Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Biochim Biophys Acta. Author manuscript; available in PMC 2010 July 1.
Published in final edited form as:
PMCID: PMC2746242

The Role of the Photoreceptor ABC Transporter ABCA4 in Lipid Transport and Stargardt Macular Degeneration


ABCA4 is a member of the ABCA subfamily of ATP binding cassette (ABC) transporters that is expressed in rod and cone photoreceptors of the vertebrate retina. ABCA4, also known as the Rim protein and ABCR, is a large 2273 amino acid glycoprotein organized as two tandem halves, each containing a single membrane spanning segment followed sequentially by a large exocytoplasmic domain, a multispanning membrane domain and a nucleotide binding domain. Over 500 mutations in the gene encoding ABCA4 are associated with a spectrum of related autosomal recessive retinal degenerative diseases including Stargardt macular degeneration, cone-rod dystrophy and a subset of retinitis pigmentosa. Biochemical studies on the purified ABCA4 together with analysis of abca4 knockout mice and patients with Stargardt disease have implicated ABCA4 as a retinylidene-phosphatidylethanolamine transporter that facilitates the removal of potentially reactive retinal derivatives from photoreceptors following photoexcitation. Knowledge of the genetic and molecular basis for ABCA4 related retinal degenerative diseases is being used to develop rationale therapeutic treatments for this set of disorders.

1. Introduction

The transport of lipids across biological membranes is critical for the structure, function and survival of cells. This process is typically carried out by a variety of integral membrane proteins which specifically bind and actively or passively translocate lipids across the membrane lipid bilayer. Some lipid transporters import or export lipids from cells or intracellular organelles, while others simply flip lipids such as phospholipids across the lipid bilayer to generate or maintain transbilayer lipid asymmetry critical for cell membrane structure and function [1]. Lipid substrates known to be transported across membranes include vitamins, fatty acids, phospholipids, glycolipids, cholesterol, bile salts, steroids, toxins, drugs, and metabolites.

ATP binding cassette (ABC) transporters comprise a large superfamily of proteins found in essentially all living organisms [2]. The human genome encodes 48 ABC transporters which have been organized into 7 subfamilies designated ABCA through ABCG[3]. A significant number of ABC transporters function in lipid trafficking in cells and many have been linked to severe genetic diseases [4, 5]. For example, inherited defects in ALD (ABCD1), a transporter of very long chain fatty acids, is associated with adrenoleukodystrophy[6] , defects in Sterolin1 and 2 (ABCG5/ABCG8), transporters for sterols, have been linked to sitosterolemia [7] , defects in ABC1 (ABCA1) which mediate the efflux of cholesterol and phospholipid from cells causes Tangier disease [8], and defects in MDR3 and BSEP (ABCB11) involved in the transport of phosphatidylcholine and bile acids, respectively, are known to cause liver disease [9] [10] .

Members of the ABCA subfamily of ABC transporters have been implicated in the transport of various lipids across cell membranes [5]. Of these, four ABCA transporters are now known to be associated with a various genetic disorders that result from defects in lipid transport. In this review, we will focus on the biochemical properties of one of these transporters, ABCA4, and its role in lipid transport and retinal degenerative diseases associated with severe vision loss.

2. ABCA Subfamily of ABC Transporters

The ABCA subfamily of ATP binding cassette transporters is a group of ABC transporters having a relatively high degree of sequence identity and a similar structural organization. Twelve ABCA genes designated ABCA1ABCA13 have been identified in the human genome [5, 11]. ABCA11 is now known to be a pseudogene and therefore is no longer considered as a member of this subfamily. Two subgroups have been identified. One comprises a cluster of genes (ABCA5, ABCA6, ABCA8, ABCA9, and ABCA10) that map to chromosome 17q24.2–3 and code for proteins of similar size (1543 – 1642 amino acids) and sequence (53 – 78% similarity). The other group consists of ABCA1, ABCA2, ABCA3, ABCA4, ABCA7, ABCA12, and ABCA13 genes. A cluster of 3 additional genes, Abca14, Abca15, and Abca16, have been found in the mouse but not the human genome [12]. Homology searches have revealed the presence of ABCA homologues in a wide variety of other eukaryotic and prokaryotic organisms [13].

