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PubMed2000

Anion modulation of calcium current voltage dependence and amplitude in salamander rods.

Hofmeister anions were used to investigate the ability of Cl(-) replacement to produce inhibition and a hyperpolarizing activation shift in L-type Ca(2+) currents (I(Ca)) of rod photoreceptors. Inhibition of I(Ca) largely followed the Hofmeister sequence: Cl(-)=Br(-)<NO(-)(3)<I(-)<ClO(-)(4) (ClO(-)(4) caused the greatest suppression). Anion-induced hyperpolarizing activation shifts also followed the Hofmeister sequence: Cl(-)<Br(-)<NO(-)(3)<I(-)<ClO(-)(4) (ClO(-)(4) caused the largest shift). Agreement with the Hofmeister sequence suggests that these effects are due to anion interactions at the membrane surface. Hofmeister anions also caused similar hyperpolarizing shifts in the voltage dependence of inwardly rectifying cation currents (I(h)) and outward K(+) currents (I(K)) consistent with the hypothesis that hyperpolarizing shifts arise from anion effects on membrane surface potential. Sulfate and phosphate inhibited rod I(Ca) and phosphate caused a significant leftward activation shift suggesting these anions are strongly adsorbed to the membrane. Because of the overlap between the physiological voltage range and the lower part of the I(Ca) activation curve, anion effects on amplitude and activation may influence synaptic transmission at the first retinal synapse.

Animals↗
PubMedDate not supplied

A 221-bp fragment of the mouse opsin promoter directs expression specifically to the rod photoreceptors of transgenic mice.

Mutations in the human rod opsin gene have been shown to segregate with autosomal dominant retinitis pigmentosa (ADRP) and photoreceptor degeneration in transgenic mice. While these degenerations are characterized by the primary degeneration of rods, cones eventually die as well. To determine whether this subsequent cone degeneration is the result of expression of mutant rod opsin in the cones, the retinal cell-type specificity of a 221-bp fragment of the mouse rod opsin promoter was evaluated. Two transgenic mouse lines generated by injecting a fusion gene comprised of a 221-bp fragment of the mouse rod opsin promoter and the simian virus 40 large tumor antigen gene (Tag) were examined. The expression of Tag causes photoreceptor cell degeneration in members of both transgenic lines. However, the two lines differed with respect to the level of Tag expression and the rate and extent of photoreceptor cell degeneration. Immunocytochemical localization of opsin and Tag in surviving photoreceptor cells was determined and the results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Rod- and cone-mediated function was evaluated by electroretinography (ERG). In the higher Tag-expressing transgenic line only one row of nuclei remained in the outer nuclear layer at postnatal day (P) 150. While these nuclei showed no antigenicity for rod opsin or Tag, they did stain with an antibody that reacts with both rod and cone S-antigens (arrestins), indicating that these cells were surviving photoreceptor nuclei. Positive staining with peanut agglutinin, which uniquely decorates matrix domains surrounding cones in the normal retina, confirmed that the surviving photoreceptor nuclei were of cone origin. RT-PCR substantiated the results from immunostaining; amplification product was obtained using blue cone opsin transcripts but not from either Tag or rod opsin transcripts. The second transgenic mouse line exhibited a much slower photoreceptor cell death that was associated with low levels of Tag transgene transcript. At P120, approximately 50% of photoreceptors remained and an approximately 45% reduction in the rod ERG a-wave was observed. Cone-mediated ERGs, however, were normal. The results demonstrate the rod-specific expression of Tag as directed by the 221-bp fragment of the mouse rod opsin promoter and suggest that the cone degeneration in ADRP or transgenic mice associated with mutations in the rod opsin gene is a secondary effect of rod degeneration.

