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PubMed1988

Analysis of the promoter of the Rh2 opsin gene in Drosophila melanogaster.

We have analyzed the cis-acting regulatory sequences of the Drosophila melanogaster Rh2 gene that encodes the protein component of a rhodopsin which is expressed in ocellar photoreceptor cells. DNA fragments containing the start point of transcription of the Rh2 gene were fused to either the Escherichia coli chloramphenicol acetyltransferase (CAT) or lacZ (beta-galactosidase) genes and introduced into the Drosophila germline by P-element-mediated transformation. Expression of the E. coli genes was then used to assay the ability of various sequences from the Rh2 gene to confer upon the indicator genes the Rh2 pattern of expression. Fragments containing between 4.3 kb and 183 bp upstream of the start of transcription plus the first 32 bp of the 5'-untranslated leader were found to result in nearly identical levels of head-specific CAT expression. Deletion of Rh2 sequences distal to position -112 bp resulted in loss of detectable CAT expression from these Rh2/CAT fusion constructs. We have, therefore, defined a region essential for head-specific expression of the Rh2 gene to a region extending from -183 to -112. We have determined the DNA sequence of the Rh2 promoter from -448 to +32 and have found an 11-bp sequence which is also present in the upstream flanking sequences of two other photoreceptor-specific genes (ninaE and ninaC). By histochemical staining of beta-galactosidase expressed under the control of the Rh2 promoter and by analyzing the effect of the ocelliless mutation on the expression of an Rh2/CAT fusion gene, we have been able to demonstrate that this promoter is active in ocelli.

Animals↗
PubMed2002

Trans-specific evolution of opsin alleles and the maintenance of trichromatic colour vision in Callitrichine primates.

Many New World (NW) primates possess a remarkable polymorphism in an X-linked locus, which encodes for the visual pigments (opsins) used for colour vision. Females that are heterozygous for opsin alleles of different spectral sensitivity at this locus have trichromatic colour vision, whereas homozygous females and males are dichromatic, with poor colour discrimination in the red-green range. Here we describe an extensive survey of allelic variation in both exons and introns at this locus within and among species of the Callitrichines (marmosets and tamarins). All five genera of Callitrichines have the X-linked polymorphism, and only the three functional allelic classes described previously (with maximum wavelength sensitivities at about 543 nm, 556 nm and 563 nm) were found among the 16 species and 233 or more X-chromosomes sampled. In spite of the homogenizing effects of gene conversion, phylogenetic analyses provide direct evidence for trans-specific evolution of alleles over time periods of at least 5-6 million years, and up to 14 million years (estimated from independent phylogenies). These conclusions are supported by the distribution of insertions and deletions in introns. The maintenance of polymorphism over these time periods requires an adaptive explanation, which must involve a heterozygote advantage for trichromats. The lack of detection of alleles that are recombinant for spectral sensitivity suggests that such alleles are suboptimal. The two main hypotheses for the selective advantage of trichromacy in primates are frugivory for ripe fruits and folivory for young leaves. The latter can be discounted in Callitrichines, as they are not folivorous.

Alleles↗
PubMed2005

Light and darkness regulate melanopsin in the retinal ganglion cells of the albino Wistar rat.

Circadian rhythms are daily adjusted to the environmental day/night cycle by photic input via the retinohypothalamic tract (RHT). Recent studies indicate that melanopsin, a newly identified opsin-like molecule, is involved in the light responsiveness of retinal ganglion cells (RGCs) constituting the RHT. In the present study, we examined the expression of melanopsin at the mRNA and protein level during a day/night cycle and during prolonged periods of light and darkness in the retina of albino Wistar rats. We observed a diurnal change in melanopsin, with mRNA level being highest at early subjective night and protein level highest at late subjective day. Prolonged exposure to darkness significantly increased melanopsin mRNA level as early as the first day, and the expression continued to increase during 5 d in darkness. The decrease in mRNA level during exposure to constant light was slower. After 48 h of light, the melanopsin mRNA level was significantly reduced, and an almost undetectable level was found after 5 d. The induction of melanopsin by darkness was even more pronounced if darkness was preceded by light suppression for 5 d. By use of immunohistochemistry, we showed that darkness increased the amount of protein in the dendritic processes, resulting in a dense network covering the entire retina. Constant light decreased melanopsin immunostaining time dependently, beginning in the distal dendrites and progressing to the proximal dendrites and the soma. Our observations suggest that the intrinsic light-responsive RGCs adapt their expression of the putative circadian photopigment melanopsin to environmental light and darkness.

