1. Visual Pigment Phosphorylation but Not Transducin Translocation Can Contribute to Light Adaptation in Zebrafish Cones 
Matthew J Kennedy, Felice A Dunn, James B Hurley 
Neuron 
Volume 41, Issue 6, Pages (March 2004)
DOI: /S (04)

2. Figure 1
Cone Opsins Are Multiply Phosphorylated in Response to Steady Illumination
(A) Elution profiles from reversed-phase chromatography are shown for cone opsin C-terminal peptides from approximately two-thirds of a dark-adapted zebrafish retina. Each chromatogram monitors a narrow mass window (1.5–2 Daltons) centered on the predicted mass to charge ratio (m/z) of each cone opsin C-terminal peptide (shown above each chromatogram). The peptides were detected in their doubly charged state with the following mass-to-charge ratios (m/z): uv, (amino acids 320–336); red, (amino acids 338–356); blue, (amino acids 336–354); green-1/2, (amino acids 330–349); green-3/4, (amino acids 330–349). Each peptide was sequenced by collision-induced dissociation to confirm its identity (data not shown). The y axis for each chromatogram represents the relative abundance of each peak and is normalized to the amplitude of the maximum signal. The x axis represents the elution time from the C18 reversed-phase column.
(B) Ion chromatograms monitoring mass windows corresponding to green-1/2 opsin C-terminal peptides with 0, 1, 2, 3, 4, and 5 phosphates from a dark-adapted retina (left) or from a retina that was exposed to 4 min of steady 6.8 mW/cm2 white light (right). Note the appearance of peaks in mass windows corresponding to phosphorylated green opsin (shaded peaks) that are absent from the dark-adapted sample. The numbers to the left of each peak represent the relative fraction of each species calculated by integrating the area under each peak and applying a correction factor. Very similar results were obtained for blue opsin.
(C and D) Peptides eluting from the column with masses corresponding to the different phosphorylated forms of blue (C) or green (D) cone opsin were fragmented using collision-induced dissociation (CID). Elution profiles for singly charged fragments (shown by brackets above the peptide sequences) that are produced specifically upon dissociation of cone opsin C-terminal peptides containing 1, 2, or 3 phosphates are shown in the bottom three chromatograms (peaks shaded in black). We monitored the production of a 15 amino acid fragment from the N-terminal side of the blue opsin peptide (b15 ion) and either a 17 or 18 amino acid fragment from the N-terminal side of the green opsin peptide (b17 or b18 ion). This method allowed us to quickly and reliably identify cone opsin peptides and to exclude signals from contaminating peptides that have a similar mass. The peaks shaded in gray in the upper three chromatograms are confirmed cone opsin C-terminal peptides. These samples are from dissected retinas that were exposed to 6.8 mW/cm2 white light for 3 min.
Neuron  , DOI: ( /S (04) )

3. Figure 2
The Kinetics of Cone Opsin Phosphorylation and Dephosphorylation
(A) The level of blue cone opsin phosphorylation in dark-adapted retinas or in retinas that were exposed to light (6.8 mW/cm2) for various times from 30 s to 4 min was quantified by integrating the area under peaks corresponding to the blue opsin peptide. The level of each species is expressed as the fraction of the total blue opsin peptide detected. Each time point is the average of at least three separate determinations. Standard deviations were typically ±5%.
(B) The rate of cone opsin phosphorylation was measured following a flash that bleached ∼20% of the visual pigment. The rate of blue cone opsin phosphorylation from zebrafish cones (filled circles) is compared to rhodopsin phosphorylation in mouse rods following a flash that bleached the same fraction of rhodopsin (open circles) (data taken from Kennedy et al., 2001). The inset shows an expanded view of the early time course of phosphorylation. The y axis represents the fraction of pigment molecules modified by at least one phosphate.
(C) The level of blue cone opsin phosphorylation was measured in vivo at various times during and following steady 12 mW/cm2 illumination for 10 min (open bar above plot). Phosphorylation and dephosphorylation of cone opsins reached a steady state within 1 (green opsins; data not shown) to 4 min (blue opsin). The rate of cone opsin dephosphorylation was determined by measuring the phosphorylation state of cone opsins after the light was extinguished following 10 min of continuous illumination (black bar above plot). Both blue and green opsins were dephosphorylated with a t1/2 of 4 min. Data are expressed as the fraction of unphosphorylated blue pigment.
Neuron  , DOI: ( /S (04) )

