1Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology and Program in Neuroscience, and 2Center for Integrative Biotechnology, Washington State University, Pullman, Washington
Submitted 8 October 2004 ; accepted in final form 21 February 2005
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ABSTRACT |
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phosphodiesterase
CNG channels are tetrameric proteins (22, 39, 65) composed of some combination of CNGA1, CNGA2, CNGA3, CNGA4, CNGB1, and CNGB3 subunits (3, 21, 34, 57). The CNGA1, CNGA2, or CNGA3 subunit can form functional homomeric channels when expressed alone (2, 11, 28, 67). While CNGB1, CNGB3, and CNGA4 subunits do not form functional channels by themselves, they can modulate the channel properties when coassembled with the other subunit types (36, 21, 37, 57). Native rod CNG channels are heteromeric proteins formed by assembly of three CNGA1 subunits and one CNGB1 subunit (66, 74, 76). Recent studies suggest that cone CNG channels adopt a different structure, being composed of CNGA3 and CNGB3 subunits in a 2 x 2 configuration (49). Each channel subunit contains six transmembrane regions, cytoplasmic NH2 and COOH termini, a conserved pore domain, and a cyclic nucleotide-binding domain (Fig. 1A) (for review, see Ref. 29). Loss-of-function alterations in the cone photoreceptor CNG channels due to missense mutations, deletions, or splice site disruption in the genes encoding these subunits result in abnormal cone function, leading to daylight and color vision deficiencies (for review, see Ref. 52). The general forms of color blindness, cone dystrophies, can be divided into two broad groups: stationary and progressive cone dystrophy. The stationary form of cone dystrophy is also called "achromatopsia" or "monochromatism" (58). Progressive cone dystrophies are a group of clinically heterogeneous disorders. Patients with these diseases exhibit progressive loss of visual acuity and color vision, together with photophobia and nystagmus, in late childhood or early adulthood (59). Psychophysical and electrophysiological examinations show abnormal cone function, while rod function is intact (58). The mechanisms underlying the pathophysiology of cone dystrophies are still poorly understood.
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Recently, 51 mutations have been identified in the gene encoding the human CNGA3 subunit of cone photoreceptor CNG channels and linked to achromatopsia and progressive cone dystrophy (32, 68). Three of these mutations are present in patients with severe progressive cone dystrophy: R277C (in the S4 domain), N471S (in the C-linker region), and R563H (in the cyclic nucleotide-binding domain, CNBD) (Fig. 1A). An important step toward understanding the development of this disease is to determine how individual mutations alter the functional properties of CNG channels and how abnormal channel function may lead to cone photoreceptor degeneration.
Herein we report the functional consequences of three progressive cone dystrophy-associated mutations in CNGA3 subunits. Our results suggest that these mutations disrupt plasma membrane localization, impair channel protein posttranslational modification, and/or alter the gating properties of cone CNG channels, thus leading to abnormal cone photoreceptor function and ultimately to degeneration.
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MATERIALS AND METHODS |
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Oocytes were isolated as previously described (64, 72) and microinjected with a fixed amount of mRNA for all constructs (5 ng). For efficient generation of heteromeric channels, the ratio of wild-type or mutant CNGA3 mRNA to CNGB3 mRNA was 1:5 (49). Oocytes were incubated in ND96 (in mM: 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.6, supplemented with 10 µg/ml gentamicin). For some experiments, oocytes were incubated in ND96 that also contained 5 µM tunicamycin (EMD Bioscience, La Jolla, CA).
Electrophysiology.