Animals↗
PubMed2000

Ambient hypoxia reverses retinal vascular attenuation in a transgenic mouse model of autosomal dominant retinitis pigmentosa.

PURPOSE: Loss of retinal capillaries is an inherent component of late stage autosomal dominant retinitis pigmentosa (ADRP). This study examined the hypothetical role of tissue hyperoxia in this vascular attenuation process and tested the potential of ambient hypoxia to reverse it. METHODS: Transgenic mice expressing a mutant opsin gene with a 3-bp deletion of isoleucine at codon 255/256 were used. This model is characterized by early onset of a rapidly progressing retinal degeneration that by postnatal day (P)20 results in the loss of all but one row of photoreceptor nuclei. At P20 some mice were placed in 12% oxygen until they were euthanatized at P26. The remainder were maintained in normoxia and killed at the same age. Retinas were dissected, stained for ADPase, and flat-mounted. RESULTS: Deep plexus capillary density was significantly different in normoxic normals versus transgenics at 20 days of age (P: </= 0. 005). An additional 65% reduction of capillary density occurred within the deep plexus of normoxic transgenics between P20 and P26 (P: </= 0.005). Ambient hypoxia between days P20 and P26 reversed this trend, causing an increase in deep capillary plexus density of nearly 100% (P: </= 0.001). CONCLUSIONS: This model of ADRP demonstrates two important features of human retinitis pigmentosa: photoreceptor cell death and subsequent retinal capillary atrophy. Low ambient oxygen was used to reverse the capillary atrophy and to stimulate new capillary growth, implying that retinal oxygen tension may link these two features of the pathology. The implications of this study hold importance for strategies designed to treat retinitis pigmentosa with retinal cell transplantation.

Animals↗
PubMed1999

L, M and L-M hybrid cone photopigments in man: deriving lambda max from flicker photometric spectral sensitivities.

Using heterochromatic flicker photometry, we have measured the corneal spectral sensitivities of the X-chromosome-linked photopigments in 40 dichromats, 37 of whom have a single opsin gene in their tandem array. The photopigments encoded by their genes include: the alanine variant of the normal middle-wavelength sensitive photopigment, M(A180); the alanine and serine variants of the normal long-wavelength sensitive photopigment, L(A180) and L(S180); four different L-M hybrid or anomalous photopigments, L2M3(A180), L3M4(S180), L4M5(A180) and L4M5(S180); and two variants of the L-cone photopigment, encoded by genes with embedded M-cone exon two sequences, L(M2; A180) and L(M2; S180). The peak absorbances (lambda max) of the underlying photopigment spectra associated with each genotype were estimated by correcting the corneal spectral sensitivities back to the retinal level, after removing the effects of the macular and lens pigments and fitting a template of fixed shape to the dilute photopigment spectrum. Details of the genotype-phenotype correlations are summarized elsewhere (Sharpe, L. T., Stockman, A., Jägle, H., Knau, H., Klausen, G., Reitner, A. et al. (1998). J. Neuroscience, 18, 10053-10069). Here, we present the individual corneal spectral sensitivities for the first time as well as details and a comparison of three analyses used to estimate the lambda max values, including one in which the lens and macular pigment densities of each observer were individually measured.

Adult↗
PubMed2001

Development of normal and injury-induced gene expression of aFGF, bFGF, CNTF, BDNF, GFAP and IGF-I in the rat retina.

