Bleached Rhodopsin Research Articles

Monomeric Rhodopsin Is Sufficient for Normal Rhodopsin Kinase (GRK1) Phosphorylation and Arrestin-1 Binding

G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated light-activated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.

Replacing the rod with the cone transducin α subunit decreases sensitivity and accelerates response decay

Cone vision is less sensitive than rod vision. Much of this difference can be attributed to the photoreceptors themselves, but the reason why the cones are less sensitive is still unknown. Recent recordings indicate that one important factor may be a difference in the rate of activation of cone transduction; that is, the rising phase of the cone response per bleached rhodopsin molecule (Rh*) has a smaller slope than the rising phase of the rod response per Rh*, perhaps because some step between Rh* and activation of the phosphodiesterase 6 (PDE6) effector molecule occurs with less gain. Since rods and cones have different G-protein alpha subunits, and since this subunit (Talpha) plays a key role both in the interaction of G-protein with Rh* and the activation of PDE6, we investigated the mechanism of the amplification difference by expressing cone Talpha in rod Talpha-knockout rods to produce so-called GNAT2C mice. We show that rods in GNAT2C mice have decreased sensitivity and a rate of activation half that of wild-type (WT) mouse rods. Furthermore, GNAT2C responses recover more rapidly than WT responses with kinetic parameters resembling those of native mouse cones. Our results show for the first time that part of the difference in sensitivity and response kinetics between rods and cones may be the result of a difference in the G-protein alpha subunit. They also indicate more generally that the molecular nature of G-protein alpha may play an important role in the kinetics of G-protein cascades for metabotropic receptors throughout the body.

Relationships among Visual Cycle Retinoids, Rhodopsin Phosphorylation, and Phototransduction in Mouse Eyes during Light and Dark Adaptation

Phosphorylation and regeneration of rhodopsin, the prototypical G-protein-coupled receptor, each can influence light and dark adaptation. To evaluate their relative contributions, we quantified rhodopsin, retinoids, phosphorylation, and photosensitivity in mice during a 90 min illumination followed by dark adaptation. During illumination, all-trans-retinyl esters and, to a lesser extent, all-trans-retinal accumulate and reach the steady state in <1 h. Each major phosphorylation site on rhodopsin reaches a steady state level of phosphorylation at a different time during illumination. The dominant factor that limits dark adaptation is isomerization of retinal. During dark adaptation, dephosphorylation of rhodopsin occurs in two phases. The faster phase corresponds to rapid dephosphorylation of regenerated rhodopsin present at the end of the illumination period. The slower phase corresponds to dephosphorylation of rhodopsin as it forms by regeneration. We conclude that rhodopsin phosphorylation has three physiological functions: it quenches phototransduction, reduces sensitivity during light adaptation, and suppresses bleached rhodopsin activity during dark adaptation.

Microsecond Time-Resolved Circular Dichroism of Rhodopsin Photointermediates

Time-resolved circular dichroism measurements, over a spectral range from 300 to 700 nm, were made at delays of 5, 100, and 500 micros after room-temperature photoexcitation of bovine rhodopsin in a lauryl maltoside suspension. The purpose was to provide more structural information about intermediate states in the activation of rhodopsin and other G protein-coupled receptors. In particular, information was sought about photointermediates that are isochromic or nearly isochromic in their unpolarized absorbance. The circular dichroism spectrum of lumirhodopsin, obtained after correcting the 5 micros difference CD data for the bleached rhodopsin, was in reasonable agreement with the lumirhodopsin CD spectrum obtained previously by thermal trapping at -76 degrees C. Similarly, the metarhodopsin II spectrum obtained with a 500 micros delay was also in agreement with the results of previous work on the temperature-trapped form of metarhodopsin II. However, the CD of the mixture formed with a 100 micros delay after photoexcitation, whose only visible absorbing component is lumirhodopsin, could not be accounted for near 480 nm in terms of the initially formed, 5 micros lumirhodopsin CD spectrum. Thus, the CD spectrum of lumirhodopsin changes on the time scale from 5 to 100 micros, showing reduced rotational strength in its visible band, possibly associated with either a process responsible for a small spectral shift that occurs in the lumirhodopsin absorbance spectrum at earlier times or the Schiff base deprotonation-reprotonation which occurs during equilibration of lumirhodopsin with the Meta I(380) photointermediate. Either explanation suggests a chromophore conformation change closely associated with deprotonation which could be the earliest direct trigger of activation.