All members of the human ABCA subfamily are large full transporters organized in two tandem halves. Both the N-terminal and C-terminal half consists of a multi-spanning transmembrane domain followed by a nucleotide binding domain. A characteristic feature of ABCA proteins is the presence of a large extracellular domain that separates the first transmembrane segment from a cluster of five transmembrane segments in each half of the protein [14, 15].

The substrates transported by most members of the ABCA subfamily remain to be identified. However, biochemical analyses of the ABCA1, ABCA3, ABCA4, and ABCA12 transporters together with their association with inherited diseases have implicated these proteins in the active transport of lipids across cell membranes. ABCA1 plays a crucial role in the efflux of cholesterol and phospholipids from macrophage and other cells [16]. Defects in ABCA1 are known to cause Tangier disease and familial high density lipoprotein deficiency, disorders associated with defective cholesterol and phospholipid efflux from cells and a deficiency in circulating high density lipoproteins [8, 1719]. ABCA3 has been implicated in phospholipid transport required for the formation of normal lamellar bodies and surfactant secretion into the alveoli of the lungs [2022]. Mutations in ABCA3 cause neonatal surfactant deficiency associated with abnormal lamellar bodies in the lungs and a milder respiratory disease known as pediatric interstitial lung disease. Mutations in ABCA12 are responsible for harlequin and lamellar ichthyosis, diseases of the skin generally resulting from defective lipid transport [2325]. ABCA7 and ABCA2 have been implicated in lipid transport although these transporters have yet to be linked to any inherited diseases.

3. Cellular Localization and Structural Features of ABCA4

ABCA4 was first cloned in 1997 using two different strategies. In one approach, the gene for the Rim protein, an abundant high molecular weight photoreceptor membrane protein of unknown function first described in the late 1970’s, was cloned and shown to encode a novel photoreceptor ATP binding cassette protein [14, 26, 27]. In another approach, Allikmets and coworkers identified the gene defective in Stargardt disease, a relatively common, early onset macular dystrophy [28, 29]. This gene located on chromosome 1p22.1 was found to encode a retina specific ABC transporter which was named ABCR. Sequence comparison of ABCR and the Rim protein indicate that they are the same ABC transporter [30], now generally known as ABCA4 for the fourth identified member of the ABCA subfamily.

3.1. Localization of ABCA4 in Rod and Cone Photoreceptor Cells

ABCA4 is almost exclusively expressed in the retina, the light sensing neural tissue at the back of the eye [28, 29]. The retina consists of 5 major neuronal cell types (photoreceptors, bipolar cells, horizontal cells, amacrine cells and ganglion cells) and one glial cell type known as Mueller cells. ABCA4 gene expression is confined to the photoreceptor cells as determined by in situ hybridization [28, 29]. Immunofluorescence microscopic studies have localized the ABCA4 protein to the photoreceptor outer segment, a specialized compartment of the photoreceptor cell that functions in the capture of light and its conversion into an electrical signal in a process known as phototransduction [14, 31].

There are two main types of photoreceptors in the vertebrate retina, rod cells that function under dim lighting conditions and cones that are responsible for vision under normal lighting conditions, color vision, and high visual acuity. Initial immunolabeling studies suggested that ABCA4 was only present in the outer segments of mammalian rod photoreceptors [14, 31] . However, in subsequent studies, ABCA4 has been localized to the outer segments of foveal and peripheral cone cells as well as rod photoreceptors of the human retina [32]. This finding is in agreement with the early studies of Papermaster et al. [33] showing that the Rim protein is present in outer segment discs of frog rod and cone photoreceptors.

The outer segment of a rod cell consists of a stack of over 1000 discs surrounded by a separate plasma membrane. Each disc is composed of two adjacent, flattened membranes that are circumscribed by a hairpin loop known as the rim region. The continuous disc membrane encloses a space known as the intradiscal space or disc lumen. The circumference of the disc is interrupted by one or more incisures that penetrate toward the center of the disc. Immunoelectron microscopic studies have localized ABCA4 to the rim and incisures of the disc membrane in both mammalian and amphibian photoreceptor cells [14, 33].

Although ABCA4 is primarily expressed in retinal photoreceptors, mRNA and protein expression have been found in the choroid plexus of rat brain [34]. The functional significance of ABCA4 expression in the brain, however, remains to be established.