4. Figure 3 Blue and Green Cone Opsin Phosphorylation Sites
(A) Monophosphorylated blue opsin peptides from retinas that were exposed to steady illumination for 3 min resolve into 4 different species under optimal chromatography conditions. Each peak represents blue opsin C-terminal peptide phosphorylated at a different site. We elucidated the major site of phosphorylation for each species using collision-induced dissociation (data not shown). The sites of phosphorylation (from earliest eluting peak) were serine 349, 348, 341 or 342, and 339 or 340.
(B) Monophosphorylated green-1/2 opsin peptides from retinas that were exposed to steady illumination for 3 min resolve into 3 different peaks under optimal chromatography conditions (peaks a–c). Our CID analysis indicated that the sites of phosphorylation (from earliest eluting peak) were serine 334, serine 344 or threonine 341, and serine 333.
(C) Analysis of the phosphorylation state of blue cone opsin in vivo before (upper chromatogram) and after (lower chromatrogram) steady-state phosphorylation/dephosphorylation was achieved. Note that before phosphorylation reaches steady state there is significant accumulation of phosphate groups closer to the C-terminal side of the peptide at serine 348 (peak b). During steady-state conditions and during subsequent dark adaptation, sites farthest from the C terminus predominated.
Neuron  , DOI: ( /S (04) )

5. Figure 4
The Extent of Light-Stimulated Pigment Phosphorylation Is Reduced in cTα Null Cones Demonstrating the Presence of a Ca2+-Dependent Mechanism that Regulates Phosphorylation
(A) The amount of blue cone opsin phosphorylation following 4 min of steady illumination (12 mW/cm2) was measured in vivo from wt and cTα null fish. We observed approximately 7-fold more cone opsin phosphorylation in wt eyes compared with cTα null eyes. Results are expressed as the fraction of blue pigment containing at least 1 phosphate.
(B) The level of blue opsin phosphorylation in dissected retinas from wild-type (black bars) and cTα null fish (white bars) that were bleached for either 30 s (left panel) or 3 min (right panel). Note that the amount of cone pigment phosphorylation is nearly identical between wt and cTα null retinas that were bleached for only 30 s, but after 3 min, the level of phosphorylation was much higher in wt retinas. When cTα null retinas were bleached in a solution designed to lower the level of Ca2+ in photoreceptors (0 Ca2+ solution), we observed wt levels of cone opsin phosphorylation (gray bars, right panel).
(C) Typical elution profiles for blue opsin C-terminal peptides from a wild-type retina bleached in Ringer solution (left), a cTα null retina bleached in Ringer solution (center), and a cTα null retina bleached in a modified physiological solution designed to specifically remove intracellular Ca2+ from photoreceptors (0 Ca2+ solution) (right). Retinas were bleached for 3 min at 6.8 mW/cm2. The number above each peak represents the relative fraction of each species compared to the total amount of blue C terminus detected. Note the absence of significant accumulation of blue opsin modified with 3, 4, or 5 phosphates in cTα null retinas bleached in Ringer solution. The y axis for each chromatogram represents the relative abundance of each peak and is normalized to the amplitude of the maximum signal.
Neuron  , DOI: ( /S (04) )

6. Figure 5 Ca2+ Regulates the Sites of Cone Opsin Phosphorylation
Monophosphorylated blue opsin elutes from the reversed phase column in four different peaks, each containing blue opsin modified at different sites under optimal chromatography. The major phosphorylation sites are designated by gray circles behind the phosphorylated residue on the blue opsin C-terminal peptide sequences to the right of each chromatogram. In cases where two residues are marked, the site of phosphorylation was narrowed down to these two residues, but the exact site could not be determined. In dissected retinas from wild-type fish, a significant amount of phosphorylation accumulated at serine 348, but no phosphorylation at this site was observed in cTα null cones. Phosphorylation at serine 348 did accumulate in cTα null retinas if they were bleached in a modified physiological solution designed to lower the intracellular concentration of Ca2+ in photoreceptors (0 Ca2+ solution), showing that Ca2+ influences the sites of light-triggered phosphorylation. Retinas were bleached for 2 min at 6.8 mW/cm2, but very similar results were obtained when retinas were bleached for only 30 s.
Neuron  , DOI: ( /S (04) )

7. Figure 6
Cone Opsin Dephosphorylation Is Blocked in Wild-Type Retinas in a Light- and cTα-Dependent Fashion
(A) Dephosphorylation of cone opsin was complete after 20 min of dark adaptation following a 10 min bleach that drove ∼60% phosphorylation of blue opsin. Green-1/2 opsin was dephosphorylated at a similar rate.
(B) The level of green-1/2 opsin phosphorylation was measured in dissected retinas that were illuminated with 11 mW/cm2 light for 30 s. Samples were quenched either immediately following light exposure or following a 10 min period of dark adaptation in oxygenated Ringer solution. In vivo, cone opsin dephosphorylation is nearly complete after 10 min, but very little dephosphorylation occurs after 10 min of dark adaptation in dissected retinas. When a similar amount of cone pigment was phosphorylated at the same sites in dark-adapted retinas by bathing them in 0 Ca2+ solution, dephosphorylation of pigment was almost complete after a 5 min incubation in normal Ringer solution.
(C) Very little blue cone opsin was dephosphorylated after 20 min of dark adaptation in dissected retinas incubated in Ringer solution (following a 30 s light exposure at 11 mW/cm2). Incubation of dissected retinas with 9-cis retinal (data not shown) or the PDE inhibitor IBMX during dark adaptation failed to stimulate cone opsin dephosphorylation. Pigment was dephosphorylated at a normal rate in cTα null cones that were bleached in 0 Ca2+ solution and then dark adapted in either Ringer solution or 0 Ca2+ solution. Because long incubations in 0 Ca2+ solution stimulates significant levels of light-independent phosphorylation, the basal value of phosphorylation from unbleached cTα null retinas incubated in 0 Ca2+ solution was subtracted from the level of phosphorylation in bleached cTα null retinas incubated in 0 Ca2+ solution during dark adaptation.
Neuron  , DOI: ( /S (04) )

