March 2003
Volume 44, Issue 3
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Retina  |   March 2003
Cone Deactivation Kinetics and GRK1/GRK7 Expression in Enhanced S Cone Syndrome Caused by Mutations in NR2E3
Author Affiliations
  • Artur V. Cideciyan
    From the Department of Ophthalmology and the
  • Samuel G. Jacobson
    From the Department of Ophthalmology and the
  • Nisha Gupta
    From the Department of Ophthalmology and the
  • Shoji Osawa
    Department of Cell and Developmental Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; the
  • Kristin G. Locke
    Retina Foundation of the Southwest, Dallas, Texas; and the
  • Ellen R. Weiss
    Department of Cell and Developmental Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; the
  • Alan F. Wright
    Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, United Kingdom.
  • David G. Birch
    Retina Foundation of the Southwest, Dallas, Texas; and the
  • Ann H. Milam
    From the Department of Ophthalmology and the
    F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania; the
Investigative Ophthalmology & Visual Science March 2003, Vol.44, 1268-1274. doi:10.1167/iovs.02-0494
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      Artur V. Cideciyan, Samuel G. Jacobson, Nisha Gupta, Shoji Osawa, Kristin G. Locke, Ellen R. Weiss, Alan F. Wright, David G. Birch, Ann H. Milam; Cone Deactivation Kinetics and GRK1/GRK7 Expression in Enhanced S Cone Syndrome Caused by Mutations in NR2E3. Invest. Ophthalmol. Vis. Sci. 2003;44(3):1268-1274. doi: 10.1167/iovs.02-0494.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To determine the relationship between cone deactivation kinetics in patients with the enhanced S cone syndrome (ESCS) caused by mutations in NR2E3 and the immunoreactivity to G-protein-coupled receptor kinase 1 (GRK1) and GRK7.

methods. Electroretinogram (ERG) photoresponses were used to investigate activation kinetics of cones with a model of cone phototransduction. Deactivation kinetics of cones after bright flashes was quantified with a paired-flash ERG paradigm. Immunocytochemistry was performed with antibodies against cone opsins and kinases GRK1 and GRK7 in postmortem normal and ESCS retinal tissue.

results. Activation kinetics of long/middle-wavelength-sensitive (L/M) cone-mediated responses in patients with ESCS were similar to those of normal L/M cones. Activation kinetics of ESCS short-wavelength-sensitive (S) cones, when compared with normal L/M cone responses evoked by the same stimulus, were slower by an amount consistent with the expected differences in spectral sensitivities. After bright flashes chosen to evoke identical activation kinetics, ESCS S cones deactivated much more slowly than ESCS or normal L/M cones. Normal human retina revealed strongly labeled cone outer segments with anti-GRK1 and anti-GRK7. In an ESCS retina, outer segments positive for L/M opsin were strongly labeled with anti-GRK1, whereas outer segments positive for S opsin showed no detectable GRK1 reactivity. GRK7 labeling was absent in all photoreceptors of the ESCS retina.

conclusions. The cone-dominant human retina resulting from NR2E3 mutations affords greater understanding of the physiological roles of GRK1 and GRK7 in human cone photoreceptors. Normal deactivation kinetics in human L/M cones can occur without GRK7 when GRK1 is present in ESCS, but does not occur when GRK7 is present but GRK1 is deficient in Oguchi disease. Lack of both GRK1 and GRK7 in S cones of patients with ESCS results in a more pronounced abnormality in deactivation kinetics and suggests the existence of partial compensation by either GRK when the other is deficient.

Most environmental stimuli, such as light and odor, lead to a response in sensory neurons by activating G-protein-coupled receptors (GPCRs), which form the largest known family of signal-transducing proteins. Phosphorylation by G-protein-coupled receptor kinases (GRK) initiates timely termination of GPCR signaling by homologous desensitization targeted specifically to activated receptors. 1 Photoreceptors in the human retina are sensory neurons where activation of GPCR followed by quick deactivation results in a highly reproducible signal, the photoresponse. 2 Unlike in rod photoreceptor cells and unlike in most other mammals, human (and monkey) cone photoreceptors express two GRKs, GRK1 and GRK7. 3 4 The relative contributions of these two molecules to human cone cell deactivation are currently unknown. 
We have sought understanding of cone photoreceptor function in humans through naturally occurring, molecularly defined human disease. Abnormally slow cone deactivation kinetics in Oguchi disease caused by a homozygous null mutation in the GRK1 gene has provided evidence that GRK1 contributes to human cone function. 5 Grk1 −/− mice also show slowed kinetics of cone deactivation. The extent of the abnormality in mice appears more exaggerated than in humans. 6 Contribution of phosphorylation by GRK7 expressed in human, but not in mouse, cones has been proposed to explain the apparent interspecies differences in phenotype resulting from a lack of functional GRK1. 3 4 In the current work, we took advantage of the large ERG signals originating from cone-dominant human retinas in patients with the enhanced S cone syndrome (ESCS) to further investigate cone deactivation. Mutations in NR2E3 cause ESCS, in which an abnormality in cell-fate determination leads to a photoreceptor mosaic dominated by cones expressing short-wavelength-sensitive (S) opsin, and fewer cones expressing long/middle-wavelength-sensitive (L/M) opsin. 7 8 9 10 11 12 13 Postmortem human retinal tissue from a patient with ESCS gave us the rare opportunity to relate the expression of GRK1 and GRK7 to physiology in cones. 
