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PubMed1997

Synthesis of Retinals Fluorinated at Odd-Numbered Side-Chain Positions and of the Corresponding Fluorobacteriorhodopsins.

Conventional Horner-Wadsworth-Emmons and Wittig condensations were used to fluorinate the odd-numbered positions of the retinal side chain past C(7). The stereochemically labile cis-fluororetinals were easily converted into the most stable trans-fluororetinals, which were incubated with bacterio-opsin. Contrary to expectations, the fluorinated retinals provided artificial pigments with near normal absorption properties, showing that any electrostatic interactions between the fluorine atoms and protein groups were insufficient to prevent normal binding. The new artificial pigments had smaller opsin shifts than did native bacteriorhodopsin, which is interpreted as due either to greater electrostatic interaction between the protonated imine and its counterion, or to local interactions between the fluorine substituents and nearby polar protein groups.

Journal Article↗
PubMed1997

Identification and localization of an immunoreactive AMPA-type glutamate receptor subunit (GluR4) with respect to identified photoreceptor synapses in the outer plexiform layer of goldfish retina.

L-glutamate, the main excitatory synaptic transmitter in the retina, is released from photoreceptors and evokes responses in second-order retinal neurons (horizontal, bipolar cells) which utilize both ionotropic and metabotropic types of glutamate receptors. In the present study, to elucidate the functional roles of glutamate receptors in synaptic transmission, we have identified a specific ionotropic receptor subunit (GluR4) and determined its localization with respect to photoreceptor cells in the outer plexiform layer of the goldfish retina by light and pre-embedding electron-microscopical immunocytochemistry. We screened antisera to mammalian AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate)-preferring ionotropic glutamate receptors (GluR 1-4) of goldfish retina by light- and electron-microscopical immunocytochemistry. Only immunoreactive (IR) GluR4 was found in discrete clusters in the outer plexiform layer. The cones contacted in this manner were identified as long-wavelength ("red") and intermediate-wavelength ("green") cones, which were strongly immunoreactive to monoclonal antibody FRet 43 and antisera to goldfish red and green-cone opsins; and short-wavelength ("blue") cones, which were weakly immunoreactive to FRet 43 but strongly immunoreactive with antiserum to blue-cone opsin. Immunoblots of goldfish retinal homogenate with anti-GluR4 revealed a single protein at M(r) = 110 kDa. Preadsorption of GluR4 antiserum with either the immunizing rat peptide, or its goldfish homolog, reduced or abolished staining in retinal sections and blots. Therefore, we have detected and localized genuine goldfish GluR4 in the outer plexiform layer of the goldfish retina. We characterized contacts between photoreceptor cells and GluR4-IR second-order neurons in the electron microscope. IR-GluR4 was localized to invaginating central dendrites of triads in ribbon synapses of red cones, semi-invaginating dendrites in other cones and rods, and dendrites making wide-cleft basal junctions in rods and cones; the GluR4-IR structures are best identified as dendrites of OFF-bipolar cells. The results of our studies indicate that in goldfish retina GluR4-expressing neurons are postsynaptic to all types of photoreceptors and that transmission from photoreceptors to OFF-bipolars is mediated at least in part by AMPA-sensitive receptors containing GluR4 subunits.

Amino Acid Sequence↗
PubMed2001

Two-step process for photoreceptor formation in Drosophila.

The formation of photoreceptor cells (PRCs) in Drosophila serves as a paradigm for understanding neuronal determination and differentiation. During larval stages, a precise series of sequential inductive processes leads to the recruitment of eight distinct PRCs (R1-R8). But, final photoreceptor differentiation, including rhabdomere morphogenesis and opsin expression, is completed four days later, during pupal development. It is thought that photoreceptor cell fate is irreversibly established during larval development, when each photoreceptor expresses a particular set of transcriptional regulators and sends its projection to different layers of the optic lobes. Here, we show that the spalt (sal) gene complex encodes two transcription factors that are required late in pupation for photoreceptor differentiation. In the absence of the sal complex, rhabdomere morphology and expression of opsin genes in the inner PRCs R7 and R8 are changed to become identical to those of outer R1-R6 PRCs. However, these cells maintain their normal projections to the medulla part of the optic lobe, and not to the lamina where outer PRCs project. These data indicate that photoreceptor differentiation occurs as a two-step process. First, during larval development, the photoreceptor neurons become committed and send their axonal projections to their targets in the brain. Second, terminal differentiation is executed during pupal development and the photoreceptors adopt their final cellular properties.

Animals↗
PubMed1994

Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness.