Animals↗
PubMed1995

Characterization of guanylate cyclase activity in single retinal rod outer segments.

cGMP mediates vertebrate phototransduction by directly gating cationic channels on the plasma membrane of the photoreceptor outer segment. This second messenger is produced by a guanylate cyclase and hydrolyzed by a light-activated cGMP-phosphodiesterase. Both of these enzyme activities are Ca2+ sensitive, the guanylate cyclase activity being inhibited and the light-activated phosphodiesterase being enhanced by Ca2+. Changes in these activities due to a light-induced decrease in intracellular Ca2+ are involved in the adaptation of photoreceptors to background light. We describe here experiments to characterize the guanylate cyclase activity and its modulation by Ca2+ using a truncated rod outer segment preparation, in order to evaluate the enzyme's role in light adaptation. The outer segment of a tiger salamander rod was drawn into a suction pipette to allow recording of membrane current, and the remainder of the cell was sheared off with a probe to allow internal dialysis. The cGMP-gated channels on the surface membrane were used to monitor conversion of GTP, supplied from the bath, into cGMP by the guanylate cyclase in the outer segment. At nominal 0 Ca2+, the cyclase activity had a Km of 250 microM MgGTP and a Vmax of 25 microM cGMP s-1 in the presence of 1.6 mM free Mg2+; in the presence of 0.5 mM free Mg2+, the Km was 310 microM MgGTP and the Vmax was 17 microM cGMP s-1. The stimulation by Mg2+ had an EC50 of 0.2 mM Mg2+ for MgGTP at 0.5 mM. Ca2+ inhibited the cyclase activity. In a K+ intracellular solution, with 0.5 mM free Mg2+ and 2.0 mM GTP, the cyclase activity was 13 microM cGMP s-1 at nominal 0 Ca2+; Ca2+ decreased this activity with a IC50 of approximately 90 nM and a Hill coefficient of approximately 2.0.

Animals↗
PubMed2005

Organization of rhodopsin molecules in native membranes of rod cells--an old theoretical model compared to new experimental data.

It has been shown that rhodopsin forms an oligomer in the shape of long double rows of monomers. Because of the importance of rhodopsin as a template for all G protein-coupled receptors, its dimeric, tetrameric and higher-oligomeric structures also provide a useful pattern for similar structures in GPCRs. New experimental data published recently are discussed in the context of a proposed model of the rhodopsin oligomer 1N3M deposited in the protein data bank. The new rhodopsin structure at 2.2 A resolution with all residues resolved as well as an electron cryomicroscopy structure from 2D crystals of rhodopsin are in agreement with the 1N3M model. Accommodation of movement of transmembrane helix VI, regarded as a major event during the activation of rhodopsin, in a steady structure of the oligomer is also discussed. [Figure: see text]. Superimposition of the 1U19 (red wire), 1GZM (purple wire) and 1N3M (blue wire) rhodopsin structures. Size of the wires is proportional to thermal factors of backbone C(alpha) atoms, view parallel to the membrane.

Cell Membrane↗
PubMed1998

Light-dependent activation of rod transducin by pineal opsin.

The pineal gland expresses a unique member of the opsin family (P-opsin; Max, M., McKinnon, P. J., Seidenman, K. J., Barrett, R. K., Applebury, M. L., Takahashi, J. S., and Margolskee, R. F. (1995) Science 267, 1502-1506) that may play a role in circadian entrainment and photo-regulation of melatonin synthesis. To study the function of this protein, an epitope-tagged P-opsin was stably expressed in an embryonic chicken pineal cell line. When incubated with 11-cis-retinal, a light-sensitive pigment was formed with a lambdamax at 462 +/- 2 nm. P-opsin bleached slowly in the dark (t1/2 = 2 h) in the presence of 50 mM hydroxylamine. Purified P-opsin in dodecyl maltoside activated rod transducin in a light-dependent manner, catalyzing the exchange of more than 300 mol of GTPgammaS (guanosine 5'-O-(3-thiotriphosphate))/mol of P-opsin. The initial rate for activation (75 mol of GTPgammaS bound/mol of P-opsin/min at 7 microM) increased with increasing concentrations of transducin. The addition of egg phosphatidylcholine to P-opsin had little effect on the activation kinetics; however, the intrinsic rate of decay in the absence of transducin was accelerated. These results demonstrate that P-opsin is an efficient catalyst for activation of rod transducin and suggest that the pineal gland may contain a rodlike phototransduction cascade.