Methods
Subjects
Normal subjects and 11 patients with ESCS caused by NR2E3 mutations were included in the study. 11 12 13 Previously published 5 data from a patient with Oguchi disease caused by a homozygous null mutation in the GRK1 gene were reanalyzed and presented as a comparison to the ESCS results. Ten of 11 patients with ESCS contributed to different aspects of the electrophysiological results (not all patients had all tests; the number undergoing each test is specified later). The eye donor had nonrecordable ERGs, but her psychophysical results were typical of those with ESCS. 13 Informed consent was obtained from subjects after explanation of the procedures. All studies conformed to institutional guidelines and the Declaration of Helsinki. Student’s t-test was used for testing the significance of the difference of means. 
Electroretinography
The magnitude of the photoreceptor dark-current immediately before the presentation of a light flash and the initial kinetics of its suppression can be estimated in vivo by recording the leading edge (<15 ms) of ERG photoresponses evoked with high-energy stimuli 14 and analyzing it with biochemically based quantitative models. 15 The magnitude of the dark current at longer (>15 ms) times, including the kinetics of the restoration of the dark current, can be estimated in vivo with the paired-flash paradigm, by using a test flash and a probe flash. 16 17 18 The details of our methods for recording and analyzing single- and paired-flash ERG photoresponses evoked with high-energy (saturating) stimuli in human subjects have been published. 5 9 19 20 21 22 23 24  
In the current set of experiments, full-field (ganzfeld) flash (∼1 ms) stimuli were presented, and the resultant ERG photoresponses were recorded with unipolar Burian-Allen (Hansen Ophthalmics, Iowa City, IA) contact lens electrodes (referenced to the forehead) and a computerized ERG apparatus (Pathfinder II; Nicolet Instruments Corp., Madison, WI). In most experiments, recording bandwidth was 1 kHz, and waveforms were digitized at 2.5 kHz. In the remaining experiments, higher bandwidths and higher sampling frequencies were used. In normal subjects, patients with ESCS, and the patient with Oguchi disease, single-flash ERG photoresponse families were recorded with red (Wratten 26 filter; Eastman Kodak Co., Rochester, NY) flashes (most subjects had series from 2.2–4.1 log troland-seconds [td · s]; some subjects had 3.6 and 4.1 log td · s) under light adaptation (white, 3.2 log td). Current evidence suggests that these responses are dominated by L/M cone activity. 5 9 19 23 In patients with ESCS, additional photoresponse families were recorded with violet (Wratten 98 filter; 1.0–2.5 log td · s) or white (3.8–5.1 log td · s) flash stimuli under light adaptation (white, 3.2 log td). Responses evoked by violet and white flashes in ESCS are dominated by S cone activity. 7 8 9 11 To date, we have been unable to record an unequivocal S cone-mediated component within the leading edge of photoresponses in normal subjects 9 11 or in pigs with an S cone density higher than that in normal humans. 24 Leading edges (≤10 ms) of all photoresponse families were fit with the alternative model of cone phototransduction activation 19 23 by allowing two free parameters of this model: maximum amplitude (R max) and sensitivity (σ) to change. Other parameters were fixed at values previously published for normal subjects. 19 23 Flash luminances were measured with a calibrated photometer (model IL1700; International Light, Newburyport, MA). 
Paired-flash ERG photoresponses were used to estimate the kinetics of cone deactivation under light-adaptation (white, 3.2 log td). In normal subjects and in the patient with Oguchi disease, a white (4.1 log td · s) test flash was followed by a red (4.1 log td · s) probe flash for a range of interstimulus intervals (100–400 ms). In patients with ESCS L/M cone deactivation was estimated with red test and red probe flashes (both 4.1 log td · s), and S cone deactivation was estimated with white test and white probe stimuli. Based on pilot experiments estimating differences in sensitivity of responses dominated by L/M and S cones in ESCS (described later), the test and probe flashes for S cone deactivation were chosen to be 5.1 log td · s. Amplitude of the photoresponse evoked by each probe was measured at a fixed time (10 ms), and deactivation kinetics was quantified by fitting an S-shaped function of the form R(t)/R 0 = 1/{1 + exp[− (tt 1/2)/τ]}, where R(t) is the amplitude of the response evoked by the probe presented at time t after the test flash, R 0 is the amplitude of the probe-alone response, t 1/2 is the half-recovery time, and τ is a parameter that defines the rate of recovery. 25  
Estimation of Isomerizations in Normal Cones
For the interpretation of the results of the current work, we assume that a given number of isomerizations in human cones produce a stereotypical photoresponse with invariant kinetics independent of cone type. This assumption is based on evidence from single cells stimulated with dim flashes in primate L, M, and S cones. 26 Because of the lack of evidence to the contrary, we parsimoniously assume that the invariance also extends to the activation and deactivation phases of saturating photoresponses in vivo. This assumption allows us to compare the S cone-mediated responses in patients with ESCS to L/M-cone-mediated responses in normal subjects and patients, as long as the responses are evoked by an equal number of isomerizations in respective cone types. 
