, we examined the development of the neonatal mouse retina in an organotypic culture system. The segregation of MWS and SWS cones into dorsal and ventral fields in the mouse retina offers an opportunity to isolate a phenotypically homogeneous population of immature cones prior to opsin expression. Retinae were harvested from mice ranging in age from birth (P0) to P18 and maintained in vitro for up to 4 weeks. Cones from newborn mice were first immunoreactive to SWS opsin-specific antibodies (OS-2 and JH455) after 5 days in vitro, which corresponds to a time course similar to that in vivo. The topographic separation of SWS cones into distinct dorsal and ventral fields was also obvious in retinal explants from newborn mice. However, the MWS opsin, identified by polyclonal antibody JH492, was expressed only in vitro when dorsal explants were harvested from P3 or older pups. Despite the absence of MWS opsin expression in newborn retinal cultures, there was no evidence of an increase in the numbers of SWS cones. To test if local diffusable cues could induce immature cones to express an aberrant opsin, dorsal and ventral retinal explants at different stages of maturation were cocultured during the incubation period. Neither the emergence of the cone fields nor the difference in the regional and temporal development of the MWS and SWS opsins was affected in these experiments. These results suggest that positional information in the retina and the opsin identity of cones is determined prior to birth and argue against the hypothesis that postnatal cones can be induced to express an aberrant opsin.

Animals↗