Focal mechanical injury to the retina substantially increases basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF) mRNA expression, accompanied by a transient increase in FGFR-1 mRNA, and this response is thought to protect photoreceptors near the injury site from inherited and light-induced retinal degenerations. We have now examined retinal gene expression of the principal survival factors involved in the response to injury in normal rats as a function of postnatal age both in normal and injured retinas. Sprague-Dawley rats were injured in one eye by needle incision through the retina at postnatal day (P) 10, 22, 35, 60, 90, 120 and 180. The other eye was uninjured and served as the control. Retinas were taken 1 day post-injury. Northern blot analysis was performed to determine the mRNA expression of the following factors and receptors: bFGF and acidic fibroblast growth factors (aFGF) and FGF receptor-1 (FGFR-1); CNTF and CNTF receptor alpha (CNTFR-alpha); brain-derived neurotrophic factor (BDNF) and its receptor trk B; and insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR); glial fibrillary acidic protein (GFAP) and opsin. In the uninjured control eyes, mRNA expression of most of the factors increased with postnatal age, with little expression at P10 and maximal expression levels reached at P22 (opsin), P35 (aFGF), P60 (BDNF) or P90 (bFGF, FGFR-1, CNTF and GFAP). In contrast, IGF-1 mRNA rapidly decreased from a high level of expression at P10 to about 55% of that level by P22, reaching a stable 45-50% of the P10 level at P35 and thereafter. The response to injury of bFGF, FGFR-1, CNTF and GFAP mRNAs increased with postnatal age. Unexpectedly, only minimal increases in bFGF, FGFR-1, CNTF and GFAP over those seen in the control eyes were observed before P35. Thereafter, the increase of bFGF mRNA after injury reached a maximum of three-fold at P60, maintained this level to P120, and slightly decreased to 2.5-fold by P180. Expression of FGFR-1 mRNA showed a maximum increase of 2.6-fold at P90. Expression of CNTF and GFAP mRNAs followed a time course similar to that of bFGF. Mechanical injury did not alter the mRNA levels of aFGF, BDNF, IGF-I, and receptors, CNTFR-alpha, trk B and IGF-IR. These data show that the response to injury is minimal at early postnatal ages but increases with age and peaks at P60-90 for most potential survival factors.

Analysis of Variance↗
PubMed2005

Gene replacement therapy rescues photoreceptor degeneration in a murine model of Leber congenital amaurosis lacking RPGRIP.

PURPOSE: Retinitis pigmentosa GTPase regulator (RPGR) is a photoreceptor protein anchored in the connecting cilia by an RPGR-interacting protein (RPGRIP). Loss of RPGRIP causes Leber congenital amaurosis (LCA), a severe form of photoreceptor degeneration. The current study was an investigation of whether somatic gene replacement could rescue degenerating photoreceptors in a murine model of LCA due to a defect in RPGRIP. METHODS: An RPGRIP expression cassette, driven by a mouse opsin promoter, was packaged into recombinant adeno-associated virus (AAV). The AAV vector was delivered into the right eyes of RPGRIP(-/-) mice by a single subretinal injection into the superior hemisphere. The left eyes received a saline injection as a control. Full-field electroretinograms (ERGs) were recorded from both eyes at 2, 3, 4, and 5 months after injection. After the final follow-up, retinas were analyzed by immunostaining or by light and electron microscopy. RESULTS: Delivery of the AAV vector led to RPGRIP expression and restoration of normal RPGR localization at the connecting cilia. Photoreceptor preservation was evident by a thicker cell layer and well-developed outer segments in the treated eyes. Rescue was more pronounced in the superior hemisphere coincident with the site of delivery. Functional preservation was demonstrated by ERG. CONCLUSIONS: AAV-mediated RPGRIP gene replacement preserves photoreceptor structure and function in a mouse model of LCA, despite ongoing cell loss at the time of intervention. These results indicate that gene replacement therapy may be effective in patients with LCA due to a defect in RPGRIP and suggest that further preclinical development of gene therapy for this disorder is warranted.

Animals↗
PubMed1995

Histidine tagging both allows convenient single-step purification of bovine rhodopsin and exerts ionic strength-dependent effects on its photochemistry.