The relative effectiveness of an external stimulus in causing isomerization in different cone types can be estimated with the spectral energy distribution of the stimulus, spectral sensitivity, quantum efficiency, peak optical density and light-funneling factor for each photoreceptor type, and transmission spectra of preretinal filters. We used recent estimates of relative spectral sensitivity of S, L, and M cones 27 28 and assumed equal quantum efficiency. 29 For peak optical densities, we used 0.15 for peripheral S cones and 0.3 for peripheral L and M cones. 27 28 We assumed that light-funneling is proportional to the axial taper of the inner segment cross-sectional area. We estimated this ratio to be 1.6 times greater for peripheral L and M cones than for S cones, based on published mean diameters. 30 31 We used a recent estimate of average lens-density spectrum 28 as the most significant preretinal filter for stimulating the peripheral retina. Spectral energy distribution of the flash (measured with a calibrated spectrometer model USB2000; Ocean Optics, Dunedin, FL) was multiplied by the lens-transmission spectrum and the sensitivity spectra of respective cone types and integrated with respect to wavelength. Each integral was multiplied with the light-transmission and funneling factors of respective cone types to arrive at relative estimates of isomerization efficiency for the three cone types. For the white flash used in the current work, the resultant estimate shows 0.98 and 1.11 log unit lower isomerizations in S cones than in M and L cones, respectively. 
Considering a white flash of 1 cd · s/m2 integrated luminance has been previously estimated to cause approximately 3000 isomerizations per peripheral L/M cone when viewed by a normal human eye with a dilated pupil, 32 33 34 our relative isomerization efficiency can be used to estimate an absolute value of approximately 300 isomerizations per peripheral S cone per 1 cd · s/m2 full-field white flash viewed by a dilated pupil. In terms of the stimuli used in the current work, the 5.1 log td · s white flash would result in 8 × 105 isomerizations per S cone, which would be matched by 4.1 log td · s flashes in L/M cones. Because of the small (<10%) amplitudes expected, we disregarded the contribution of L/M cones to white flashes, the contribution of S cones to red flashes in ESCS, and the contribution of S cones to red or white flashes in the patient with Oguchi disease and normal subjects (see the Results section). 
Immunocytochemistry
Cryosections of three normal adult human retinas, one retina with ESCS caused by NR2E3 mutations, 13 and one retina with autosomal dominant retinitis pigmentosa caused by the T17M mutation in rhodopsin 35 were processed for immunofluorescence, as described previously. 36 Primary antibodies were mouse anti-GRK1 (1:400, Affinity Bioreagents, Inc., Golden, CO), rabbit anti-GRK7 (1:25 3 ), rabbit anti-L/M cone opsin (1:500, UW-161; John Saari, University of Washington, Seattle, WA); rabbit anti-S cone opsin (1:5000; JH 455; Jeremy Nathans, Johns Hopkins University, Baltimore, MD), and mouse anti-S cone opsin (1:1000, OS-2; Ágoston Szel, Semmelweis University Medical School, Budapest, Hungary). Cell nuclei were stained blue (T0-PRO-3; Molecular Probes, Eugene, OR). Sections were examined by epifluorescence or confocal microscopy. Images were processed as described previously. 13  
Results
Activation Kinetics
Leading edges of an ERG photoresponse family recorded under light adaptation in a representative normal human subject demonstrated the electrical activity that is believed to be dominated by simultaneous activation of L/M cones across the retina (Fig. 1A , top). The model of cone phototransduction activation, fit as an ensemble, quantitatively describes the response family with two parameters, R max (82 μV) and σ (2.28 log td−1 · s−3), which relate to the outer segment membrane area and the amplification during activation, respectively, averaged across the retina. 37 Statistics of these parameters in a group of normal subjects have been previously defined (mean ± 95% CI, R max = 85.3 ± 4.3 μV, σ = 2.26 ± 0.07 log td−1 · s−3, n = 16). In a representative ESCS patient (Fig. 1A , middle) ERG photoresponses evoked by red flashes appear qualitatively similar to normal results, although of smaller amplitude. Using spectrally distinct stimuli, previous work has demonstrated that these responses are dominated by the activity of the L/M cone system in patients with ESCS. 7 8 9 10 11 Not unexpectedly, the model of cone phototransduction describes the leading edges of the responses. Patients with ESCS showed a significantly (P < 0.001) abnormal R max (37.4 ± 8.8 μV, n = 8); but σ parameter (2.20 ± 0.11 log phot td−1 · s−3, n = 8) was not significantly different from normal (P = 0.33). These results would be consistent with an abnormally reduced number of L/M cones (or shortened L/M cone outer segments) in ESCS retinas containing normal molecular components necessary for signal amplification during activation. 
ERG photoresponses evoked with violet or white stimuli under light adaptation in patients with ESCS can be much larger in amplitude than normal responses, albeit showing similar kinetics (Fig. 1A , bottom). Leading edges (<10 ms) of these responses were well described by the model of cone phototransduction activation. The R max of white-flash responses was 368 μV (±94 μV, n = 6) and that of violet responses was 356 ± 58 μV, n = 6). Their similarity was consistent with previous work suggesting that both white and violet flash responses are dominated by the activity of the S cone system in patients with ESCS. 7 8 9 10 11 The ratio of R max in responses evoked by white flash to that evoked by red stimuli was 9.29 ± 1.15 (n = 6), which in turn was consistent with the approximately 10:1 ratio of S:L/M photoreceptor cell density found in an ESCS eye. 13  
The sensitivity parameter of white flash photoresponses in patients with ESCS (σ = 1.08 ± 0.17 log phot td−1 · s−3, n = 6) was significantly (P < 0.001) different from normal photoresponses. The difference in apparent sensitivities of S and L/M cones in patients with ESCS compared favorably with that expected from a theoretical analysis of normal S and L/M cones (see the Methods section). 