Two to seven days after microinjection of mRNA, patch-clamp experiments were performed in the inside-out configuration with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Recordings were made at 2023°C. Data were acquired using Pulse software (HEKA Elektronik, Lambrecht, Germany). Current traces were elicited by voltage steps from a holding potential of 0 to +80 mV, then to 80 mV, and back to 0 mV. Initial pipette resistances were 0.40.8 M. Intracellular and extracellular solutions contained (in mM) 130 NaCl, 0.2 EDTA, and 3 HEPES (pH 7.2). Intracellular solutions were changed using an RSC-160 rapid solution changer (Molecular Kinetics, Pullman, WA). Currents in the absence of cyclic nucleotide were subtracted. For channel activation by cGMP or cAMP, dose-response data were fitted to the Hill equation I/Imax = {[cNMP]nH/(KnH + [cNMP]nH)}, where I is the current amplitude, Imax is the maximum current elicited by saturating concentration of ligand, [cNMP] is the ligand concentration, K1/2 is the apparent ligand affinity, and nH is the Hill slope. For current block by tetracaine, data were fitted to a modified Hill equation in the form Itetracaine/I = {K1/2nH/(K1/2nH + [tetracaine]nH)}. To confirm the formation of heteromeric CNGB3 plus CNGA3 channels, we measured sensitivity to block by applying 25 µM L-cis-diltiazem (RBI, Natick, MA) to the intracellular face of the patch in the presence of 1 mM cGMP. Data were analyzed using Igor (Wavemetrics, Lake Oswego, OR), SigmaPlot, and SigmaStat software (SPSS, Chicago, IL). All values are reported as means ± SE of n experiments (patches) unless otherwise indicated. Statistical significance was determined using Student's t-test or the Mann-Whitney rank-sum test, and P < 0.05 was considered significant.
To describe the gating of homomeric CNGA3 channels, we used a simplified linear allosteric model in which independent ligand-binding steps are followed by a single allosteric transition from the liganded but closed state to the open state (18, 23, 36, 64):
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In this kinetic scheme, K is the equilibrium constant for the initial binding of cyclic nucleotide and L is the equilibrium constant of the allosteric conformational transition. We used the local anesthetic tetracaine (Sigma, St. Louis, MO), a known state-dependent blocker of CNG channels that binds to closed channels with nearly 1,000-fold greater affinity compared with open channels (18), to investigate the altered gating properties of mutant homomeric channels. By applying a saturating concentration of cGMP (fully ligand-bound state), we isolated the allosteric conformational change associated with channel opening. Tetracaine sensitivity is a reporter for this equilibrium; thus channels that spend more time in the open state are less sensitive to tetracaine block. We calculated the equilibrium constant for the allosteric transition (L) for cGMP-bound channels using the equation of Fodor et al. (18):
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In this equation, Kdc (220 nM) and Kdo (170 µM) are dissociation constants for tetracaine binding to closed or open channels, respectively (18). The L value for cAMP-bound channels was determined using the relative agonist efficacy of cAMP compared with cGMP. K was determined from fits of the simple allosteric model described above to the dose-response data using L values calculated from tetracaine apparent affinity and relative agonist efficacy. The free energy differences for L and for K were calculated as follows: GX = RT x ln (X). Also, we calculated
G =
Gmutant
Gwt. Tetracaine apparent affinity was not used to analyze the gating properties of heteromeric channels because CNGB3 subunits lack the negatively charged residue in the pore thought to be critical for state-dependent binding of tetracaine (17), and tetracaine binding to channels containing CNGB3 subunits has not been characterized. Thus the model of Fodor et al. (18) is not expected to apply to this channel type.
Confocal microscopy.
As described previously (48), confocal images of oocytes expressing GFP-tagged CNGA3 were obtained using a Bio-Rad MRC-1024 confocal laser-scanning system and a krypton-argon laser with a Nikon Eclipse TE 300 inverted microscope with a x10 lens objective. Four days after injection of mRNA, oocytes expressing homomeric or heteromeric channels were placed in borosilicate coverglass chambers such that the equator was approximately perpendicular to the plane of imaging. GFP fluorescence was determined using an excitation wavelength of 488 nm and a 522 DF 32 emission filter. Laser intensity, pinhole aperture, and photomultiplier gain were the same for all experiments. Images were analyzed using NIH ImageJ software. Surface fluorescence for each oocyte was determined from an area within the animal hemisphere representing 5% of the circumference in a single plane and expressed as intensity of signal per unit area. Background fluorescence was determined for an equivalent area using a blank region of the same image and subtracted. Values are reported as means ± SE of n oocytes tested.