The Dependence of Retinal Degeneration Caused by the Rhodopsin P23H Mutation on Light Exposure and Vitamin A Deprivation

To characterize the influence of light and vitamin A on retinal degeneration in an animal model of retinitis pigmentosa caused by the rhodopsin P23H mutation. Retinal degeneration was examined in transgenic Xenopus laevis expressing P23H rhodopsin, in which retinal degeneration is completely rescued by preventing light exposure. The sensitivity of this retinal degeneration to varying intensities, wavelengths, and durations of light exposure, and to vitamin A deprivation was characterized. Green light was the most effective inducer of retinal degeneration in this model. Retinal degeneration was induced by prolonged exposure to green light and was prevented by filters that block short wavelengths. Reducing the duration of light exposure prevented retinal degeneration, even when the light intensity was proportionally increased. Vitamin A deprivation also induced retinal degeneration associated with defects in P23H rhodopsin biosynthesis. Vitamin A deprivation did not cause retinal degeneration in nontransgenic animals. The mechanism of retinal degeneration in this animal model of RP involves the interaction of light with rhodopsin rather than with free chromophore or bleached rhodopsin. These results may explain the clinical benefits of vitamin A for patients with retinitis pigmentosa and may indicate that pharmacological chaperones are a viable approach to RP therapy. Results also suggest strategies for minimizing RD in patients through controlling light exposure duration or wavelengths.

Formation Of All-Trans Retinol In Mouse Rod Photoreceptors

Light detection destroys the visual pigment of vertebrate rod photoreceptors, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. Rhodopsin is regenerated through a series of reactions that begin in the rod outer segment with the release of the all-trans retinal and its reduction to all-trans retinol. All-trans retinol is then transported to the neighboring retinal pigment epithelial cells where it is used to remake 11-cis retinal. The reduction of all-trans retinal to all-trans retinol is catalyzed by retinol dehydrogenase and requires metabolic input in the form of NADPH. We have used the fluorescence of all-trans retinol to monitor its concentration in isolated mouse rod photoreceptors. After the bleaching of rhodopsin, all-trans retinol formation proceeds with a rate of ∼0.06 min−1, which is faster than the rate of rhodopsin regeneration in whole animals; this would allow recycled chromophore to contribute to the 11-cis retinal used for regeneration. Inner segment metabolic pathways appear to make a significant contribution to the pool of NADPH needed for the reduction of all-trans retinal, as formation of all-trans retinol is suppressed in rod outer segments separated from the cell body. Finally, generation of all-trans retinol is suppressed in the absence of glucose, indicating a critical dependence of all-trans retinol formation on the level of metabolic activity.

Affinity of transducin for photoactivated rhodopsin: dependence on nucleotide binding state

The interaction of the rod GTP binding protein, Transducin (G(t)), with bleached Rhodopsin (R(*)) was investigated by measuring radiolabeled guanine nucleotide binding to and release from soluble and/or membrane-bound G(t) by reconstituting G(t) containing bound GDP (G(t-)GDP) or the hydrolysis-resistant GTP analog guanylyl imidodiphosphate (G(t-)p[NH]ppG) with R* under physiological conditions. Release of GDP and p[NH]ppG from G(t) occurred to the same extent and with the same light sensitivity both in the presence and absence of added GTP. Significant amounts of G(t) without bound nucleotide (G(t-)) were generated. When ROS containing bleached rhodopsin (R(*)) were centrifuged in low ionic strength buffer, G(t-) remained associated with the membrane fraction, whereas G(t-)GDP remained in the soluble fraction. These results suggest that G(t-)GDP and G(t-)p[NH]ppG have similar affinities for R(*). The results also suggest that G(t-), rather than G(t-)GDP, is the moiety which exhibits tight, "light-induced" binding to rhodopsin.