For rapid single-step purification of recombinant rhodopsin, a baculovirus expression vector was constructed containing the bovine opsin coding sequence extended at the 3'-end by a short sequence encoding six histidine residues. Recombinant baculovirus-infected Spodoptera frugiperda cells produce bovine opsin carrying a C-terminal histidine tag (v-opshis6x). The presence of this tag was confirmed by immunoblot analysis. Incubation with 11-cis-retinal produced a photosensitive pigment (v-Rhohis6x) at a level of 15-20 pmol/10(6) cells. The histidine tag was exploited to purify v-Rhohis6x via immobilized metal affinity chromatography. Optimized immobilized metal affinity chromatography yielded a binding capacity of > or = 35 nmol of v-Rhohis6x per ml of resin and purification factors up to 500. Best samples were at least 85% pure, with an average purity of 70% (A280 nm/A500 nm = 2.5 +/- 0.4, n = 7). Remaining contamination was largely removed upon reconstitution into lipids, yielding rhodopsin proteoliposomes with a purity over 95%. Spectral analysis of v-Rhohis6x showed a small but significant red shift (501 +/- 1 nm) compared to wild type rhodopsin (498 +/- 1 nm). The pK alpha of the Meta I<==>Meta II equilibrium in v-Rhohis6x is down-shifted from 7.3 to 6.4 resulting in a significant shift at pH 6.5 toward the Meta I photointermediate. Both effects are reversed upon increasing the ionic strength. FT-IR analysis of the Rho-->Meta II transition shows that the corresponding structural changes are identical in wild type and v-Rhohis6x.

Amino Acid Sequence↗
PubMed1995

Drosophila in vision research. The Friedenwald Lecture.

A genetic approach involving the use of ERG-defective Drosophila mutants, described in this article, was developed with the explicit goal of elucidating phototransduction and related photoreceptor events at the molecular level. Advances in the past 30 years have made this goal a reality. Although the phototransduction process in Drosophila is not yet fully understood, enormous strides are being made by many investigators now working in this field. In addition to elucidating phototransduction vents in invertebrate photoreceptors, works of these investigators are providing insights into the functional machinery of signaling systems in general. For example, mobilization of Ca2+ and replenishment of intracellular Ca2+ stores after their depletion is a current topic of intense interest (see, for example, references 93, 136 and 137). Study of trp mutants is providing insights into this process. Similarly, the mechanisms of inactivation of G-protein coupled receptors appear to be highly conserved in diverse transduction systems, and inactivation of Drosophila metarhodopsin is proving a valuable model system for this study. The identification of the NinaA protein and its role as chaperon and/or foldase for opsin would not have been possible without the genetic approach described in this article. In fact, study of the NinaA gene is providing fresh insights not only into the rhodopsin maturation process but also into the role of cyclophilins in general. Moreover, recent results suggest that related retina-specific proteins are also present in mammals. As valuable as the NinaA protein has been, it may be only the first of a number of such novel proteins or novel isozymes of known proteins required in the retina to be discovered through this approach. For two of the Drosophila proteins discussed in this article, NorpA and NinaA, related mammalian proteins expressed in photoreceptors have been identified, even though it seemed unlikely that such proteins would have any role in mammalian photoreceptors. A recent report suggests that the Trp protein has human homologs expressed most heavily in the brain (C. Montell, cited in reference 93). It may well be that almost any protein identified in the Drosophila retina has its counterpart(s) in mammals. Discovery of these mammalian homologs of Drosophila proteins will probably spur new lines of investigation in mammalian photoreceptor function. For example, because no role had been assigned to PLC in cone phototransduction, the discovery that NorpA-homologous PLCs are present in cone outer segments raises questions about what role(s) these PLCs might play in signal transduction in cone outer segments.

Amino Acid Isomerases↗
PubMed2003

Mosaic regularity of horizontal cells in the mouse retina is independent of cone photoreceptor innervation.