ABCA4 has been transiently expressed in several mammalian culture cell lines including HEK 293 and COS cells [35, 36]. Immunofluorescence microscopic studies have localized ABCA4 to intracellular vesicular structures of varying sizes [37, 38], a distribution that has also been observed for peripherin/rds and rom-1 [39, 40], photoreceptor specific proteins that also localize to the rim and incisures of photoreceptor disc membranes [40, 41]. The intracellular localization of heterologously expressed ABCA4 is in marked contrast to the plasma membrane localization of heterologously expressed ABCA1, a protein that is 50% identical in amino acid sequence to ABCA4. These studies suggest that ABCA4 and ABCA1 contain distinct signals that target these transporters to intracellular membranes in the case of ABCA4 and the plasma membrane for ABCA1.

3.2. General Structural Organization

The human ABCA4 gene consisting of 50 exons encodes a 2,273 amino acid protein having a molecular mass of 256 kDa [29, 30]. ABCA4 is organized into two tandem halves, with each half containing a transmembrane segment followed sequentially by a large exocytoplasmic (extracellular/lumen) domain (ECD), a multi-spanning membrane domain and a nucleotide binding domain (NBD) in both the N and C halves of the protein (Figure 1a,b) [14, 15]. Other members of the ABCA subfamily have a similar membrane topology and domain organization [15, 42].

Figure 1
Structural features of the ABCA4 transporter. (a) A linear diagram showing the full transporter arranged as two tandem halves. Each half consists of six membrane spanning segments which together make up the transmembrane domain (TMD). A large exocytoplasmic ...

The short 24 amino acid N-terminal segment containing positively charged arginine and lysine residues is predicted to reside within the cytoplasm. The large ECDs in the N-half and C-half have multiple N-linked oligosaccharide chains as confirmed by site-directed mutagenesis in combination with lectin binding and endoglycosidase digestion [15]. Numerous conserved cysteine residues in the ECDs form intramolecular disulfide bonds within each domains as well as between domains [15]. The function of the ECD domains in ABCA4 is not known, although in the case of ABCA1, these domains serve as binding sites for apoA1 [42, 43]. Both the N and C halves of ABCA4 are predicted to contain five hydrophobic membrane spanning segments connected by relatively short hydrophilic loops. These membrane segments, together with the single transmembrane segments preceding each of the ECDs, most likely form the substrate binding site and translocation pathway.

In addition to these domains, ABCA4 contains an extended C-terminal segment harboring a conserved six amino acid VFVNFA motif, also found in ABCA1, but not other ABCA proteins [44]. Recent deletion and substitution mutagenesis studies indicate that this conserved motif plays an important role in the proper folding of ABCA4 and ABCA1 into a functional protein [38].

3.3 Nucleotide Binding Domains

The nucleotide binding domains (NBDs) of ABCA4 consisting of approximately 200 amino acid residues display a motif organization found in other ABC transporters. This includes a conserved A-loop, Walker A, Q-loop, ABC signature, Walker B, and an H-loop [45, 46]. The Walker A motif (GxxGxGKS/T; x- any amino acid) and Walker B motif (hhhhD; h- any hydrophobic amino acid) form the ATP nucleotide binding fold (Figure 2a) [45]. The A-loop consists of a tyrosine or a hydrophobic residue that may be involved in stacking interactions with the adenine ring of ATP [47].

Figure 2
(a). Alignment of the nucleotide binding domains of ABCA4. Conserved motifs including the A-loop, Walker A, signature, Walker B, Q-loop, and the H-loop are in bold. Disease mutations, which are substituted in Stargardt disease, are shown in red italics ...

Homology modeling suggests that the NBDs of ABCA4 have a similar structural organization as other ABC transporters of known structure (Figure 2b). Each NBD consists of two arms forming an L-shaped structure. Arm I, termed the RecA-like core domain, has β-sheets flanked by α-helices and harbors the Walker A and Walker B sites, while arm II is α-helical and contains the signature motif [48]. A conserved glutamine in the flexible Q-loop connects the RecA-like core domain to the α-helical domain. This interaction has been implicated in transmitting conformational changes between the TMD and NBDs [49].