Recovery of Rod Photoresponses in ABCR-Deficient Mice

The ABCR protein of the rod outer segment is thought to facilitate movement of the all-trans retinal photoproduct of rhodopsin bleaching out of the disc lumen. This study was undertaken to investigate the extent to which ABCR deficiency affects the post-bleach recovery of the rod photoresponse in ABCR-deficient (abcr-/-) mice. Electroretinographic (ERG) a-wave responses were recorded from abcr-/- mice and two control strains. A bright probe flash was used to examine the course of rod recovery after fractional rhodopsin bleaches of approximately 10(-6), approximately 3 x 10(-5), approximately 0.03, and approximately 0.30 to approximately 0.40. Dark-adapted abcr-/- mice and control animals exhibited similar normalized near-peak amplitudes of the paired-flash-ERG-derived, weak-flash response. Response recovery after approximately 10(-6) bleaching exhibited an average exponential time constant of 319, 171, and 213 ms, respectively, in the abcr-/- and the two control strains. Recovery time constants determined for approximately 3 x 10(-5) bleaching did not differ significantly among strains. However, those determined for the approximately 0.03 bleach indicated significantly faster recovery in abcr-/- mice (2.34 +/- 0.74 minutes) than in the controls (5.36 +/- 2.20 and 5.92 +/- 2.44 minutes). After approximately 0.30 to approximately 0.40 bleaching, the initial recovery in the abcr-/- mice was, on average, faster than in control mice. By comparison with control animals, abcr-/- mice exhibit faster rod recovery after a bleach of approximately 0.03. The data suggest that ABCR in normal rods may directly or indirectly prolong all-trans retinal clearance from the disc lumen over a significant bleaching range, and that the essential function of ABCR may be to promote the clearance of residual amounts of all-trans retinal that remain in the discs long after bleaching.

The Proteome of the Mouse Photoreceptor Sensory Cilium Complex

Primary cilia play critical roles in many aspects of biology. Specialized versions of primary cilia are involved in many aspects of sensation. The single photoreceptor sensory cilium (PSC) or outer segment elaborated by each rod and cone photoreceptor cell of the retina is a classic example. Mutations in genes that encode cilia components are common causes of disease, including retinal degenerations. The protein components of mammalian primary and sensory cilia have not been defined previously. Here we report a detailed proteomics analysis of the mouse PSC complex. The PSC complex comprises the outer segment and its cytoskeleton, including the axoneme, basal body, and ciliary rootlet, which extends into the inner segment of photoreceptor cells. The PSC complex proteome contains 1968 proteins represented by three or more unique peptides, including approximately 1500 proteins not detected in cilia from lower organisms. This includes 105 hypothetical proteins and 60 proteins encoded by genes that map within the critical intervals for 23 inherited cilia-related disorders, increasing their priority as candidate genes. The PSC complex proteome also contains many cilia proteins not identified previously in photoreceptors, including 13 proteins produced by genes that harbor mutations that cause cilia disease and seven intraflagellar transport proteins. Analyses of PSC complexes from rootletin knock-out mice, which lack ciliary rootlets, confirmed that 1185 of the identified PSC complex proteins are derived from the outer segment. The mass spectrometry data, benchmarked by 15 well characterized outer segment proteins, were used to quantify the copy number of each protein in a mouse rod outer segment. These results reveal mammalian cilia to be several times more complex than the cilia of unicellular organisms and open novel avenues for studies of how cilia are built and maintained and how these processes are disrupted in human disease.