Animals↗
PubMed2004

Interaction with transducin depletes metarhodopsin III: a regulated retinal storage in visual signal transduction?

In the phototransduction pathway of rhodopsin, the metarhodopsin (Meta) III retinal storage form arises from the active G-protein binding Meta II by a slow spontaneous reaction through the Meta I precursor or by light absorption and photoisomerization, respectively. Meta III is a side product of the Meta II decay path and holds its retinal in the original binding site, with the Schiff base bond to the apoprotein reprotonated as in the dark ground state. It thus keeps the retinal away from the regeneration pathway in which the photolyzed all-trans-retinal is released. This study was motivated by our recent observation that Meta III remains stable for hours in membranes devoid of regulatory proteins, whereas it decays much more rapidly in situ. We have now explored the possibility of regulated formation and decay of Meta III, using intrinsic opsin tryptophan fluorescence and UV-visible and Fourier transform infrared spectroscopy. We find that a rapid return of Meta III into the regeneration pathway is triggered by the G-protein transducin (G(t)). Depletion of the retinal storage is initiated by a novel direct bimolecular interaction of G(t) with Meta III, which was previously considered inactive. G(t) thereby induces the transition of Meta III into Meta II, so that the retinylidene bond to the apoprotein can be hydrolyzed, and the retinal can participate again in the normal retinoid cycle. Beyond the potential significance for retinoid metabolism, this may provide the first example of a G-protein-catalyzed conversion of a receptor.

Animals↗
PubMed2004

Developmental regulation of calcium-dependent feedback in Xenopus rods.

The kinetics of activation and inactivation in the phototransduction pathway of developing Xenopus rods were studied. The gain of the activation steps in transduction (amplification) increased and photoresponses became more rapid as the rods matured from the larval to the adult stage. The time to peak was significantly shorter in adults (1.3 s) than tadpoles (2 s). Moreover, adult rods recovered twice as fast from saturating flashes than did larval rods without changes of the dominant time constant (2.5 s). Guanylate cyclase (GC) activity, determined using IBMX steps, increased in adult rods from approximately 1.1 s(-1) to 3.7 s(-1) 5 s after a saturating flash delivering 6,000 photoisomerizations. In larval rods, it increased from 1.8 s(-1) to 4.0 s(-1) 9 s after an equivalent flash. However, the ratio of amplification to the measured dark phosphodiesterase activity was constant. Guanylate cyclase-activating protein (GCAP1) levels and normalized Na+/Ca2+, K+ exchanger currents were increased in adults compared with tadpoles. Together, these results are consistent with the acceleration of the recovery phase in adult rods via developmental regulation of calcium homeostasis. Despite these large changes, the single photon response amplitude was approximately 0.6 pA throughout development. Reduction of calcium feedback with BAPTA increased adult single photon response amplitudes threefold and reduced its cutoff frequency to that observed with tadpole rods. Linear mathematical modeling suggests that calcium-dependent feedback can account for the observed differences in the power spectra of larval and adult rods. We conclude that larval Xenopus maximize sensitivity at the expense of slower response kinetics while adults maximize response kinetics at the expense of sensitivity.

Adaptation, Ocular↗
PubMed2002

Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina.

It has recently been shown that bone marrow cells can differentiate into various lineage cells including neural cells in vitro and in vivo. We therefore examined whether bone marrow stem cells can differentiate into retinal neural cells in adult rats. PKH-67-labeled stem cell-enriched bone marrow cells (BMCs) were injected into the vitreous space of eyes in which the retinas had been mechanically injured using a hooked needle. Two weeks after the injection of these cells, immunohistochemical examinations were carried out. The stem cell-enriched BMCs had been incorporated and had differentiated into retinal neural cells in the injured retina. The stem cell-enriched BMCs had accumulated mainly in the outer nuclear layer around the injured sites. The incorporated cells expressed glial fibrillary acidic protein, calbindin, rhodopsin, and vimentin. These results raise the possibility that stem cell-enriched BMCs have the ability to differentiate into retinal neural cells, and that the injection of stem cell-enriched BMCs into the retina would help repair damaged retinal cells.