Deactivation Kinetics
The deactivation kinetics of normal human L/M-cone photoresponses was demonstrated with waveforms evoked by the paired-flash paradigm, in which a white test flash is followed at fixed times with a red probe flash (Fig. 1B , left panel). The leading edge amplitude of the probe response was approximately half of the probe-alone response by 120 ms and fully recovered between 140 and 200 ms. Some normal subjects showed probe-response amplitudes larger than probe-alone responses over the interval 150 to 300 ms. Other subjects did not show this overshoot. The early phase of the deactivation function from a group of normal subjects is fit with τ = 12 ms and t 1/2= 110 ms (Fig. 1D)
Deactivation in ESCS cones was studied with the test flashes chosen to evoke activation phases identical with the test flashes used to determine deactivation kinetics in normal human L/M cones. For ESCS-L/M cones, red test flashes were used, and for ESCS-S cones, white test flashes were used (Fig. 1C) . ESCS-L/M cones showed normal deactivation phase kinetics, although in this sample, they appeared not to have the overshoot seen in some normal subjects (Fig. 1D) . The deactivation kinetics of ESCS-S cones was much slower than normal and ESCS L/M cones (Figs. 1B 1D ; τ = 85 ms, t 1/2= 300 ms), although the activation phase to the test flash was matched in kinetics to normal and ESCS L/M cones (Fig. 1C) and thus was assumed to have resulted in the same number of isomerizations. 
Abnormally delayed cone deactivation kinetics has been reported to date in only one other human retinopathy, Oguchi disease due to a null mutation in the GRK1 gene. 5 We reanalyzed our previously published data with current methods and present the activation and deactivation kinetics for comparison with the current results (Figs. 1C 1D) . With the test flash used, L/M-cone-dominated responses in the patient with Oguchi disease showed activation kinetics similar to those in normal subjects (Fig. 1C) , but the deactivation was delayed (Fig. 1D ; τ = 13 ms, t 1/2 = 135 ms). Specifically, recovery of amplitude at probe times of 100 and 120 ms after the test flash falls outside the 95% CI of a group of normal results. 
GRK1/7 Expression
We performed immunocytochemistry to document GRK1 and GRK7 in photoreceptors of a patient with ESCS. Normal adult human retina sections showed labeling of all rod outer segments with anti-GRK1 (Fig. 2A 2B 2C) , and of all cone outer segments with both anti-GRK1 and anti-GRK7 (Figs. 2A 2B 2C 2D 2E 2F) . The cones in the ESCS retina were present in layers of rather uniform thickness, and there was no scalloping of the outer nuclear layer. 38 The cone outer segments ranged from short to absent. ESCS outer segments expressing L/M opsin showed strong GRK1 labeling but no GRK7 labeling. Outer segments expressing S opsin showed no immunoreactivity for GRK1 or GRK7 (Figs. 2G 2H 2I 2J 2K 2L) . To rule out downregulation of GRK1 and GRK7 expression secondary to a degenerative process, we also investigated a human retina that was previously shown to lack expression of some cytoplasmic proteins. 39 The retina with retinitis pigmentosa due to a mutation in the rhodopsin gene showed labeling of all rod outer segments with anti-GRK1 (Fig. 2M) and of all cone outer segments with both anti-GRK1 and anti-GRK7 (Figs. 2M 2N)
Discussion
Colocalization of two members of the GRK family in human cone photoreceptors led to the hypothesis of shared enzymatic activity by GRK1 and GRK7 in phosphorylating photoisomerized cone opsins, thus initiating deactivation. 3 4 We were able to test this hypothesis in vivo in cone-dominant retinas of patients with ESCS, in a patient with Oguchi disease, and in normal subjects and correlate the pathophysiology to GRK1 and GRK7 expression in retinas from donor eyes. In electrophysiological experiments, our stimuli evoked a-waves with saturated amplitudes (ERG photoresponses) dominated by L/M cones in the patient with Oguchi disease and normal subjects, and by L/M or S cones (depending on stimulus color) in patients with ESCS. It is well accepted that kinetics of activation, duration of saturation, and kinetics of deactivation resulting from a bright flash in a given photoreceptor type depend strongly on the number of visual pigment molecules isomerized. Specifically, an increase in the number of isomerizations results in faster activation, longer saturation, and slower deactivation kinetics. 16 17 18 26 Thus, to be able to compare deactivation kinetics in the four different photoreceptor types, we had to consider possible differences in their sensitivity to external light stimuli. We used a model of cone phototransduction activation and determined that L/M cone dominated ERG photoresponse sensitivities in patients with ESCS or Oguchi disease were normal. We therefore assumed that a given flash would produce the same number of isomerizations in the three groups of L/M cones. Photoresponses of ESCS S cones were approximately 1 log unit less sensitive than normal L/M cones. This was consistent with the difference expected between normal S and L/M cones, suggesting that the anatomy and phototransduction activation reactions of ESCS S cones could not be dramatically different from normal S cones. A direct test of this hypothesis would require better definition of the anatomy of ESCS S cones and the physiology of normal S cones. For the current work, we empirically chose test flash intensities that would be expected to result in an equal number of isomerizations in ESCS S and L/M cones during deactivation experiments. 
Deactivation of L/M cones in patients with ESCS showed normal kinetics. Unexpectedly, L/M cones from the ESCS retina showed no detectable GRK7 immunoreactivity, although they were positive for GRK1. Deactivation of L/M cones in Oguchi disease due to a null mutation in the GRK1 gene, however, was abnormally slow. Although direct evidence is lacking, it is reasonable to assume that the retina in Oguchi disease does not have functional GRK1 but does possess GRK7. Results from patients with ESCS or Oguchi disease, taken together, argue against a predominant physiological role for GRK7-mediated phosphorylation in human L/M cones. In contrast, GRK1-mediated phosphorylation appears necessary for the attainment of the normal rate of deactivation. The possibility of both GRKs contributing to human cone deactivation is not inconsistent with our results. Under this scenario, the observed absence of physiological abnormality in L/M cones in ESCS could be due to upregulation of GRK1 to compensate for the lack of GRK7. Upregulation of GRK7 in Oguchi disease would then not be as physiologically effective and would result in the abnormal recovery detectable with our methods. 