Interphotoreceptor Retinoid-Binding Protein Is the Physiologically Relevant Carrier That Removes Retinol from Rod Photoreceptor Outer Segments

Light detection by vertebrate rod photoreceptor outer segments results in the destruction of the visual pigment, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. The regeneration of rhodopsin is necessary for vision and begins with the release of the all-trans retinal and its reduction to all-trans retinol. Retinol is then transported out of the rod outer segment for further processing. We used fluorescence imaging to monitor retinol fluorescence and quantify the kinetics of its formation and clearance after rhodopsin bleaching in the outer segments of living isolated frog (Rana pipiens) rod photoreceptors. We independently measured the release of all-trans retinal from bleached rhodopsin in frog rod outer segment membranes and the rate of all-trans retinol removal by the lipophilic carriers interphotoreceptor retinoid binding protein (IRBP) and serum albumin. We find that the kinetics of all-trans retinol formation in frog rod outer segments after rhodopsin bleaching are to a good first approximation determined by the kinetics of all-trans retinal release from the bleached pigment. For the physiological concentrations of carriers, the rate of retinol removal from the outer segment is determined by IRBP concentration, whereas the effect of serum albumin is negligible. The results indicate the presence of a specific interaction between IRBP and the rod outer segment, probably mediated by a receptor. The effect of different concentrations of IRBP on the rate of retinol removal shows no cooperativity and has an EC50 of 40 micromol/L.

Evidence for Specificity in Lipid-Rhodopsin Interactions

The interaction of bovine rhodopsin with poly- and monounsaturated lipids was studied by (1)H MAS NMR with magnetization transfer from rhodopsin to lipid. Experiments were conducted on bovine rod outer segment (ROS) disks and on recombinant membranes containing lipids with polyunsaturated, docosahexaenoyl (DHA) chains. Poly- and monounsaturated lipids interact specifically with different sites on the rhodopsin surface. Rates of magnetization transfer from protein to DHA are lipid headgroup-dependent and increased in the sequence PC < PS < PE. Boundary lipids are in fast exchange with the lipid matrix on a time scale of milliseconds or shorter. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids, but highest rates were observed for Meta-III rhodopsin. The experiments show clearly that the surface of rhodopsin has sites for specific interaction with lipids. Current theories of lipid-protein interaction do not account for such surface heterogeneity.

Cyclic Nucleotide-gated Ion Channels in Rod Photoreceptors Are Protected from Retinoid Inhibition

In vertebrate rods, photoisomerization of the 11-cis retinal chromophore of rhodopsin to the all-trans conformation initiates a biochemical cascade that closes cGMP-gated channels and hyperpolarizes the cell. All-trans retinal is reduced to retinol and then removed to the pigment epithelium. The pigment epithelium supplies fresh 11-cis retinal to regenerate rhodopsin. The recent discovery that tens of nanomolar retinal inhibits cloned cGMP-gated channels at low [cGMP] raised the question of whether retinoid traffic across the plasma membrane of the rod might participate in the signaling of light. Native channels in excised patches from rods were very sensitive to retinoid inhibition. Perfusion of intact rods with exogenous 9- or 11-cis retinal closed cGMP-gated channels but required higher than expected concentrations. Channels reopened after perfusing the rod with cellular retinoid binding protein II. PDE activity, flash response kinetics, and relative sensitivity were unchanged, ruling out pharmacological activation of the phototransduction cascade. Bleaching of rhodopsin to create all-trans retinal and retinol inside the rod did not produce any measurable channel inhibition. Exposure of a bleached rod to 9- or 11-cis retinal did not elicit channel inhibition during the period of rhodopsin regeneration. Microspectrophotometric measurements showed that exogenous 9- or 11-cis retinal rapidly cross the plasma membrane of bleached rods and regenerate their rhodopsin. Although dark-adapted rods could also take up large quantities of 9-cis retinal, which they converted to retinol, the time course was slow. Apparently cGMP-gated channels in intact rods are protected from the inhibitory effects of retinoids that cross the plasma membrane by a large-capacity buffer. Opsin, with its chromophore binding pocket occupied (rhodopsin) or vacant, may be an important component. Exceptionally high retinoid levels, e.g., associated with some retinal degenerations, could overcome the buffer, however, and impair sensitivity or delay the recovery after exposure to bright light.