Mutations in the gene for the visual pigment rhodopsin cause retinitis pigmentosa (RP) and congenital night blindness. Inheritance of the diseases is generally autosomal dominant and about 40 different rhodopsin mutations have been documented. Although the cell death and retinal degeneration associated with RP have been suggested to result from improper folding and accumulation of the mutant proteins in rod photoreceptor cells, this may not account for the disease in all cases. For example, RP mutations at Lys 296, site of Schiff base linkage to the retinal chromophore, result in constitutive activation of the protein in vitro; that is, the mutants can catalytically activate the G protein transducin in the absence of chromophore and in the absence of light. Similarly, mutation of Ala 292-->Glu activates opsin in vitro and causes night blindness. We show here that the mutation Gly 90-->Asp (G90D) in the second transmembrane segment of rhodopsin, which causes congenital night blindness, also constitutively activates opsin. Furthermore, we show that Asp 90 can substitute for the Schiff base counterion, Glu 113, which is located in the third transmembrane segment of the protein. This demonstrates the proximity of Asp 90 and Lys 296 in the three-dimensional structure of rhodopsin and suggests that the constitutively activating mutations operate by a common molecular mechanism, disrupting a salt bridge between Lys 296 and the Schiff base counterion, Glu 113.

Cell Line↗
PubMed2000

Protection of Rpe65-deficient mice identifies rhodopsin as a mediator of light-induced retinal degeneration.

Light-induced apoptosis of photoreceptors represents an animal model for retinal degeneration. Major human diseases that affect vision, such as age-related macular degeneration (AMD) and some forms of retinitis pigmentosa (RP), may be promoted by light. The receptor mediating light damage, however, has not yet been conclusively identified; candidate molecules include prostaglandin synthase, cytochrome oxidase, rhodopsin, and opsins of the cones and the retinal pigment epithelium (PE). We exposed to bright light two groups of genetically altered mice that lack the visual pigment rhodopsin (Rpe65-/- and Rho-/-). The gene Rpe65 is specifically expressed in the PE and essential for the re-isomerization of all-trans retinol in the visual cycle and thus for the regeneration of rhodopsin after bleaching. Rho-/- mice do not express the apoprotein opsin in photoreceptors, which, consequently, do not contain rhodopsin. We show that photoreceptors lacking rhodopsin in these mice are completely protected against light-induced apoptosis. The transcription factor AP-1, a central element in the apoptotic response to light, is not activated in the absence of rhodopsin, indicating that rhodopsin is essential for the generation or transduction of the intracellular death signal induced by light.

Animals↗
PubMed2001

A photic visual cycle of rhodopsin regeneration is dependent on Rgr.

During visual excitation, rhodopsin undergoes photoactivation and bleaches to opsin and all-trans-retinal. To regenerate rhodopsin and maintain normal visual sensitivity, the all-trans isomer must be metabolized and reisomerized to produce the chromophore 11-cis-retinal in biochemical steps that constitute the visual cycle and involve the retinal pigment epithelium (RPE; refs. 3-8). A key step in the visual cycle is isomerization of an all-trans retinoid to 11-cis-retinol in the RPE (refs. 9-11). It could be that the retinochrome-like opsins, peropsin, or the retinal G protein-coupled receptor (RGR) opsin12-16 are isomerases in the RPE. In contrast to visual pigments, RGR is bound predominantly to endogenous all-trans-retinal, and irradiation of RGR in vitro results in stereospecific conversion of the bound all-trans isomer to 11-cis-retinal. Here we show that RGR is involved in the formation of 11-cis-retinal in mice and functions in a light-dependent pathway of the rod visual cycle. Mutations in the human gene encoding RGR are associated with retinitis pigmentosa.

Animals↗
PubMed1993

Heterozygous missense mutation in the rhodopsin gene as a cause of congenital stationary night blindness.

A number of mutations in the rhodopsin gene have been shown to cause both dominant and recessive retinitis pigmentosa. Here we describe another phenotype associated with a defect in this gene. We discovered a patient with congenital stationary night blindness who carries the missense mutation Ala292Glu. When coupled with 11-cis-retinal in vitro, Ala292Glu rhodopsin is able to activate transducin in a light-dependent manner like wild-type rhodopsin. However, without a chromophore, Ala292Glu opsin anomalously activates transducin. We speculate that the rod dysfunction in this patient is due to an abnormal, continuous activation of transducin by mutant opsin molecules in photoreceptor outer segments.

Adult↗
PubMed2004

Inner retinal photoreceptors (IRPs) in mammals and teleost fish.