8. Figure 7
Ca2+ Regulates Phosphorylation of Unbleached Cone Opsin in Dark-Adapted Cones
(A) Zebrafish (left side) or mouse (right side) retinas were incubated in the dark in either normal Ringer solution or 0 Ca2+ solution for the times specified. Significant amounts of phosphorylated cone opsin accumulate when retinas are bathed in 0 Ca2+ solution in complete darkness. Both blue and green opsins are phosphorylated in the dark in 0 Ca2+ solution with an apparent rate of ∼3%/min. Rhodopsin phosphorylation in mouse retinas in 0 Ca2+ solution was only modestly elevated under the same conditions. Dark phosphorylation of cone opsins in 0 Ca2+ solution was fully reversible if the retinas were incubated in 0 Ca2+ solution for 5 min and then moved to normal Ringer solution for 5 min (black bar). Retinas were also incubated with excess 9-cis retinal (∼70 μM) for 10–15 min in normal Ringer solution before being moved to 0 Ca2+ solution (also containing 70 μM 9-cis retinal) for 5 min (white bar). The y axis represents the fraction of pigment molecules modified by at least one phosphate.
(B) Frozen sections from retinas bathed in normal Ringer or 0 Ca2+ solution were stained with antibodies recognizing red, blue, and uv cone opsins. Cone outer segment morphology appeared normal after zebrafish retinas were bathed in 0 Ca2+ solution for 5 min.
(C) Monophosphorylated blue opsin from retinas bathed in 0 Ca2+ solution (top chromatogram) and from retinas that were bleached for 30 s (6.8 mW/cm2) in normal Ringer solution with a similar amount of phosphorylation (bottom chromatogram). Note that the distribution of blue opsin peptides phosphorylated at different sites appears the same under both conditions. Similar results were obtained for green opsin.
(D) Monophosphorylated blue opsin from wt or cTα null retinas bleached for 30 s. Note that very little phosphorylation occurs on serine 348 in cTα null retinas.
Neuron  , DOI: ( /S (04) )

9. Figure 8
Transducin Localization in Dark- and Light-Adapted Zebrafish Cones
Cone transducin was localized in dark- and light-adapted zebrafish cones using a polyclonal antibody that specifically recognizes cone transducin (Brockerhoff et al., 2003). Zebrafish were exposed to bright light (10–12 mW/cm2) for 15 min. Under these conditions, approximately 60%–70% of the blue and green visual pigments are phosphorylated and obvious retinomotor movements have occurred. Light microscopy images are merged with cone transducin staining (red) and zpr-1 staining (green) in (A) and (B). zpr-1 specifically labels double (red/green) cone cell bodies and synapses. Note that in light-adapted retinas, the RPE envelops the double cone outer segments. Panels (C)–(F) show transducin staining in red and zpr-1 staining in green (C and D) in dark-adapted and light-adapted eyes. Note that the transducin staining pattern in blue (ls) cone outer segments is identical in light- and dark-adapted eyes. Red/green cone outer segments cannot be seen in light-adapted retinal sections because the RPE quenches immunofluorescence. Panels (E) and (F) show labeling of cone transducin alone (red). RPE, retinal pigment epithelium; dc os, double cone (red/green) outer segments; ls os, long single cone (blue) outer segments; ss os, single short cone (uv) outer segments; opl, outer plexiform layer.
Neuron  , DOI: ( /S (04) )

10. Figure 9
Hypotheses for the Roles of Cone Visual Pigment Phosphorylation in Light Adaptation
(A) Ca2+ alters the site specificity of cone pigment phosphorylation. In bright light (low Ca2+), sites closest to the C terminus of cone opsin are phosphorylated, while in dim light, (high Ca2+) sites further away from the C terminus of cone opsin are phosphorylated. Studies using rhodopsin have shown that opsins phosphorylated at sites closest to the C terminus bind arrestin more effectively than pigments phosphorylated closer to the membrane. In bright light (low Ca2+) the lifetime of active pigment will be shortened by enhanced arrestin binding, reducing the amount of transducin stimulated per photoisomerization. Phosphorylation at different sites could also have different effects on the gain of G protein activation, but further investigation is needed to define the roles of phosphorylation at each site.
(B) A light-triggered drop in Ca2+ increases the amount of phosphorylated cone pigment by stimulating phosphorylation of unbleached pigment. Phosphorylated G protein-coupled receptors are less efficient at stimulating their partner G proteins when activated, so signaling is desensitized as phosphorylated pigment accumulates. Arrestin binding to phosphorylated G protein-coupled receptors further attenuates their activity.
Neuron  , DOI: ( /S (04) )