PURPOSE: To determine whether the density and the mosaic regularity of the population of horizontal cells is dependent on innervation from the cone photoreceptors by comparing these features in wild-type and transgenic mice expressing an attenuated diphtheria toxin in the cones. METHODS: Retinal wholemounts from coneless transgenic mice and their wild-type littermates were immunostained for calbindin or for cone opsins, and labeled cells and outer segments were counted to determine horizontal cell and photoreceptor density. The x-y positional coordinates of each horizontal cell were also determined, from which the geometrical properties of the horizontal cell mosaic were examined using nearest-neighbor and Voronoi domain analyses. Autocorrelation and density-recovery profile analyses were also conducted to identify the presence of exclusion zones within the population of horizontal cells. For each sampled field, random simulations of matched density, constrained by the physical size of the horizontal cells, were generated in parallel and analyzed with the real data. RESULTS: Coneless mice were confirmed to contain only 3% of the normal cone photoreceptor population. Despite the loss of these afferents, horizontal cell density did not differ between the wild-type and coneless retinas. Mosaic regularity in wild-type and coneless retinas did not differ, but each differed significantly from random simulations of identical density. Horizontal cells in both the wild-type and coneless mouse retina exhibited exclusion zones extending beyond the physical size of the soma, suggested to reflect intercellular interactions during early development that drive tangential dispersion; these were slightly larger in the wild-type retina. CONCLUSIONS: Cone innervation is not a necessary condition for horizontal cell survival during postnatal development. The resiliency of the regularity in the horizontal cell mosaic is consistent with the hypothesis that such global patterning is an emergent property of these cells as they engage in local interactions that are largely independent of their afferent innervation.

Animals↗
PubMed1987

Analysis of the promoter of the ninaE opsin gene in Drosophila melanogaster.

We have analyzed the cis-acting regulatory sequences of the ninaE gene. This gene encodes the major Drosophila melanogaster opsin, the protein component of the primary chromophore of photo-receptor cells R1-R6 of the adult eye. DNA fragments containing the start point of transcription of the ninaE gene were fused to either the Escherichia coli chloramphenicol acetyltransferase or lacZ (beta-galactosidase) gene and introduced into the Drosophila germline by P-element-mediated transformation. Expression of the E. coli genes was then used to assay the ability of various sequences from the ninaE gene to confer the ninaE pattern of expression. Fragments containing between 2.8 kb and 215 bp of the sequences upstream of the start of transcription plus the first 67 bp of the untranslated leader were able to direct nearly wild-type expression. We have identified three separable control regions in the ninaE promoter. The first, which has the properties of an enhancer element, is located between nucleotides -501 and -219. The removal of this sequence had little effect on promoter function; this sequence appears to be redundant. However, it appears to be able to substitute for the second control region which is located between nucleotides -215 and -162, and which also affects the level of output from this promoter. Removal of these two control regions resulted in a 30-fold decrease in expression; however tissue specificity was not affected. The third control region, located downstream from nucleotide -120, appears to be absolutely necessary for promoter function in the absence of the first two regulatory sequences. Examination of larvae containing fusion genes expressing beta-galactosidase suggests that the ninaE gene is also expressed in a subset of cells in the larval photoreceptor organ.

Acetyltransferases↗
PubMed2005

Molecular cloning, localization and circadian expression of chicken melanopsin (Opn4): differential regulation of expression in pineal and retinal cell types.

The avian retina and pineal gland contain autonomous circadian oscillators and photo-entrainment pathways, but the photopigment(s) that mediate entrainment have not been definitively identified. Melanopsin (Opn4) is a novel opsin involved in entrainment of circadian rhythms in mammals. Here, we report the cDNA cloning of chicken melanopsin and show its expression in retina, brain and pineal gland. Like the melanopsins identified in amphibians and mammals, chicken melanopsin is more similar to the invertebrate retinaldehyde-based photopigments than the retinaldehyde-based photopigments typically found in vertebrates. In retina, melanopsin mRNA is expressed in cells of all retinal layers. In pineal gland, expression was strong throughout the parenchyma of the gland. In brain, expression was observed in a few discrete nuclei, including the lateral septal area and medial preoptic nucleus. The retina and pineal gland showed distinct diurnal expression patterns. In pineal gland, melanopsin mRNA levels were highest at night at Zeitgeber time (ZT) 16. In contrast, transcript levels in the whole retina reached their highest levels in the early morning (ZT 0-4). Further analysis of melanopsin mRNA expression in retinal layers isolated by laser capture microdissection revealed different patterns in different layers. There was diurnal expression in all retinal layers except the ganglion cell layer, where heavy expression was localized to a small number of cells. Expression of melanopsin mRNA peaked during the daytime in the retinal pigment epithelium and inner nuclear layer but, like in the pineal, at night in the photoreceptors. Localization and regulation of melanopsin mRNA in the retina and pineal gland is consistent with the hypothesis that this novel photopigment plays a role in photic regulation of circadian function in these tissues.