Research over the past decade has provided overwhelming evidence that photoreception in the vertebrate eye is not confined to the rods and cones. The discovery of non-rod, non-cone ocular photoreceptors in mammals and fish arose from quite different lines of investigation. In transgenic mice entirely lacking functional rod and cone photoreceptors a range of responses to light, including the regulation of the circadian system and a pupillary light reflex, are preserved. Electrophysiological and imaging approaches were then able to characterise a coupled plexus of directly light sensitive ganglion cells. Most recently action spectroscopy has shown that a novel 'blue-light' sensitive photopigment based upon opsin/vitamin A (OP480) mediates these responses to light. Several candidate genes have emerged for OP480, with melanopsin being by far the strongest. A definitive link, however, between this gene and OP480 has still to be established. In contrast to the mammals, the discovery of inner retinal photoreceptors (IRPs) in fish started with the discovery of a new gene family (VA opsin). The teleost VA opsins form functional photopigments and are expressed in several different types of inner retinal neuron, including retinal horizontal cells. Recent studies have investigated the electrical properties of these photosensitive neurones, but their light-sensing role remains a matter of speculation. Thus the study of IRP is developing along quite separate lines. In the mammals the research is directed towards a molecular identification of the photopigment (OP480) and its cascade, whilst in fish the major effort is directed towards identifying a role for these novel photoreceptors using physiological approaches. The discovery of IRPs in the vertebrates tells us that despite 150 years of research, we still have much to learn about how the eye processes light.

Animals↗
PubMed2002

Activation of rhodopsin kinase.

The present study confirms our original assertion that peptides corresponding to the C-terminal sequence of rhodopsin are phosphorylated by rhodopsin kinase (RK), but only in the presence of photo-activated rhodopsin [Rho*, which is functionally equivalent to metarhodopsin II (Meta II)]. Under optimized conditions, the extent of peptide phosphorylation reached up to 60% that of Rho*. Rho* phosphorylation began to plateau within 15 min of the initiation of photolysis, whereas the peptide phosphorylation continued linearly for >60 min. This lack of co-ordination in the phosphorylation of the physiological (Rho*) and synthetic (peptide) substrates necessitated the present study, which showed that RK is activated for peptide phosphorylation not only by Meta II but also by Meta III, as well as by the phosphorylated derivatives of these species, but not by opsin. These results led to the conclusion that all the derivatives of opsin, which contain the Schiff base linkage with the all-trans-retinylidene moiety, retain the ability to activate RK.

Animals↗
PubMed1978

Topography of the rhodopsin molecule. Identification of the domain phosphorylated.

Studies on the light-stimulated phosphorylation of rod outer segments by [gamma-32P]ATP showed that although nearly 1 mol of [32P]phosphate was incorporated/mol of total opsin, only a small fraction of the molecules were phosphorylated, and these contained at least 2-3 mol of phosphate/mol. Rod outer segments containing the phosphorylated opsin were incubated with 11-cis-retinal to generate phosphorylated rhodopsin and then digested with papain to produce a cleaved complex comprising three fragments, heavy (H), medium (M) and light (L). It was shown that L-fragment of apparent mol.wt. 6000 contained all the phosphorylation sites. This suggests that one specific domain of rhodopsin is susceptible to multiple phosphorylation.

Adenosine Triphosphate↗
PubMed1979

The incorporation of tritiated retinyl moiety into the active-site lysine residue of bacteriorhodopsin.

Purple membranes were isolated from Halobacterium halobium bleached and regenerated with all-trans-[15-3H]retinal. The incorporation of label was 1.2 mol of retinal/mol of bacterio-opsin. The [3H]retinyl-bacterio-opsin obtained from regeneration was hydrolysed to give tritiated retinyl-lysine, which, on hydrogenation to N-epsilon-perhydro[3H]retinyl-lysine and reaction with 1-fluoro-2,4-dinitrobenzene, gave bis-(2,4-dinitrophenyl)-N-epsilon-perhydro[3H]retinyl-lysine. This result confirmed that the retinyl moiety of the chromophore is attached to an epsilon-amino group of lysine.

Bacteriorhodopsins↗
PubMed2003

Demonstration of a sensory rhodopsin in eubacteria.