Effect of Visible Light on Normal and P23H‐3 Transgenic Rat Retinas: Characterization of a Novel Retinoic Acid Derivative Present in the P23H‐3 Retina

Transgenic rats with the P23H mutation in rhodopsin exhibit increased susceptibility to light damage, compared with normal animals. It is known that light-induced retinal damage requires repetitive bleaching of rhodopsin and that photoreceptor cell loss is by apoptosis; however, the underlying molecular mechanism(s) leading to photoreceptor cell death are still unknown. Photoproducts, such as all-trans retinal or other retinoid metabolites, released by the extensive bleaching of rhodopsin could lead to activation of degenerative processes, especially in animals genetically predisposed to retinal degenerations. Using wild-type and transgenic rats carrying the P23H opsin mutation, we evaluated the effects of acute intense visible light on retinoid content, type and distribution in ocular tissues. Rats were exposed to green light (480-590 nm) for 0, 5, 10, 30 and 120 min. Following light treatment, rats were sacrificed and neural retinas were dissected free of the retinal pigment epithelium. Retinoids were extracted from retinal tissues and then subjected to HPLC and mass spectral analysis. We found that the light exposure affected relative levels of retinoids in the neural retina and retinal pigment epithelium of wild-type and P23H rat eyes similarly. In the P23H rat retina but not the wild-type rat retina, we found a retinoic acid-like compound with an absorbance maximum of 357 nm and a mass of 304 daltons. Production of this retinoic acid-like compound in transgenic rats is influenced by the age of the animals and the duration of light exposure. It is possible that this unique retinoid may be involved in the process of light-induced retinal degeneration.

Physiological Features of the S- and M-cone Photoreceptors of Wild-type Mice from Single-cell Recordings

Cone cells constitute only 3% of the photoreceptors of the wild-type (WT) mouse. While mouse rods have been thoroughly investigated with suction pipette recordings of their outer segment membrane currents, to date no recordings from WT cones have been published, likely because of the rarity of cones and the fragility of their outer segments. Recently, we characterized the photoreceptors of Nrl −/− mice, using suction pipette recordings from their “inner segments” (perinuclear region), and found them to be cones. Here we report the use of this same method to record for the first time the responses of single cones of WT mice, and of mice lacking the α-subunit of the G-protein transducin (G tα−/−), a loss that renders them functionally rodless. Most cones were found to functionally co-express both S- (λmax = 360 nm) and M- (λmax = 508 nm) cone opsins and to be maximally sensitive at 360 nm (“S-cones”); nonetheless, all cones from the dorsal retina were found to be maximally sensitive at 508 nm (“M-cones”). The dim-flash response kinetics and absolute sensitivity of S- and M-cones were very similar and not dependent on which of the coexpressed cone opsins drove transduction; the time to peak of the dim-flash response was ∼70 ms, and ∼0.2% of the circulating current was suppressed per photoisomerization. Amplification in WT cones (A ∼4 s−2) was found to be about twofold lower than in rods (A ∼8 s−2). Mouse M-cones maintained their circulating current at very nearly the dark adapted level even when >90% of their M-opsin was bleached. S-cones were less tolerant to bleached S-opsin than M-cones to bleached M-opsin, but still far more tolerant than mouse rods to bleached rhodopsin, which exhibit persistent suppression of nearly 50% of their circulating current following a 20% bleach. Thus, the three types of mouse opsin appear distinctive in the degree to which their bleached, unregenerated opsins generate “dark light.”