Amino Acid Sequence↗
PubMed1999

Photoactivation of rhodopsin: interplay between protein and chromophore.

Data in the literature suggest a finely tuned interaction between ligand (11-cis-retinal) and protein (opsin) in order to allow very efficient photoactivation of the ligand and highly vectorial rhodopsin activation with a huge increase in receptor activity. We have further investigated this interaction using ligand homologues, 13C-ligand labelling or 15N-protein labelling, in combination with Fourier transform infrared (FT-IR) and solid-state magic angle spinning (ss-MAS)-NMR spectroscopy. Using 1D rotational resonance (RR) or double-quantum heteronuclear local field (2Q-HLF) ss-MAS-NMR we report the first structure refinement of the rhodopsin chromophore in situ. These measurements yield a specification of the torsional strain in the for isomerization essential C10-C13 segment of the chromophore. This strain is thought to contribute to the high rate and stereospecificity of the photoisomerization reaction. In agreement with previous data, the C10-C13 segment region reaches a relaxed all-trans configuration at the lumirhodopsin photointermediate. MAS-NMR analysis of [15N]lysine-labelled rhodopsin reveals the presence of a 'soft' counterion, requiring intermediate water molecules for stabilization. FT-IR studies on [2H]tyrosine-labelled rhodopsin demonstrate participation of several tyrosin(at)e residues in receptor activation. One of these, probably Tyr268, is already active at the bathorhodopsin stage. Finally, the effect of ligands with single additional methyl substituents in the C10-C12 region has been investigated. They do not affect the general activation pathway, but perturb the activation kinetics of rhodopsin, suggesting steric interference with protein residues. Possible implications of these results for a structural role of water residues will be discussed, as well.

Animals↗
PubMed2003

The cone visual pigments of an Australian marsupial, the tammar wallaby (Macropus eugenii): sequence, spectral tuning, and evolution.

Studies on marsupial color vision have been limited to very few species. There is evidence from behavioral, electroretinographic (ERG), and microspectrophotometric (MSP) measurements for the existence of both dichromatic and trichromatic color vision. No studies have yet investigated the molecular mechanisms of spectral tuning in the visual pigments of marsupials. Our study is the first to determine the mRNA sequence, infer the amino acid sequence, and determine, by in vitro expression, the spectra of the cone opsins of a marsupial, the tammar wallaby (Macropus eugenii). This yielded some information on mechanisms and evolution of spectral tuning of these pigments. The tammar wallaby retina contains only short-wavelength sensitive (SWS) and middle-wavelength sensitive (MWS) pigment mRNAs. This predicts dichromatic color vision, which is consistent with conclusions from previous behavioral studies ( Hemmi 1999). We found that the wallaby has a SWS1 class pigment of 346 amino acids. Sequence comparison with eutherian SWS pigments predicts that this SWS1 pigment absorbs maximally (lambdamax) at 424 nm and, therefore, is a blue rather than a UV pigment. This (lambdamax) is close to that of the in vitro-expressed wallaby SWS pigment (lambdamax of 420 +/- 2 nm) and to that determined behaviorally (420 nm). The difference from the mouse UV pigment (lambdamax of 359 nm) is largely accounted for by the F86Y substitution, in agreement with in vitro results comparing a variety of other SWS pigments. This suggests that spectral tuning employing F86Y substitution most likely arose independently in the marsupials and ungulates as a result of convergent evolution. An apparently different mechanism of spectral tuning of the SWS1 pigments, involving five amino acid positions, evolved in primates. The wallaby MWS pigment has 363 amino acids. Species comparisons at positions critical to spectral tuning predict a lambdamax near 530 nm, which is close to that of the in vitro-expressed pigment (529 +/- 1 nm), but quite different from the value of 539 nm determined by microspectrophotometry. Introns interrupt the coding sequences of the wallaby, mouse, and human MWS pigment sequences at the same corresponding nucleotide positions. However, the length of introns varies widely among these species.