We report the first sensory rhodopsin observed in the eubacterial domain, a green light-activated photoreceptor in Anabaena (Nostoc) sp. PCC7120, a freshwater cyanobacterium. The gene encoding the membrane opsin protein of 261 residues (26 kDa) and a smaller gene encoding a soluble protein of 125 residues (14 kDa) are under the same promoter in a single operon. The opsin expressed heterologously in Escherichia coli membranes bound all-trans retinal to form a pink pigment (lambda max 543 nm) with a photochemical reaction cycle of 110 ms half-life (pH 6.8, 18 degrees C). Co-expression with the 14 kDa protein increased the rate of the photocycle, indicating physical interaction with the membrane-embedded rhodopsin, which we confirmed in vitro by affinity enrichment chromatography and Biacore interaction. The pigment lacks the proton donor carboxylate residue in helix C conserved in known retinylidene proton pumps and did not exhibit detectable proton ejection activity. We detected retinal binding to the protein in Anabaena membranes by SDS-PAGE and autofluorography of 3H-labelled all-trans retinal of reduced membranes from the organism. We conclude that Anabaena rhodopsin functions as a photosensory receptor in its natural environment, and suggest that the soluble 14 kDa protein transduces a signal from the receptor. Therefore, unlike the archaeal sensory rhodopsins, which transmit signals by transmembrane helix-helix interactions with membrane-embedded transducers, the Anabaena sensory rhodopsin may signal through a soluble cytoplasmic protein, analogous to higher animal visual pigments.

Amino Acid Sequence↗
PubMed1999

A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii.

The use of Chlamydomonas reinhardtii as a model system has been hindered by difficulties encountered in expressing foreign genes. We have synthesised a gene encoding green fluorescent protein (GFP) adapted to the codon usage of C. reinhardtii (cgfp). After verifying the gene was functional in Escherichia coli, the cgfp was fused in frame to the phleomycin resistance gene ble from Streptoalloteichus hindustanus and expressed in C. reinhardtii under control of the rbcS2 promoter and intron sequences. The GFP-fluorescence was seen only in the nucleus demonstrating the nuclear accumulation of the Ble-GFP fusion protein. The cgfp was also fused to the chlamyopsin gene, cop, and expressed in C. reinhardtii under control of the cop promoter. The eyespot became fluorescent indicating that the opsin-GFP fusion protein was correctly directed into the eyespot along with the endogenous unmodified opsin. We conclude that cgfp provides a useful tool to visualize protein synthesis and localisation in vivo in C. reinhardtii and possibly in related green algal species.

Algal Proteins↗
PubMed2003

The immunocytochemical localization of connexin 36 at rod and cone gap junctions in the guinea pig retina.

Connexin 36 (Cx36) is a channel-forming protein found in the membranes of apposed cells, forming the hexameric hemichannels of intercellular gap junction channels. It localizes to certain neurons in various regions of the brain including the retina. We characterized the expression pattern of neuronal Cx36 in the guinea pig retina by immunocytochemistry using specific antisera against Cx36 and green/red cone opsin or recoverin. Strong Cx36 immunoreactivity was visible in the ON sublamina of the inner plexiform layer and in the outer plexiform layer, as punctate labelling patterns. Double-labelling experiments with antibody directed against Cx36 and green/red cone opsin or recoverin showed that strong clustered Cx36 immunoreactivity localized to the axon terminals of cone or close to rod photoreceptors. By electron microscopy, Cx36 immunoreactivity was visible in the gap junctions as well as in the cytoplasmic matrices of both sides of cone photoreceptors. In the gap junctions between cone and rod photoreceptors, Cx36 immunoreactivity was only visible in the cytoplasmic matrices of cone photoreceptors. These results clearly indicate that Cx36 forms homologous gap junctions between neighbouring cone photoreceptors, and forms heterologous gap junctions between cone and rod photoreceptors in guinea pig retina. This focal location of Cx36 at the terminals of the photoreceptor suggests that rod photoreceptors can transmit rod signals to the pedicle of a neighbouring cone photoreceptor via Cx36, and that the cone in turn signals to corresponding ganglion cells via ON and OFF cone bipolar cells.

Animals↗
PubMed2002

The nuclear receptor NR2E3 plays a role in human retinal photoreceptor differentiation and degeneration.

Normal human retinal development involves orderly generation of rods and cones by complex mechanisms. Cell-fate specification involves progenitor cell lineage and external signals such as soluble factors and cell-cell interactions. In most inherited human retinal degenerations, including retinitis pigmentosa, a mutant gene causes loss of visual function, death of mature rods, and eventually death of all cone subtypes. Only one inherited retinal disorder, the enhanced S cone syndrome (ESCS), shows increased visual function, involving the minority S (blue) cones, and decreased rod and L/M (red/green) cone function. This autosomal recessive disease is caused by mutations in NR2E3, a photoreceptor nuclear receptor transcription factor, and may result from abnormal cell-fate determination, leading to excess S cones at the expense of other photoreceptor subtypes. In 16 ESCS patients with the most common NR2E3 mutation, R311Q, we documented an abnormal ratio of S to L/M cone function and progressive retinal degeneration. We studied the postmortem retina of an ESCS patient homozygous for NR2E3 R311Q. No rods were identified, but cones were increased approximately 2-fold, and 92% were S cones. Only 15% of the cones expressed L/M cone opsin, and some coexpressed S cone opsin. The retina was disorganized, with densely packed cones intermixed with inner retinal neurons. The retina was also degenerate, retaining photoreceptors in only the central and far peripheral regions. These observations suggest a key role for NR2E3 in regulation of human photoreceptor development. Degeneration of the NR2E3 retina may result from defective development, known S cone fragility, or abnormal maintenance of mature photoreceptors.