Functional ABC transporters require the formation of NBD dimers. There are three models of NBD-dimer formation. A head-to-tail dimer formation aligns the Walker A domain of one subunit with the signature domain from the opposing subunit, producing a composite of two nucleotide binding sites [50]. A back-to-back arrangement shows ATP molecules being solvent exposed and facing away from each other with their Arm I domains forming hydrophobic interactions [51]. The third arrangement demonstrates a mirror image of Walker A and signature motifs facing each other. Whether dimerization of the NBDs in ABCA4 proceeds in a head-to-tail arrangement is uncertain. Furthermore, the importance of the signature motif needs to be investigated for analysis of the dimer conformation.

4. Functional Properties of ABCA4

4.1. ATP Binding and Hydrolysis

Specific binding of nucleotides to ABCA4 was first demonstrated using photoaffinity labeling techniques. The photoreactive 8-azido ATP or 8-azido ADP covalently labeled purified ABCA4 or ABCA4 in photoreceptor membranes upon irradiation with UV light [14]. Excess unlabeled ATP or GTP inhibited 8-azido ATP labeling indicating that the labeled nucleotide binding site of ABCA4 can accommodate either adenine or guanine nucleotides. When photoaffinity labeled ABCA4 in photoreceptor membranes was cleaved with trypsin into its N and C halves, or when the N and C halves were co-expressed in culture cells, ATP labeling was detected only in the C-half of ABCA4 [37].

The ATPase activity of many ABC transporters such as P-glycoprotein has been shown to be activated by compounds known to be transported by these proteins [5254]. This strategy was used to identify possible substrates for ABCA4 [55]. For these studies ABCA4 was immunoaffinity-purified from detergent solubilized rod outer segment membranes employing a specific monoclonal antibody (Rim 3F4) directed against a well-defined nine amino acid epitope near the C-terminus of ABCA4 [14]. Purified ABCA4 in detergent or reconstitution into brain polar lipid vesicles had a relatively low basal ATPase activity [36, 55]. Over 40 different compounds were tested for their capacity to activate the ATPase activity of ABCA4. Of these compounds, two physiologically relevant retinoid compounds, all-trans retinal and 11-cis retinal, stimulated the ATPase activity of detergent solubilized and reconstituted ABCA4 several fold above the basal activity. Kinetic analysis indicated that all-trans retinal increased both the Vm and Km for ATP. Other retinoid and aliphatic aldehyde derivatives including retinol, retinyl esters, nonyl aldehyde, and others failed to stimulate the ATPase activity of ABCA4. These studies provided the first clue that a retinal compound, a key molecule required for vision, may serve as the substrate for ABCA4.

In addition to retinal, the ATPase activity of ABCA4 was shown to be strongly dependent on the phospholipid composition [36, 55]. Basal and retinal stimulated ATPase activity was observed for ABCA4 reconstituted into membranes consisting of soybean, brain and native photoreceptor outer segment lipids. However, the highest activity was observed for ABCA4 reconstituted into vesicles made from rod outer segment lipids known to have a high content of PE (>40%) [36]. The ATPase activity of ABCA4 reconstituted in brain polar lipid could be elevated to levels comparable to that found for outer segment lipids by the addition of excess synthetic PE. The importance of PE was further confirmed by the finding that the ATPase activity of ABCA4 reconstituted into vesicles lacking PE was not stimulated by retinal. The length or degree of unsaturation of the fatty acyl chain of PE had no significant effect on the ATPase activity of ABCA4. Taken together, these studies indicated that retinal stimulated ATPase activity is not only dependent of retinal, but also PE.

The ATPase activity of the NBD1 and NBD2 of ABCA4 expressed in E.coli has been studied [56, 57]. Both domains exhibited low ATPase activity. The kinetic parameters (Km and Vmax) for the NBD2 were comparable to that reported for reconstituted ABCA4. The ATPase activity of NBD1 was considerably lower than that of NBD2. Significant differences in nucleotide specificity were reported with NBD1 capable of hydrolyzing GTP, CTP and UTP as well as ATP and NBD2 specific for ATP.

The basal and retinal activated activities of both NBDs were also investigated by expressing and analyzing ABCA4 containing Walker A mutations (K969M for NBD1) and K1978M for NBD2) known to inhibit ATP hydrolysis [35, 37]. The single and double mutants expressed at wild-type levels and bound ATP as indicated by photoaffinity labeling measurements. The ATPase activities, however, were strongly affected by these mutations [35]. The double K969M/K1978M double mutant and the K969M single mutant showed no basal or retinal stimulated ATPase activity, whereas the K1978M mutant in NBD2 exhibited basal ATPase activity but not retinal stimulated activity. These studies indicate that NBD1 and NBD2 are both important, but play distinct mechanistic roles.