Amino Acid Sequence↗
PubMed1998

An additional methyl group at the 10-position of retinal dramatically slows down the kinetics of the rhodopsin photocascade.

The present study focuses on ligand-protein interactions in a rhodopsin analogue generated from bovine opsin and the 10-methyl homologue of 11-cis-retinal. The analogue pigment displays a reduced alpha-band at 506 +/- 2 and a stronger beta-band at 325 nm. Remarkably, the rotational strength of these bands observed in visible circular dichroism spectra was found to be similar for both native and 10-methyl rhodopsin. The quantum yield of the analogue pigment was determined to be 0.55. All photointermediates were analyzed by Fourier transform infrared difference spectroscopy. At the batho stage, strong hydrogen-out-of-plane vibrations were observed, indicating that the 10-methyl chromophore also adopts a distorted all-trans conformation at this stage. In contrast to native rhodopsin, the batho intermediate of the 10-methyl pigment is stable up to 180 K and only slowly decays to the next intermediate between 180 and 210 K. As in native rhodopsin, the 10-methyl metarhodopsin I intermediate is generated at about 220 K, but its transition to the metarhodopsin II state is again shifted to a much higher temperature (> 293 K) than for the native pigment (> 260 K). Infrared analysis, nevertheless, shows that the conformational changes in the photointermediates of the 10-methyl pigment are basically identical with those observed in the native pigment. This is supported by a signal function assay, showing that the analogue pigment is able to activate transducin. The dual effect of the 10-methyl group on the photocascade is attributed to steric interactions which, initially, hamper the relaxation of strain in the polyene chain of the chromophore and, eventually, interfere with the conformational rearrangements of the protein moiety required to adopt the active conformation of the receptor. Our data provide direct support for the concept that the relaxation of strain in the retinal polyene chain acts as the major driving force of the photocascade dark reaction.

Animals↗
PubMed2000

Expression of a recombinant human RGR opsin in Lentivirus-transduced cultured cells.

PURPOSE: Our goals were to produce a functional recombinant RPE retinal G protein-coupled receptor (RGR) opsin for biochemical studies and to test the efficiency of a lentiviral vector for transgene expression of human RGR. METHODS: A human RGR cDNA was cloned into a replication-defective lentiviral vector, and recombinant hRGR-Lentivirus was prepared for transduction of the ARPE-19, a human retinal pigment epithelium (RPE) cell line, and COS-7 cells. Recombinant RGR expression was detected by Western blot analysis, and functionality of the protein was tested by a [3H]all-trans-retinal binding assay. RESULTS: RGR protein was detected in each cell type after transduction with recombinant virus and was not observed in untreated cells. RGR expression in ARPE-19 cells increased steadily for up to 10 days after transduction and was stable for at least 6 months. The transduced ARPE-19 cells produced approximately 100-fold higher amounts of RGR protein than the transduced COS-7 cells. When cell membranes from the ARPE-19 cells were incubated with [3H]all-trans-retinal, the chromophore bound specifically to the expressed protein. Uptake of [3H]all-trans-retinol into the ARPE-19 cells was followed by specific binding of radiolabeled retinoid to RGR. CONCLUSIONS: Using a Lentivirus-derived gene delivery system, we were able to express high amounts of human RGR protein in the ARPE-19 human RPE cell line. The transduced ARPE-19 cells remain able to process all-trans-retinol, and the expressed protein is capable of binding to the all-trans-retinal chromophore. The Lentivirus-based expression of functional RGR can be used to study RGR in cultured cells and to test in vivo transduction of quiescent RPE cells.

Animals↗
PubMed1990

Phototransduction: different mechanisms in vertebrates and invertebrates.