Amacrine Cells↗
PubMed2000

Mechanism of transducin activation of frog rod photoreceptor phosphodiesterase. Allosteric interactiona between the inhibitory gamma subunit and the noncatalytic cGMP-binding sites.

The rod photoreceptor phosphodiesterase (PDE) is unique among all known vertebrate PDE families for several reasons. It is a catalytic heterodimer (alphabeta); it is directly activated by a G-protein, transducin; and its active sites are regulated by inhibitory gamma subunits. Rod PDE binds cGMP at two noncatalytic sites on the alphabeta dimer, but their function is unclear. We show that transducin activation of frog rod PDE introduces functional heterogeneity to both the noncatalytic and catalytic sites. Upon PDE activation, one noncatalytic site is converted from a high affinity to low affinity state, whereas the second binding site undergoes modest decreases in binding. Addition of gamma to transducin-activated PDE can restore high affinity binding as well as reducing cGMP exchange kinetics at both sites. A strong correlation exists between cGMP binding and gamma binding to activated PDE; dissociation of bound cGMP accompanies gamma dissociation from PDE, whereas addition of either cGMP or gamma to alphabeta dimers can restore high affinity binding of the other molecule. At the active site, transducin can activate PDE to about one-half the turnover number for catalytic alphabeta dimers completely lacking bound gamma subunit. These results suggest a mechanism in which transducin interacts primarily with one PDE catalytic subunit, releasing its full catalytic activity as well as inducing rapid cGMP dissociation from one noncatalytic site. The state of occupancy of the noncatalytic sites on PDE determines whether gamma remains bound to activated PDE or dissociates from the holoenzyme, and may be relevant to light adaptation in photoreceptor cells.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2001

Calcium-activated potassium current clamps the dark potential of vertebrate rods.

Vertebrate photoreceptors respond to light with a graded hyperpolarization from a membrane potential in the dark of approximately -35 mV. The present work investigates the physiological role of the Ca2+-activated K+ current in the photovoltage generation in mechanically isolated rods from salamander retina. Membrane current or voltage in isolated rods was recorded from light- and dark-adapted rods under voltage- or current-clamp conditions, respectively. In light-adapted rods of the salamander, selective blockade of Ca2+-activated K+ channels by means of charybdotoxin depolarized the plasma membrane of current-clamped rods by approximately 30 mV, from a resting potential of approximately -35 mV. A similar depolarization was observed if external Ca2+ (1 mM) was substituted with Ba2+ or Sr2+. Under control conditions, the injection of currents of increasing amplitude (up to -100 pA, to mimic the current entering the rod outer segment) could not depolarize the membrane potential beyond a saturating value of approximately -20 mV. However, in the presence of charybdotoxin, rods depolarized up to +20 mV. In experiments with dark-adapted current-clamped rods, charybdotoxin perfusion lead to transient depolarizations up to 0 mV and steady-state depolarizations of approximately 5 mV above the dark resting potential. Finally, the recovery phase of the voltage response to a flash of light in the presence of charybdotoxin showed a transient overshoot of the membrane potential. It was concluded that Ca2+-activated K+ current is necessary for clamping the rod photovoltage to values close to the dark potential, thus allowing faithful single photon detection and correct synaptic transmission.

Ambystoma mexicanum↗
PubMed2005

Peripheral expression of rod photoreceptor arrestin induces an epitope-specific, protective response against experimental autoimmune uveoretinitis.