Protein biochemistry. To assess the overall abundance and processing of CNGA3 subunits expressed in Xenopus oocytes, we performed Western blot analysis of proteins from oocytes expressing GFP-tagged CNGA3. Oocyte lysates were prepared as previously described (47, 53, 54). Briefly, oocytes were placed in lysis buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100 (Surfact-Amps X-100; Pierce Biotechnology, Rockford, IL), and a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Oocytes were subjected to trituration followed by cup sonication, repeated a total of three times. The soluble cell lysate was then separated from yolk and other insoluble material by performing centrifugation at 20,000 g and 4°C for 10 min, which was repeated three times. Lysate representing approximately one oocyte per lane was loaded and separated by SDS-PAGE using NuPage 412% Tris acetate gels (Invitrogen, Carlsbad, CA). Proteins were then transferred to nitrocellulose membrane using the NuPage transfer buffer system (Invitrogen). Immunoblots were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 (TTBS; Bio-Rad) for 2 h and then probed with anti-GFP Aequorea victoria peptide polyclonal antibody (Clontech, Palo Alto, CA) at a concentration of 1:2,500 in TTBS buffer with 1% nonfat dry milk. GFP-tagged channel subunits were visualized using SuperSignal West Dura substrate (Pierce Biotechnology) and autoradiographic film (Kodak X-Omat Blue XB-1; Eastman Kodak). The molecular weights of the GFP-tagged subunits were estimated using protein standards (SeeBlue Plus2; Invitrogen). The relative amounts of CNGA3 protein (band density) for all GFP-A3 immunoreactive bands (glycosylated and unglycosylated) were estimated using NIH ImageJ software. To verify that approximately equal amounts of total protein were loaded in each lane, the same blots were probed with MAB1501 pan-actin antibody (Chemicon International, Temecula, CA). The variation in actin signal between lanes, determined using densitometry, was <10% (n = 4 immunoblots).
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RESULTS |
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Surface membrane fluorescence levels in oocytes were determined by performing confocal microscopy after 4 days of incubation. Figure 1, B and C, shows representative confocal images and relative surface membrane fluorescence (F) levels normalized to the levels of wild-type homomeric CNGA3 channels. For both homomeric and heteromeric channels, cell surface expression levels were significantly reduced (P < 0.01) for R277C (Fhomo = 0.06 ± 0.01, n = 39; Fhetero = 0.04 ± 0.01, n = 26) and R563H (Fhomo = 0.15 ± 0.04, n = 42; Fhetero = 0.12 ± 0.02, n = 26) (Fig. 1, B and C) compared with the corresponding wild-type channels. Surprisingly, N471S did not interfere with plasma membrane localization of homomeric or heteromeric channels. Maximum patch-current (Imax) levels, determined using patch-clamp recording at a saturating concentration of cGMP (1 mM), were consistent with the observed plasma membrane fluorescence levels (Fig. 1C). Compared with wild-type channels, mean Imax for both homomeric and heteromeric channels was reduced for R563H-containing channels (n = 1213; P < 0.01) but did not change significantly for N471S-containing channels. No current was elicited by a saturating concentration of cGMP for R277C-containing channels. These results suggest that R277C and R563H mutations impaired the plasma membrane expression of cone CNG channel subunits.
Cone dystrophy mutations altered CNG channel protein processing. There are two possible explanations for the reduced plasma membrane localization observed for R277C and R563H subunits. One is that protein folding and/or stability are impaired by the mutations. Another possibility is that the channel proteins are not properly assembled and/or targeted to the plasma membrane. To address these possibilities, we conducted Western blot analysis of oocyte lysates. Immunoblots revealed a reduction in overall protein amount and a change in the processing and maturation of R277C subunits (Fig. 2, A and B). The reduced protein levels for R277C compared with wild-type subunits (Fig. 2, A and B) indicate that the mutation disrupted channel biogenesis and/or stability, which likely accounts for much of the reduction in cell surface expression of these subunits (Fig. 1). In contrast, R563H subunits exhibited only a slight, nonstatistically significant reduction in protein levels, suggesting that this mutation primarily impaired plasma membrane targeting of CNGA3 subunits. Confocal images of intact oocytes display GFP-tagged subunits located at the surface membrane but not the fraction of subunits that remains intracellular (see Ref. 75). Thus, compared with wild-type and N471S subunits, there is a much larger fraction of R563H subunits retained intracellularly and/or retrieved to intracellular compartments.