Adult↗
PubMed2004

Vertebrate ultraviolet visual pigments: protonation of the retinylidene Schiff base and a counterion switch during photoactivation.

For visual pigments, a covalent bond between the ligand (11-cis-retinal) and receptor (opsin) is crucial to spectral tuning and photoactivation. All photoreceptors have retinal bound via a Schiff base (SB) linkage, but only UV-sensitive cone pigments have this moiety unprotonated in the dark. We investigated the dynamics of mouse UV (MUV) photoactivation, focusing on SB protonation and the functional role of a highly conserved acidic residue (E108) in the third transmembrane helix. On illumination, wild-type MUV undergoes a series of conformational changes, batho --> lumi --> meta I, finally forming the active intermediate meta II. During the dark reactions, the SB becomes protonated transiently. In contrast, the MUV-E108Q mutant formed significantly less batho that did not decay through a protonated lumi. Rather, a transition to meta I occurred above approximately 240 K, with a remarkable red shift (lambda(max) approximately 520 nm) accompanying SB protonation. The MUV-E108Q meta I --> meta II transition appeared normal but the MUV-E108Q meta II decay to opsin and free retinal was dramatically delayed, resulting in increased transducin activation. These results suggest that there are two proton donors during the activation of UV pigments, the primary counterion E108 necessary for protonation of the SB during lumi formation and a second one necessary for protonation of meta I. Inactivation of meta II in SWS1 cone pigments is regulated by the primary counterion. Computational studies suggest that UV pigments adopt a switch to a more distant counterion, E176, during the lumi to meta I transition. The findings with MUV are in close analogy to rhodopsin and provides further support for the importance of the counterion switch in the photoactivation of both rod and cone visual pigments.

Amino Acid Sequence↗
PubMed2004

Structural origins of constitutive activation in rhodopsin: Role of the K296/E113 salt bridge.

The intramolecular interactions that stabilize the inactive conformation of rhodopsin are of primary importance in elucidating the mechanism of activation of this and other G protein-coupled receptors. In the present study, site-directed spin labeling is used to explore the role of a buried salt bridge between the protonated Schiff base at K296 in TM7 and its counterion at E113 in TM3. Spin-label sensors are placed at positions in the cytoplasmic surface of rhodopsin to monitor changes in the structure of the helix bundle caused by point mutations that disrupt the salt bridge. The single point mutations E113Q, G90D, and A292E, which were previously reported to cause constitutive activation of the apoprotein opsin, are found to cause profound movements of both TM3 and TM6 in the dark state, the latter of which is similar to that caused by light activation. The mutant M257Y, which constitutively activates opsin but does not disrupt the salt bridge, is shown to cause related but distinguishable structural changes. The double mutants E113Q/M257Y and G90D/M257Y produce strong activation of the receptor in the dark state. In the E113Q/M257Y mutant investigated with spin labeling, the movement of TM6 and other changes are exaggerated relative to either E113Q or M257Y alone. Collectively, the results provide structural evidence that the salt bridge is a key constraint maintaining the resting state of the receptor, and that the disruption of the salt bridge is the cause, rather than a consequence, of the TM6 motion that occurs upon activation.

Animals↗
PubMed2001

Preservation of light signaling to the suprachiasmatic nucleus in vitamin A-deficient mice.

To investigate the role of retinal-based pigments (opsins) in circadian photoreception in mice, animals mutated in plasma retinol binding protein were placed on a vitamin A-free diet and tested for photic induction of gene expression in the suprachiasmatic nucleus. After 10 months on the vitamin A-free diet, the majority of mice contained no detectable retinal in their eyes. These mice demonstrated fully intact photic signaling to the suprachiasmatic nucleus as measured by acute mPer mRNA induction in the suprachiasmatic nucleus in response to bright or dim light. The data suggest that a non-opsin pigment is the primary circadian photoreceptor in the mouse.

Animals↗