In terms of GRK1 and GRK7 expression, ESCS L/M cones are similar to normal mouse and rat cones, 3 4 in that they all have no GRK7 and have only GRK1. Activation and deactivation kinetics of normal mouse cones have been reported, using a paired-flash ERG method similar to that used in the current work. 6 40 For test flashes that produce activation kinetics approximately 1.5 ms slower than the test flashes used in the current work, deactivation kinetics of mice are much slower than ESCS (and normal) L/M cones. 6 40 It is reasonable to assume that brighter test flashes in mice that evoke responses with a time course similar to the current experiments would result in even slower deactivation kinetics and even larger differences between ESCS L/M cones and normal mouse cones. This argument leads to the speculation that major differences between human and mouse (in addition to expression of GRK) must exist in molecular components contributing to cone recovery. Further studies isolating the activation and deactivation components of mouse UV and M cones would provide a better interspecies comparison. 40 A similar line of reasoning can be made for GRK1-associated Oguchi L/M cones and normal cones in the dog and pig—that is, that they all have no functional GRK1 and presumably have only GRK7 contributing to phosphorylation of activated cone pigments. 3 A comparison of deactivation kinetics in dogs and pigs with that found in the patient with Oguchi disease would be of strong interest. 
Deactivation kinetics of ESCS S cones were much slower than L/M cones in normal, ESCS or Oguchi retinas. ESCS S cones unexpectedly showed no immunoreactivity to both GRK1 and GRK7. Assumption of the stereotypy of phototransduction in all human cones allows the parsimonious conclusion that the absence of both GRK1 and GRK7 causes a greater degree of abnormality in cone deactivation than that caused by the deficiency of either GRK alone. This in turn is consistent with the speculation that both GRKs contribute to deactivation in human cones. This hypothesis appears more tenable than the alternative that neither GRK1 nor GRK7 contributes significantly to human cone deactivation and that the slow deactivation observed in Oguchi L/M cones and ESCS S cones is due to abnormalities in other components of the cone deactivation process. It is important to note that, ideally, the pathophysiology in ESCS S cones should be interpreted by direct comparison to normal human S cones. However, estimates of activation and deactivation in normal S cones have not been recordable to date in humans 9 11 or in animals known to have a higher density of S cones. 24  
One of the major physiological differences between cones and rods is the dramatically faster recovery observed in cones after flashes of light. 41 The existence of a cone-specific kinase, such as GRK7, could contribute significantly to this fast recovery. 3 4 41 42 Our in vivo results suggest that human cones without either GRK1 or GRK7 or both still recover much faster than normal human rods (t 1/2 < 0.3 seconds for cones compared with t 1/2 > 10 seconds for rods 16 ) when matched by the estimated number of isomerizations resulting from the test flash. Assuming that results from Oguchi and ESCS cones can be extrapolated to normal human cones, we can speculate that there are molecular components, other than GRK1/7 phosphorylation, contributing significantly to the human cone recovery. Among the candidates are those involved in the deactivation of the activated cone pigment (for example, cone arrestin 43 ), the activated transducin (for example, RGS9-1 44 ), and cone pigment regeneration. Future studies in naturally occurring molecularly defined human diseases could be useful in furthering our understanding in this area. For example, measurement of cone deactivation kinetics in patients with fundus albipunctatus due to null mutations in RDH5, who show a dramatic slowing in cone pigment regeneration, 22 would allow estimation of the contribution of cone pigment regeneration to cone deactivation. 
 
Figure 1.
 
Activation and deactivation kinetics of phototransduction in human patients with ESCS. (A) Leading edges of ERG photoresponse family (4.1–2.2 log td · s red flashes) dominated by L/M cone activity in a normal subject (top) and in ESCS (middle) showed similarity of activation kinetics. Further photoresponses in the same subject (bottom) illustrated the higher stimulus energies (traces, 5.1–2.5 log td · s white flashes) or shorter wavelengths (symbols and traces, 2.5–1.9 log td · s violet flashes) needed to equate activation kinetics of S- and L/M-cone-dominated responses. All waveforms recorded on a 3.2-log td white background. Model of cone phototransduction activation (gray lines) was fit to each series as an ensemble (R max= 82, 39, and 302 μV, σ = 2.3, 2.1, and 1.1 log td−1 · s−3, respectively for top, middle, and bottom). (B) Representative waveforms recorded with a paired-flash paradigm in which the test flash is at time 0 and the probe flash is at the time indicated with the arrow. White test flashes were 4.1 log td · s in the normal subject and 5.1 log td · s in the patient with ESCS. The probe flashes were red (4.1 log td · s) in the normal subject and white (5.1 log td · s) in the patient. All waveforms recorded on a 3.2-log td white background. (C) Activation kinetics of leading edges evoked by test flashes averaged over all trials for each subject, normalized to amplitude at 10 ms, and averaged across subjects (mean ± 95% CI shown at 1-ms intervals for clarity of presentation). Previously published data from a patient with Oguchi disease were reanalyzed and shown for comparison. Symbols are described in (D). (D) Normalized amplitude of the response evoked by probe flashes (4.1 log td · s red for L/M cones, 5.1 log td · s white for S cones) as a function of the time interval between test and probe flashes. Photoresponse amplitudes evoked by probe flashes with preceding test flashes were normalized by response amplitudes evoked by probe only. Results shown with amplitudes measured at 10 ms. Results are essentially identical when amplitudes are measured at 8 or 12 ms. Error bars represent 95% CI. Smooth gray lines: S-shaped curves fit by eye.