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Altered gating properties of homomeric CNGA3 channels.
The electrophysiological properties of the channels were investigated using patch-clamp recording. Because CNGA3 subunits can form functional homomeric channels when expressed alone, we first examined the effect of these three mutations on the behavior of homomeric channels. Expression of R277C subunits alone did not give rise to cyclic nucleotide-dependent currents even after application of a saturating concentration of cGMP, suggesting that R277C subunits did not form functional CNG channels. Homomeric channels containing N471S or R563H mutations exhibited cyclic nucleotide-activated currents (Fig. 3A) with properties that differed from those of wild-type channels. cAMP is a partial agonist for recombinant and native rod and cone photoreceptor CNG channels, exhibiting a lower efficacy compared with cGMP (29). The initial binding of cAMP is comparable to that of cGMP, but cAMP is much less capable of promoting channel opening once bound (23). For both mutant channels, the relative agonist efficacy for channel activation by a saturating concentration of cAMP compared with maximal activation by cGMP (Imax,cAMP/Imax,cGMP) was increased significantly (N471S: 0.35 ± 0.12, n = 18, P < 0.01; R563H: 0.64 ± 0.20, n = 12, P < 0.01) compared with wild-type channels (0.12 ± 0.01, n = 23) (Fig. 3, A and B). Furthermore, we calculated the apparent ligand affinity for cGMP and cAMP by fitting the Hill equation to the dose-response relationships for the activation of channels formed by wild-type or mutant subunits (Fig. 3C). The results demonstrated a significant increase in apparent affinity for both cGMP and cAMP with R563H (K= 3.28 ± 0.63 µM, nH = 1.7 ± 0.2; K
= 0.64 ± 0.09 mM, nH = 1.1 ± 0.2; n = 13; P < 0.01) and an increase in the apparent affinity for cGMP with N471S (K
= 5.76 ± 1.66 µM, nH = 2.0 ± 0.3, n = 19; P < 0.01) compared with activation of wild-type homomeric channels (K
= 9.26 ± 1.77 µM, nH = 1.9 ± 0.06; K
= 1.07 ± 0.27 mM, nH = 1.0 ± 0.2; n = 18) (Fig. 3, D and E). The Hill coefficients showed no significant change compared with that of wild-type channels. These results suggest that the N471S and R563H mutations made the channel more sensitive to activation by cyclic nucleotide.
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Figure 4A shows block of wild-type and mutant CNG channels (in 1 mM cGMP) by 50 µM tetracaine. We calculated tetracaine apparent affinity (K) by fitting the dose-response relationships with a modified Hill equation as described in MATERIALS AND METHODS (Fig. 4B). Compared with wild-type homomeric channels (K
= 55.2 ± 5.3 µM; n = 6), N471S exhibited a statistically significant decrease in tetracaine apparent affinity (K
= 102.4 ± 12 µM, n = 8; P < 0.05), suggesting that this mutant subunit generated channels that spent more time in the open state compared with wild-type channels. R563H-containing channels also exhibited a decrease in tetracaine apparent affinity (K
= 76 ± 9.6 µM; n = 5), but this change was not statistically significant (P = 0.078).