The photoreceptor cells of invertebrate animals differ from those of vertebrates in morphology and physiology. Our present knowledge of the different structures and transduction mechanisms of the two animal groups is described. In invertebrates, rhodopsin is converted by light into a meta-rhodopsin which is thermally stable and is usually re-isomerized by light. In contrast, photoisomerization in vertebrates leads to dissociation of the chromophore from opsin, and a metabolic process is necessary to regenerate rhodopsin. The electrical signals of visual excitation have opposite character in vertebrates and invertebrates: the vertebrate photoreceptor cell is hyperpolarized because of a decrease in conductance and invertebrate photoreceptors are depolarized owing to an increase in conductance. Single-photon-evoked excitatory events, which are believed to be a result of concerted action (the opening in invertebrates and the closing in vertebrates) of many light-modulated cation channels, are very different in terms of size and time course of photoreceptors for invertebrates and vertebrates. In invertebrates, the single-photon events (bumps) produced under identical conditions vary greatly in delay (latency), time course and size. The multiphoton response to brighter stimuli is several times as long as a response evoked by a single photon. The single-photon response of vertebrates has a standard size, a standard latency and a standard time course, all three parameters showing relatively small variations. Responses to flashes containing several photons have a shape and time scale that are similar to the single-photon-evoked events, varying only by an amplitude scaling factor, but not in latency and time course. In both vertebrate and invertebrate photoreceptors the single-photon-evoked events become smaller (in size) and faster owing to light adaptation. Calcium is mainly involved in these adaptation phenomena. All light adaptation in vertebrates is primarily, or perhaps exclusively, attributable to calcium feedback. In invertebrates, cyclic AMP (cAMP) is apparently another controller of sensitivity in dark adaptation. The interaction of photoexcited rhodopsin with a G-protein is similar in both vertebrate and invertebrate photoreceptors. However, these G-proteins activate different photoreceptor enzymes (phosphodiesterases): phospholipase C in invertebrates and cGMP phosphodiesterase in vertebrates. In the photoreceptors of vertebrates light leads to a rapid hydrolysis of cGMP which results in closing of cation channels. At present, the identity of the internal terminal messenger in invertebrate photoreceptors is still unsolved.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1998

Red, green, and red-green hybrid pigments in the human retina: correlations between deduced protein sequences and psychophysically measured spectral sensitivities.

To analyze the human red, green, and red-green hybrid cone pigments in vivo, we studied 41 male dichromats, each of whose X chromosome carries only a single visual pigment gene (single-gene dichromats). This simplified arrangement avoids the difficulties of complex opsin gene arrays and overlapping cone spectral sensitivities present in trichromats and of multiple genes encoding identical or nearly identical cone pigments in many dichromats. It thus allows for a straightforward correlation between each observer's spectral sensitivity measured at the cornea and the amino acid sequence of his visual pigment. For each of the 41 single-gene dichromats we determined the amino acid sequences of the X-linked cone pigment as deduced from its gene sequence. To correlate these sequences with spectral sensitivities in vivo, we determined the Rayleigh matches to different red/green ratios for 29 single-gene dichromats and measured psychophysically the spectral sensitivity of the remaining green (middle wavelength) or red (long wavelength) cones in 37 single-gene dichromats. Cone spectral sensitivity maxima obtained from subjects with identical visual pigment amino acid sequences show up to a approximately 3 nm variation from subject to subject, presumably because of a combination of inexact (or no) corrections for variation in preretinal absorption, variation in photopigment optical density, optical effects within the photoreceptor, and measurement error. This variation implies that spectral sensitivities must be averaged over multiple subjects with the same genotype to obtain representative values for a given pigment. The principal results of this study are that (1) approximately 54% of the single-gene protanopes (and approximately 19% of all protanopes) possess any one of several 5'red-3'green hybrid genes that encode anomalous pigments and that would be predicted to produce protanomaly if present in anomalous trichromats; (2) the alanine/serine polymorphism at position 180 in the red pigment gene produces a spectral shift of approximately 2.7 nm; (3) for each exon the set of amino acids normally associated with the red pigment produces spectral shifts to longer wavelengths, and the set of amino acids normally associated with the green pigment produces spectral shifts to shorter wavelengths; and (4) changes in exons 2, 3, 4, and 5 from green to red are associated with average spectral shifts to long wavelengths of approximately 1 nm (range, -0.5 to 2.5 nm), approximately 3.3 nm (range, -0.5 to 7 nm), approximately 2.8 nm (range, -0.5 to 6 nm), and approximately 24.9 nm (range, 22.2-27.6 nm).

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