Figure 1.
 
Activation and deactivation kinetics of phototransduction in human patients with ESCS. (A) Leading edges of ERG photoresponse family (4.1–2.2 log td · s red flashes) dominated by L/M cone activity in a normal subject (top) and in ESCS (middle) showed similarity of activation kinetics. Further photoresponses in the same subject (bottom) illustrated the higher stimulus energies (traces, 5.1–2.5 log td · s white flashes) or shorter wavelengths (symbols and traces, 2.5–1.9 log td · s violet flashes) needed to equate activation kinetics of S- and L/M-cone-dominated responses. All waveforms recorded on a 3.2-log td white background. Model of cone phototransduction activation (gray lines) was fit to each series as an ensemble (R max= 82, 39, and 302 μV, σ = 2.3, 2.1, and 1.1 log td−1 · s−3, respectively for top, middle, and bottom). (B) Representative waveforms recorded with a paired-flash paradigm in which the test flash is at time 0 and the probe flash is at the time indicated with the arrow. White test flashes were 4.1 log td · s in the normal subject and 5.1 log td · s in the patient with ESCS. The probe flashes were red (4.1 log td · s) in the normal subject and white (5.1 log td · s) in the patient. All waveforms recorded on a 3.2-log td white background. (C) Activation kinetics of leading edges evoked by test flashes averaged over all trials for each subject, normalized to amplitude at 10 ms, and averaged across subjects (mean ± 95% CI shown at 1-ms intervals for clarity of presentation). Previously published data from a patient with Oguchi disease were reanalyzed and shown for comparison. Symbols are described in (D). (D) Normalized amplitude of the response evoked by probe flashes (4.1 log td · s red for L/M cones, 5.1 log td · s white for S cones) as a function of the time interval between test and probe flashes. Photoresponse amplitudes evoked by probe flashes with preceding test flashes were normalized by response amplitudes evoked by probe only. Results shown with amplitudes measured at 10 ms. Results are essentially identical when amplitudes are measured at 8 or 12 ms. Error bars represent 95% CI. Smooth gray lines: S-shaped curves fit by eye.
Figure 2.
 
Immunocytochemistry of human retinas labeled with anti-GRK1 and -GRK7. Cell nuclei have been stained (blue) with T0-PRO-3. The layer of cells across the bottom of (F) and (L) is the retinal pigment epithelium (R) that is filled with autofluorescent lipofuscin granules. (A) Normal human retina labeled with anti-GRK1 (red). Note specific labeling of all rod (⋆) and cone (arrowheads) outer segments. (B) Same field as (A) labeled with anti-L/M cone opsin (green). Note labeling of most cone outer segments (arrowheads). (C) Composite of (A) and (B) showing that most cone outer segments (gold) were positive for both GRK1 and L/M-cone opsin. One S cone outer segment (red, arrowhead) was positive for GRK1 but negative for L/M cone opsin. (⋆) Rod outer segments. (D) Normal human retina labeled with anti-GRK7 (red). Note labeling of all cone outer segments. (E) Same field as in (D), labeled with anti-S cone opsin. (F) Composite of (D) and (E) showing two S cone outer segments (gold, arrowheads) positive for GRK7. The L/M-cone outer segments were positive for GRK7 (red) but negative for S cone opsin. One S cone inner segment (middle, green) was positive for S cone opsin, but its outer segment was not in the plane of section. (G) ESCS retina labeled with anti-GRK1 (red). Several cone outer segments were GRK1 positive. (H) Same field as in (G). Several cone outer segments were labeled with anti-L/M cone opsin (green). (I) Composite of (G) and (H). Note stronger GRK1 labeling of L/M cone outer segments (arrowheads) than of most of the cone outer segments that are S cone opsin positive (see K). (J) ESCS retina labeled with anti-GRK7 (red). Note absence of outer segment labeling. (K) Same field as in (J) labeled with anti-S cone opsin (green). Most outer segments were S cone opsin positive. (L) Composite of (J) and (K) showing ESCS retina labeled with anti-GRK7 (red) and anti-S cone opsin (green). Note heavy labeling of most of the cone outer segments with anti-S cone opsin (green). No outer segments were GRK7 positive. (M) Human retina with retinitis pigmentosa caused by a mutation in the rhodopsin gene. All rod (⋆) and cone (arrowheads) outer segments were positive for GRK1 (red). (N) The human retina in (M) labeled with anti-GRK7 (red). Note that all cone outer segments (arrowheads) were GRK7 positive. Bar, 100 μm.
Figure 2.