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DISCUSSION |
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Loss-of-function phenotype. Previous studies indicate that a lack of functional CNG channels in the photoreceptor outer segment might contribute to cell degeneration. In this regard, animal models provide a useful tool for investigating the molecular mechanisms of photoreceptor degeneration. Cnga3-knockout mice exhibit a progressive loss of cone photoresponse and cone photoreceptor degeneration with intact rod function (1). This animal model closely resembles human progressive cone dystrophy. Recent studies in Cnga3/Rho double-knockout mice demonstrate normal retinal morphology and photoresponse in neonatal mice, yet rod and cone photoreceptor degeneration is observed after 4 wk and progresses to almost complete loss of photoreceptors by 3 mo (8). In addition to Cnga3-deficient mice, constitutive closure of CNG channels in continuous light resembles a loss-of-function phenotype. Progressive photoreceptor degeneration is evident in this model as well (15). Functional studies of disease-associated mutations also provide evidence that the lack of functional CNG channels in the photoreceptor outer segment might contribute to photoreceptor degeneration. Five mutations in the gene encoding the rod channel CNGA1 subunit have been linked to autosomal recessive retinitis pigmentosa, a disease characterized by impaired rod function and rod degeneration (12). Three of the five mutations are null mutations that result in either the synthesis of nonfunctional channel proteins lacking the transmembrane domain and the pore-forming region (E76stop and K139stop) or no protein synthesis in the case of complete gene deletion (12). The other two mutations (S316F and frame shift R654, 1-bp del) are thought to encode functional channels with impaired targeting to the plasma membrane (12, 40, 61).
In the present study, R277C and R563H mutations significantly reduced the availability of functional CNG channels at the cell surface, resembling a loss-of-function phenotype. R277C, which is also a common mutation in patients with complete achromatopsia (68), did not form functional homomeric or heteromeric channels. This result can be accounted for primarily by a reduction in overall protein levels and in subunit maturation. Recently, several mutations in the S4 domain of bovine CNGA3 subunits also have been shown to impair subunit stability and/or processing (13). R563H subunits exhibited decreased plasma membrane localization as well, but without a significant reduction in CNGA3 protein levels or maturation. The functional consequences of these mutations in vitro suggest that high-level expression of working CNG channels in the outer segment is critical for photoreceptor survival.
There are various mechanisms by which disease-associated mutations can impair channel protein expression at the plasma membrane. Chief among these are mutations in the channel subunits that may disturb protein folding or assembly, destabilize synthesized proteins, and/or disrupt targeting to the plasma membrane. The precise cellular mechanisms involved in photoreceptor degeneration for loss-of-function phenotypes remain to be determined. It has been shown previously that the survival of central nervous system neurons, including retinal ganglion cells, depends on physiological levels of electrical activity (42). Does the decrease or absence of functional CNG channel expression at the plasma membrane lead to a lack of proper levels of electrical activity, thus resulting in photoreceptor degeneration? Further work needs to be done to address these questions.
Gain-of-function phenotype. Functional characterization of cone CNG channels containing N471S or R563H mutations in the CNGA3 subunits revealed an increase in apparent ligand affinity and in relative agonist efficacy of cAMP compared with cGMP, consistent with a gain-of-function phenotype. A similar gain-of-function phenotype has been found for an achromatopsia-associated mutation in CNGB3 subunits (S435F) that alters the gating properties of heteromeric channels when coexpressed with CNGA3 subunits (48). CNGB3 S435F-containing heteromeric channels exhibit a more than fourfold increase in cAMP sensitivity and a modest increase in apparent affinity for cGMP. In addition, single-channel recordings reveal an increase in open probability in the presence of saturating concentration of cGMP or cAMP for mutant heteromeric channels (48). In the present study, the increased ligand sensitivity and efficacy suggest that in cone photoreceptors of patients with progressive cone dystrophy, CNG channels may fail to close appropriately as intracellular cGMP (or cAMP) levels fall in response to light stimulation.