PURPOSE: To examine the immunological basis for reduced susceptibility to experimental autoimmune uveoretinitis (EAU) in rats expressing retinal photoreceptor cell arrestin in the periphery. METHODS: Peripheral expression of arrestin in Lewis rats was achieved by engraftment of syngeneic bone marrow (BM) transduced with retroviruses encoding wild-type arrestin or a mutant arrestin lacking the immunodominant epitope Arr(273 - 289) (Delta273-Arr). EAU was induced by immunization with arrestin peptides Arr(273-289) or Arr(343-362). Cultured splenocytes and/or lymphocytes from immunized rats were assayed for antigen-induced proliferation, antibody production, and cytokines. RESULTS: Rats expressing Delta273-Arr were not protected from Arr(273 - 289)-induced EAU, showing that protection was epitope specific. Proliferation assays found little difference in the ability of draining lymph node cells from arrestin-transduced rats to proliferate in response to the antigen, indicating that antigen-responsive T cells were not deleted in BM recipients. Only rats immunized with Arr(343 - 362) elicited antibodies, but no difference in titer was found between transduced and control animals. Higher levels of IFN-gamma mRNA were made by Arr(273 - 289)-immunized rats than Arr(343 - 366)-immunized rats, but in either case, the levels did not correlate with chimeric status or EAU susceptibility. Arr(273 - 289)-immunized rats had higher levels of IL-10 mRNA than Arr(343 - 362)-immunized rats, and those levels were decreased in arrestin chimeric rats. Overall, immunization with the more potently uveitogenic Arr(343 - 362) induced lower levels of IL-10 and IFN-gamma than the less uveitogenic Arr(273 - 289). A strong correlation was found between the ability of lymphocytes to make IL-4 in the arrestin-chimeric animals and inhibition of EAU. CONCLUSIONS: Peripheral expression of arrestin in a regenerating immune system induces an epitope-specific protective response to EAU induced by arrestin peptides. Although IL-4 and IL-10 levels were altered in arrestin-chimeric mice, the outcome was not consistently T(H)2-like. Only IL-4 production was clearly associated with reduced susceptibility to EAU.

Amino Acid Sequence↗
PubMed2002

Massive light-driven translocation of transducin between the two major compartments of rod cells: a novel mechanism of light adaptation.

We report a new cellular mechanism of rod photoreceptor adaptation in vivo, which is triggered by daylight levels of illumination. The mechanism involves a massive light-dependent translocation of the photoreceptor-specific G protein, transducin, between the functional compartments of rods. To characterize the mechanism, we developed a novel technique that combines serial tangential cryodissection of the rat retina with Western blot analysis of protein distribution in the sections. Up to 90% of transducin translocates from rod outer segments to other cellular compartments on the time scale of tens of minutes. The reduction in the transducin content of the rod outer segments is accompanied by a corresponding reduction in the amplification of the rod photoresponse, allowing rods to operate in illumination up to 10-fold higher than would otherwise be possible.

Adaptation, Ocular↗
PubMed2004

Photoreceptor-specific nuclear receptor NR2E3 functions as a transcriptional activator in rod photoreceptors.

NR2E3, a photoreceptor-specific orphan nuclear receptor, is believed to play a pivotal role in the differentiation of photoreceptors. Mutations in the human NR2E3 gene and its mouse ortholog are associated with enhanced S-cones and retinal degeneration. In order to gain insights into the NR2E3 function, we performed temporal and spatial expression analysis, yeast two-hybrid screening, promoter activity assays and co-immunoprecipitation studies. The Nr2e3 expression was localized preferentially to the rod, and not to the cone, photoreceptor nuclei in rodent retina. The yeast two-hybrid screening of a retinal cDNA library, using NR2E3 as the bait, identified another orphan nuclear receptor NR1D1 (Rev-erbalpha). The interaction of NR2E3 with NR1D1 was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation experiments. In transient transfection studies using HEK 293 cells, both NR2E3 and NR1D1 activated the promoters of rod phototransduction genes synergistically with neural retina leucine zipper (NRL) and cone-rod homeobox (CRX). All four proteins, NR2E3, NR1D1, NRL and CRX, could be co-immunoprecipitated from the bovine retinal nuclear extract, suggesting their existence in a multi-protein transcriptional regulatory complex in vivo. Our results demonstrate that NR2E3 is involved in regulating the expression of rod photoreceptor-specific genes and support its proposed role in transcriptional regulatory network(s) during rod differentiation.