 
Immunocytochemistry of human retinas labeled with anti-GRK1 and -GRK7. Cell nuclei have been stained (blue) with T0-PRO-3. The layer of cells across the bottom of (F) and (L) is the retinal pigment epithelium (R) that is filled with autofluorescent lipofuscin granules. (A) Normal human retina labeled with anti-GRK1 (red). Note specific labeling of all rod (⋆) and cone (arrowheads) outer segments. (B) Same field as (A) labeled with anti-L/M cone opsin (green). Note labeling of most cone outer segments (arrowheads). (C) Composite of (A) and (B) showing that most cone outer segments (gold) were positive for both GRK1 and L/M-cone opsin. One S cone outer segment (red, arrowhead) was positive for GRK1 but negative for L/M cone opsin. (⋆) Rod outer segments. (D) Normal human retina labeled with anti-GRK7 (red). Note labeling of all cone outer segments. (E) Same field as in (D), labeled with anti-S cone opsin. (F) Composite of (D) and (E) showing two S cone outer segments (gold, arrowheads) positive for GRK7. The L/M-cone outer segments were positive for GRK7 (red) but negative for S cone opsin. One S cone inner segment (middle, green) was positive for S cone opsin, but its outer segment was not in the plane of section. (G) ESCS retina labeled with anti-GRK1 (red). Several cone outer segments were GRK1 positive. (H) Same field as in (G). Several cone outer segments were labeled with anti-L/M cone opsin (green). (I) Composite of (G) and (H). Note stronger GRK1 labeling of L/M cone outer segments (arrowheads) than of most of the cone outer segments that are S cone opsin positive (see K). (J) ESCS retina labeled with anti-GRK7 (red). Note absence of outer segment labeling. (K) Same field as in (J) labeled with anti-S cone opsin (green). Most outer segments were S cone opsin positive. (L) Composite of (J) and (K) showing ESCS retina labeled with anti-GRK7 (red) and anti-S cone opsin (green). Note heavy labeling of most of the cone outer segments with anti-S cone opsin (green). No outer segments were GRK7 positive. (M) Human retina with retinitis pigmentosa caused by a mutation in the rhodopsin gene. All rod (⋆) and cone (arrowheads) outer segments were positive for GRK1 (red). (N) The human retina in (M) labeled with anti-GRK7 (red). Note that all cone outer segments (arrowheads) were GRK7 positive. Bar, 100 μm.
The authors thank Val C. Sheffield and Edwin M. Stone for initial molecular characterization of the patients; Tomas S. Aleman, Eyal Banin, David B. Hanna, Leigh M. Gardner, Elaine B. DeCastro, Jessica M. Emmons, and Sharon B. Schwartz for help with the studies; and Linda Rose for advice on immunocytochemistry. 
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Figure 1.
 
Activation and deactivation kinetics of phototransduction in human patients with ESCS. (A) Leading edges of ERG photoresponse family (4.1–2.2 log td · s red flashes) dominated by L/M cone activity in a normal subject (top) and in ESCS (middle) showed similarity of activation kinetics. Further photoresponses in the same subject (bottom) illustrated the higher stimulus energies (traces, 5.1–2.5 log td · s white flashes) or shorter wavelengths (symbols and traces, 2.5–1.9 log td · s violet flashes) needed to equate activation kinetics of S- and L/M-cone-dominated responses. All waveforms recorded on a 3.2-log td white background. Model of cone phototransduction activation (gray lines) was fit to each series as an ensemble (R max= 82, 39, and 302 μV, σ = 2.3, 2.1, and 1.1 log td−1 · s−3, respectively for top, middle, and bottom). (B) Representative waveforms recorded with a paired-flash paradigm in which the test flash is at time 0 and the probe flash is at the time indicated with the arrow. White test flashes were 4.1 log td · s in the normal subject and 5.1 log td · s in the patient with ESCS. The probe flashes were red (4.1 log td · s) in the normal subject and white (5.1 log td · s) in the patient. All waveforms recorded on a 3.2-log td white background. (C) Activation kinetics of leading edges evoked by test flashes averaged over all trials for each subject, normalized to amplitude at 10 ms, and averaged across subjects (mean ± 95% CI shown at 1-ms intervals for clarity of presentation). Previously published data from a patient with Oguchi disease were reanalyzed and shown for comparison. Symbols are described in (D). (D) Normalized amplitude of the response evoked by probe flashes (4.1 log td · s red for L/M cones, 5.1 log td · s white for S cones) as a function of the time interval between test and probe flashes. Photoresponse amplitudes evoked by probe flashes with preceding test flashes were normalized by response amplitudes evoked by probe only. Results shown with amplitudes measured at 10 ms. Results are essentially identical when amplitudes are measured at 8 or 12 ms. Error bars represent 95% CI. Smooth gray lines: S-shaped curves fit by eye.
Figure 1.
 
Activation and deactivation kinetics of phototransduction in human patients with ESCS. (A) Leading edges of ERG photoresponse family (4.1–2.2 log td · s red flashes) dominated by L/M cone activity in a normal subject (top) and in ESCS (middle) showed similarity of activation kinetics. Further photoresponses in the same subject (bottom) illustrated the higher stimulus energies (traces, 5.1–2.5 log td · s white flashes) or shorter wavelengths (symbols and traces, 2.5–1.9 log td · s violet flashes) needed to equate activation kinetics of S- and L/M-cone-dominated responses. All waveforms recorded on a 3.2-log td white background. Model of cone phototransduction activation (gray lines) was fit to each series as an ensemble (R max= 82, 39, and 302 μV, σ = 2.3, 2.1, and 1.1 log td−1 · s−3, respectively for top, middle, and bottom). (B) Representative waveforms recorded with a paired-flash paradigm in which the test flash is at time 0 and the probe flash is at the time indicated with the arrow. White test flashes were 4.1 log td · s in the normal subject and 5.1 log td · s in the patient with ESCS. The probe flashes were red (4.1 log td · s) in the normal subject and white (5.1 log td · s) in the patient. All waveforms recorded on a 3.2-log td white background. (C) Activation kinetics of leading edges evoked by test flashes averaged over all trials for each subject, normalized to amplitude at 10 ms, and averaged across subjects (mean ± 95% CI shown at 1-ms intervals for clarity of presentation). Previously published data from a patient with Oguchi disease were reanalyzed and shown for comparison. Symbols are described in (D). (D) Normalized amplitude of the response evoked by probe flashes (4.1 log td · s red for L/M cones, 5.1 log td · s white for S cones) as a function of the time interval between test and probe flashes. Photoresponse amplitudes evoked by probe flashes with preceding test flashes were normalized by response amplitudes evoked by probe only. Results shown with amplitudes measured at 10 ms. Results are essentially identical when amplitudes are measured at 8 or 12 ms. Error bars represent 95% CI. Smooth gray lines: S-shaped curves fit by eye.