What is the likely relationship between the altered gating properties of the mutant channels in the native cone outer segment membrane and cone photoreceptor degeneration? Under physiological conditions, the free intracellular cGMP level is estimated to be 24 µM (50, 51), which is well below the concentration of cGMP expected to elicit half-maximal activation of the channels (
). This cGMP concentration is sufficient to keep only a small fraction (<10%) of the channels in the open state in the absence of light (51). A light-induced rapid decrease (10 to 20 fold) of intracellular cGMP thus results in closure of nearly all of the channels (51). Because the sensitivity of channels to cGMP is so steep, even slight alterations in apparent ligand affinity may be detrimental to normal physiological function. For example, if the apparent affinity of the channels for cGMP changes [e.g., the
decreases from
15 µM (wild-type channel) to
9 µM (mutant channel)] (see Fig. 7C), nearly 10-fold the number of channels may be open in the absence of light and/or channels may not close properly in response to the fall in cGMP levels after light activation. Similar gain-of-function phenotypes have been discovered for mutations in the genes encoding other critical proteins involved in phototransduction, adaptation, and recovery processes. Recent evidence indicates that mutations which produce constitutively active guanylyl cyclase (30, 46, 62), or loss of rod cGMP PDE activity (10, 26, 63) result in increased intracellular cGMP levels. Increased intracellular cGMP, similarly to an increase in channel sensitivity to cGMP, might lead to inappropriate opening of the channels. Thus more Ca2+ would enter the photoreceptors through the CNG channels.
We hypothesize that Ca2+ overload might explain photoreceptor degeneration with gain-of-function mutations in cone CNG channels. One important aspect of CNG channel function is that it is the major pathway for Ca2+ entry into the outer segment of photoreceptors (16). It is possible that sustained opening of CNG channels, resulting from increased ligand affinity, may lead to abnormally high levels of intracellular Ca2+. Ca2+ is a critical second messenger that participates in several intracellular signaling pathways. Investigators in numerous studies have reported that a sustained elevation of intracellular Ca2+ could result in apoptotic cell death (for review, see Refs. 7 and 43). In the retina, for example, sustained elevation of intracellular Ca2+ has been shown to trigger rod photoreceptor apoptosis and retinal degeneration (19). This general mechanism may underlie other photoreceptor degenerative diseases such as progressive cone dystrophy. At the same time, it has been suggested recently that apoptosis may be triggered by a sustained decrease in intracellular Ca2+ levels in rod photoreceptors (for review, see Ref. 35) as expected for rod channel mutations presenting loss-of-function phenotypes. Consistent with this hypothesis, Rpe65-knockout mice, which exhibit impaired synthesis of the opsin chromophore ligand 11-cis-retinal, display light-independent signaling by unliganded opsin, diminished intracellular Ca2+ in photoreceptors, and progressive photoreceptor degeneration (69). Furthermore, mutations in RPE65 have been linked to Leber congenital amaurosis (LCA), a severe, early-onset retinal dystrophy (41).
For the cone dystrophy-associated mutations characterized in the present study, both loss-of-function and gain-of-function effects were observed. For example, R563H produced both a reduction in channel cell surface expression levels and an increase in ligand sensitivity. The profound decrease in functional expression for R563H, however, suggests that loss-of-function might play a greater part than the change in channel gating in the pathogenicity of the disease in these patients.
Assembly of N471S-containing CNGA3 subunits with wild-type CNGB3 subunits rescued most functional properties associated with wild-type heteromeric channels, with the exception of increased apparent cGMP affinity. The discrepancy between the mild functional change for N471S-containing heteromeric channels and the severe cone degeneration typically exhibited by patients with this disease implies that an additional, as yet unidentified mutation is necessary for disease progression (68). This additional mutation may be present in a noncoding region of CNGA3, in CNGB3, or in some other allele or modifying gene. Thus further mutation screening is needed to address this possible phenotype-genotype inconsistency.
Overall, our results suggest that complex effects may arise from the progressive cone dystrophy-associated mutations in CNGA3 subunits, including a decrease in plasma membrane localization of the channels; a disruption of channel protein biogenesis, processing, and/or stability; and an increase in ligand sensitivity and/or efficacy. These results provide insight into the molecular pathophysiology and possible cellular mechanisms underlying cone photoreceptor degenerative disease. How these mutations affect cone photoreceptor function and survival in vivo remains an important unanswered question that needs to be addressed using animal models.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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