Arrestin Translocation Is Induced at a Critical Threshold of Visual Signaling and Is Superstoichiometric to Bleached Rhodopsin

Light induces massive translocation of major signaling proteins between the subcellular compartments of photoreceptors. Among them is visual arrestin responsible for quenching photoactivated rhodopsin, which moves into photoreceptor outer segments during illumination. Here, for the first time, we determined the light dependency of arrestin translocation, which revealed two key features of this phenomenon. First, arrestin translocation is triggered when the light intensity approaches a critical threshold corresponding to the upper limits of the normal range of rod responsiveness. Second, the amount of arrestin entering rod outer segments under these conditions is superstoichiometric to the amount of photoactivated rhodopsin, exceeding it by at least 30-fold. We further showed that it is not the absolute amount of excited rhodopsin but rather the extent of downstream cascade activity that triggers translocation. Finally, we demonstrated that the total amount of arrestin in the rod cell is nearly 10-fold higher than previously thought and therefore sufficient to inactivate the entire pool of rhodopsin at any level of illumination. Thus, arrestin movement to the outer segment leads to an increase in the free arrestin concentration and thereby may serve as a powerful mechanism of light adaptation.

Recoverin Undergoes Light-dependent Intracellular Translocation in Rod Photoreceptors

Photoreceptor cells have a remarkable capacity to adapt the sensitivity and speed of their responses to ever changing conditions of ambient illumination. Recent studies have revealed that a major contributor to this adaptation is the phenomenon of light-driven translocation of key signaling proteins into and out of the photoreceptor outer segment, the cellular compartment where phototransduction takes place. So far, only two such proteins, transducin and arrestin, have been established to be involved in this mechanism. To investigate the extent of this phenomenon we examined additional photoreceptor proteins that might undergo light-driven translocation, focusing on three Ca(2+)-binding proteins, recoverin and guanylate cyclase activating proteins 1 (GCAP1) and GCAP2. The changes in the subcellular distribution of each protein were assessed quantitatively using a recently developed technique combining serial tangential sectioning of mouse retinas with Western blot analysis of the proteins in the individual sections. Our major finding is that light causes a significant reduction of recoverin in rod outer segments, accompanied by its redistribution toward rod synaptic terminals. In both cases the majority of recoverin was found in rod inner segments, with approximately 12% present in the outer segments in the dark and less than 2% remaining in that compartment in the light. We suggest that recoverin translocation is adaptive because it may reduce the inhibitory constraint that recoverin imposes on rhodopsin kinase, an enzyme responsible for quenching the photo-excited rhodopsin during the photoresponse. To the contrary, no translocation of rhodopsin kinase itself or either GCAP was identified.

Interphotoreceptor retinoid-binding protein (IRBP) promotes the release of all-trans retinol from the isolated retina following rhodopsin bleaching illumination

All-trans retinol generated in rod photoreceptors upon the bleaching of rhodopsin is known to move from the rods to the retinal pigment epithelium (RPE), where it is enzymatically converted to 11-cis retinal in the retinoid visual cycle. Interphotoreceptor retinoid-binding protein (IRBP) contained in the extracellular compartment (interphotoreceptor matrix) that separates the retina and RPE has been hypothesized to facilitate this movement of all-trans retinol, but the precise role of IRBP in this process remains unclear. To examine the activity of IRBP in the release of all-trans retinol from the rods, initially dark-adapted isolated retinas obtained from toad (Bufo marinus) eyes were bleached and then incubated in darkness for defined periods (5–180min) in physiological saline (Ringer solution) supplemented with IRBP (here termed ‘IRBP I’) at defined concentrations (2–90μm). Retinoids present in the retina and extracellular medium were then determined by extraction and HPLC analysis. Preparations incubated with ≥10μm IRBP I showed a pronounced release of all-trans retinol with increasing period of incubation. As determined with 25μm IRBP I, the increase of all-trans retinol in the extracellular medium was accompanied by a significant decrease in the combined amount of all-trans retinal and all-trans retinol contained in the retina. This effect was not mimicked by unsupplemented Ringer solution or by Ringer solution containing 25 or 90μm bovine serum albumin. However, incubation with ‘IRBP II’, a previously described variant of IRBP with altered lectin-binding properties, led to the appearance of substantial all-trans retinol in the extracellular medium. The results suggest that in vivo, IRBP plays a direct role in the release of all-trans retinol from the rods during operation of the visual cycle.