Figure 2.
 
Immunocytochemistry of human retinas labeled with anti-GRK1 and -GRK7. Cell nuclei have been stained (blue) with T0-PRO-3. The layer of cells across the bottom of (F) and (L) is the retinal pigment epithelium (R) that is filled with autofluorescent lipofuscin granules. (A) Normal human retina labeled with anti-GRK1 (red). Note specific labeling of all rod (⋆) and cone (arrowheads) outer segments. (B) Same field as (A) labeled with anti-L/M cone opsin (green). Note labeling of most cone outer segments (arrowheads). (C) Composite of (A) and (B) showing that most cone outer segments (gold) were positive for both GRK1 and L/M-cone opsin. One S cone outer segment (red, arrowhead) was positive for GRK1 but negative for L/M cone opsin. (⋆) Rod outer segments. (D) Normal human retina labeled with anti-GRK7 (red). Note labeling of all cone outer segments. (E) Same field as in (D), labeled with anti-S cone opsin. (F) Composite of (D) and (E) showing two S cone outer segments (gold, arrowheads) positive for GRK7. The L/M-cone outer segments were positive for GRK7 (red) but negative for S cone opsin. One S cone inner segment (middle, green) was positive for S cone opsin, but its outer segment was not in the plane of section. (G) ESCS retina labeled with anti-GRK1 (red). Several cone outer segments were GRK1 positive. (H) Same field as in (G). Several cone outer segments were labeled with anti-L/M cone opsin (green). (I) Composite of (G) and (H). Note stronger GRK1 labeling of L/M cone outer segments (arrowheads) than of most of the cone outer segments that are S cone opsin positive (see K). (J) ESCS retina labeled with anti-GRK7 (red). Note absence of outer segment labeling. (K) Same field as in (J) labeled with anti-S cone opsin (green). Most outer segments were S cone opsin positive. (L) Composite of (J) and (K) showing ESCS retina labeled with anti-GRK7 (red) and anti-S cone opsin (green). Note heavy labeling of most of the cone outer segments with anti-S cone opsin (green). No outer segments were GRK7 positive. (M) Human retina with retinitis pigmentosa caused by a mutation in the rhodopsin gene. All rod (⋆) and cone (arrowheads) outer segments were positive for GRK1 (red). (N) The human retina in (M) labeled with anti-GRK7 (red). Note that all cone outer segments (arrowheads) were GRK7 positive. Bar, 100 μm.
Figure 2.
 
Immunocytochemistry of human retinas labeled with anti-GRK1 and -GRK7. Cell nuclei have been stained (blue) with T0-PRO-3. The layer of cells across the bottom of (F) and (L) is the retinal pigment epithelium (R) that is filled with autofluorescent lipofuscin granules. (A) Normal human retina labeled with anti-GRK1 (red). Note specific labeling of all rod (⋆) and cone (arrowheads) outer segments. (B) Same field as (A) labeled with anti-L/M cone opsin (green). Note labeling of most cone outer segments (arrowheads). (C) Composite of (A) and (B) showing that most cone outer segments (gold) were positive for both GRK1 and L/M-cone opsin. One S cone outer segment (red, arrowhead) was positive for GRK1 but negative for L/M cone opsin. (⋆) Rod outer segments. (D) Normal human retina labeled with anti-GRK7 (red). Note labeling of all cone outer segments. (E) Same field as in (D), labeled with anti-S cone opsin. (F) Composite of (D) and (E) showing two S cone outer segments (gold, arrowheads) positive for GRK7. The L/M-cone outer segments were positive for GRK7 (red) but negative for S cone opsin. One S cone inner segment (middle, green) was positive for S cone opsin, but its outer segment was not in the plane of section. (G) ESCS retina labeled with anti-GRK1 (red). Several cone outer segments were GRK1 positive. (H) Same field as in (G). Several cone outer segments were labeled with anti-L/M cone opsin (green). (I) Composite of (G) and (H). Note stronger GRK1 labeling of L/M cone outer segments (arrowheads) than of most of the cone outer segments that are S cone opsin positive (see K). (J) ESCS retina labeled with anti-GRK7 (red). Note absence of outer segment labeling. (K) Same field as in (J) labeled with anti-S cone opsin (green). Most outer segments were S cone opsin positive. (L) Composite of (J) and (K) showing ESCS retina labeled with anti-GRK7 (red) and anti-S cone opsin (green). Note heavy labeling of most of the cone outer segments with anti-S cone opsin (green). No outer segments were GRK7 positive. (M) Human retina with retinitis pigmentosa caused by a mutation in the rhodopsin gene. All rod (⋆) and cone (arrowheads) outer segments were positive for GRK1 (red). (N) The human retina in (M) labeled with anti-GRK7 (red). Note that all cone outer segments (arrowheads) were GRK7 positive. Bar, 100 μm.
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