Animals↗
PubMed2005

Time course of deterioration of rod and cone function in RCS rat and the effects of subretinal cell grafting: a light- and dark-adaptation study.

To examine how rod and cone function are differentially affected during retinal degeneration, and after subretinal cell grafting, we obtained light- and dark-adaptation curves by recording threshold multiunit responses from the superior colliculus of anesthetized rats. Unoperated RCS dystrophic and non-dystrophic rats were used and the effects of subretinal grafting in dystrophic rats of cells known to limit photoreceptor degeneration were examined. The adaptation curves showed that rod function was severely compromised in unoperated dystrophic RCS rats at low luminance levels, even as early as 21 days of age and that cone thresholds became gradually elevated over time. While cell transplantation preserved both rod and cone photoreceptors, rod function did not recover, although further deterioration of cone threshold responses was prevented. This raises concern that measures of outer nuclear layer thickness may not in themselves be an accurate measure of visual capabilities and efficacy of a restoration strategy.

Adaptation, Ocular↗
PubMed2005

Rhodopsin-iCre transgenic mouse line for Cre-mediated rod-specific gene targeting.

Retinal photoreceptors are highly differentiated postmitotic neurons that transduce photons into electrical signals. While the functions of many photoreceptor-specific genes can be evaluated by direct gene targeting, here we facilitate the studies of nonphotoreceptor-specific genes in these cells by developing an Opsin-iCre transgenic mouse line, iCre-75, in which a 4-kb mouse rod opsin promoter drives the expression of bacteriophage P1 Cre recombinase. Immunohistochemical analysis demonstrated that Cre recombinase is present exclusively in the outer nuclear layer of iCre75 mouse retina. Cre expression is found only in rods and not in cones. The expression level reached 188+/-44 ng per retina at postnatal day (pnd) 11 and increased to 687+/-56 ng at 2 months and older. Cre-mediated excision of floxed genomic DNA was absent at pnd 4, became detectable at pnd 7, and was completed by pnd 18. Retinal morphology and electroretinograms were normal in 8-month-old transgenic animals. The iCre-75 transgenic mice are thus suitable for future genetic studies of essential genes in retinal rod photoreceptors.

Animals↗
PubMed2005

Partial agonism in a G Protein-coupled receptor: role of the retinal ring structure in rhodopsin activation.

The visual process in rod cells is initiated by absorption of a photon in the rhodopsin retinal chromophore and consequent retinal cis/trans-isomerization. The ring structure of retinal is thought to be needed to transmit the photonic energy into conformational changes culminating in the active metarhodopsin II (Meta II) intermediate. Here, we demonstrate that cis-acyclic retinals, lacking four carbon atoms of the ring, can activate rhodopsin. Detailed analysis of the activation pathway showed that, although the photoproduct pathway is more complex, Meta II formed with almost normal kinetics. However, lack of the ring structure resulted in a low amount of Meta II and a fast decay of activity. We conclude that the main role of the ring structure is to maintain the active state, thus specifying a mechanism of activation by a partial agonist of the G protein-coupled receptor rhodopsin.

Animals↗
PubMed1999

Structure of rhodopsin.

Two-dimensional crystals of rhodopsin were studied to determine the arrangement of the transmembrane alpha helices. A combination of electron cryomicroscopy, image processing and electron crystallography was used to extract amplitudes and phases from images, and a three-dimensional map to a resolution of 7.5 A was calculated. Density peaks for all seven transmembrane helices were observed and the helix axes for all seven helices could be estimated. Near the intracellular side, which interacts with the G protein transducin, we observed three layers of helices arranged differently from bacteriorhodopsin. The arrangement opens up towards the extracellular side forming a cavity that serves as the binding pocket for the retinal. This cavity is closed towards the intracellular side by the long and highly tilted helix 3, and must be closed towards the extracellular side by the loop linking helices 4 and 5 which is linked by a disulfide bridge to the extracellular end of helix 3.

Animals↗