Reduction of All-trans Retinal to All-trans Retinol in the Outer Segments of Frog and Mouse Rod Photoreceptors

The first step in the Visual Cycle, the series of reactions that regenerate the vertebrate visual pigment rhodopsin, is the reduction of all-trans retinal to all-trans retinol, a reaction that requires NADPH. We have used the fluorescence of all-trans retinol to study this reduction in living rod photoreceptors. After the bleaching of rhodopsin, fluorescence (excitation, 360nm; emission, 457 or 540nm) appears in frog and wild-type mouse rod outer segments reaching a maximum in 30–60min at room temperature. With this excitation and emission, the mitochondrial-rich ellipsoid region of the cells shows strong fluorescence as well. Fluorescence measurements at different emission wavelengths establish that the outer segment and ellipsoid signals originate from all-trans retinol and reduced pyridine nucleotides, respectively. Using outer segment fluorescence as a measure of all-trans retinol formation, we find that in frog rod photoreceptors the NADPH necessary for the reduction of all-trans retinal can be supplied by both cytoplasmic and mitochondrial metabolic pathways. Inhibition of the reduction reaction, either by retinoic acid or through suppression of metabolic activity, reduced the formation of retinol. Finally, there are no significant fluorescence changes after bleaching in the rod outer segments of Rpe65−/− mice, which lack 11-cis retinal.

From candelas to photoisomerizations in the mouse eye by rhodopsin bleaching in situ and the light-rearing dependence of the major components of the mouse ERG

To quantify the rate at which light in a ganzfeld produces photoisomerizations in mouse rods in situ, we measured the rate of rhodopsin bleaching in eyes of recently euthanized mice with fully dilated pupils. The amount of rhodopsin declined as a first-order (exponential) function of the duration of the exposure at the luminance of 920 scot cd m −2: the rate constants of bleaching were 8.3 × 10 −6 and 2.8 × 10 −5 s −1 (scot cd −1 m 2) −1 for C57B1/6 and 129P3/J mice, respectively. When the ∼3-fold difference in effective areas of the pupils of the mice are taken into consideration, the bleaching rates for both strains become essentially the same, 2.6 × 10 −6 fraction rhodopsin (scot Td s) −1. Assuming 7 × 10 7 rhodopsin molecules per rod, this bleaching rate yields the result that a flash of 1 scot Td s produces 181 photoisomerizations per rod, a value close to that derived from analysis of the collecting area of the rod for axially propagating light. We measured the electroretinograms of mice of the two strains reared under controlled illumination conditions (2 and 100 lux), and compared their properties, using the calibrations to determine the absolute sensitivities of the b-wave and a-waves. The intensity that produces a half-saturating rod b-wave response is 0.3–0.6 photoisomerizations rod −1, and the amplification constant of the rod a-wave is 5–6 s −2 photoisomerization −1, with little dependence on the strain.

Two realms of dark adaptation

The recovery of rod responsiveness after saturating flashes is greatly retarded above a certain critical level of rhodopsin bleaching (∼0.1%). A mathematical description of the process of turn-off of the phototransduction cascade allows attributing different phases of the recovery to specific products of rhodopsin photolysis. The fast phase is determined by quenching of metarhodopsin II and activated transducin. The slow phase is controlled by decay of partially inactivated (phosphorylated and arrestin-bound) metarhodopsins, and by regeneration of rhodopsin. The transition between the two regimes of adaptation is rather abrupt, occurring within a few-fold range of stimulus intensity. This marks the border between reversal of light adaptation and dark adaptation, as it is commonly defined.