4.2 N-retinylidene-PE as the Substrate for ABCA4

Aldehydes react with primary amines to form Schiff base products. The chromophore 11-cis retinal is conjugated to lysine 296 of opsin through a Schiff base linkage. All-trans retinal released from rhodopsin following photoexcitation reacts PE in disc membranes forms the Schiff base product N-retinylidene-phosphatidylethanolamine (N-retinylidene-PE) [58, 59]. Likewise, the addition of all-trans retinal to vesicles containing PE produces an equilibrium mixture of N-retinylidene-PE and free all-trans retinal [36] (Figure 3 and Figure 4). Accordingly, the observed retinal stimulated ATPase activity of ABCA4 can result from either the free all-trans retinal or N-retinylidene-PE [55]. Mechanistically, ABCA4, could act to extrude all-trans retinal from membranes similar to the efflux of hydrophobic compounds from membranes by P-glycoprotein [60]. Alternatively, ABCA4 could transport or flip N-retinylidene-PE across the disc membrane analogous to the flipping of phospholipids across the lipid bilayer as reported for other ABC transporters [6163].

Figure 3
Role of ABCA4 in the visual cycle. Light isomerizes 11-cis retinal to all-trans retinal in rhodopsin. All-trans retinal dissociates from opsin and is reduced to all-trans retinol by all-trans retinol dehydrogenase (RDH8). All-trans retinol is esterified ...
Figure 4
Reactions involved in the formation of A2E due to loss in ABCA4 activity. All-trans retinal reacts with phosphatidylethanolamine (PE) to form N-retinylidene-PE which can condense with all-trans retinal to produce the di-retinoid pyridinium-PE compound ...

Using a solid phase assay, N-retinylidene-PE was identified as the substrate for ABCA4 [64]. In this study, various retinoid compounds in the presence of phospholipids were added to ABCA4 immobilized on an immunoaffinity matrix. After removal of unbound material, the bound retinoid was analyzed by HPLC. When all-trans retinal was added to ABCA4, stoichiometric amounts of N-retinylidene-PE bound to ABCA4 with an apparent dissociation constant of 4–5 µM. N-retinyl-PE produced by sodium borohydride reduction of N-retinylidene-PE (see Figure 4) also bound tightly to the same site on ABCA4. In contrast all-trans retinol or all-trans retinal in the absence of PE failed to bind ABCA4. The Schiff base of N-retinylidene-PE can exist in either a protonated or unprotonated state. Recent spectrophotometric measurements indicate that the unprotonated form of N-retinylidene-PE binds to ABCA4 (Zhong and Molday, unpublished results).

The effect of nucleotides on the binding of N-retinylidene-PE was studied in order to further evaluate the possible role of N-retinylidene-PE as a substrate for ABCA4 [64]. Addition of ATP or GTP quantitatively released N-retinylidene-PE from ABCA4. These studies indicate that N-retinylidene-PE binds to a high affinity site on ABCA4 in the absence of nucleotide. Addition of ATP (or GTP) induces a protein conformational change that causes N-retinylidene-PE to dissociate from ABCA4. Together, these studies provide strong biochemical evidence that N-retinylidene-PE is the lipid substrate for ABCA4.

5. Transport Mechanism

On the basis of biochemical studies and structural analysis, a general scheme has been proposed for the transport of substrates across membranes by ABC transporters [46, 6568]. In the initial step, the substrate binds to a high affinity site within the transmembrane domain (TMD) of the ABC transporter. This induces a protein conformational change which alters the affinity of the NBDs for ATP. The binding of ATP causes the NBDs to come in close contact with each ATP present in the dimer interface. Dimerization of the NBDs induces a conformational change in the TMD resulting in the conversion of the high affinity to low affinity substrate binding and the release of the substrate on the opposite side of the membrane as part of the transport process. ATP hydrolysis results in the loss of NBD dimerization. Finally, the dissociation of ADP from the NBDs returns the transporter to it high affinity substrate binding state.

The mechanism by which N-retinylidene-PE is transported across the disc membrane by ABCA4 remains to be determined. However, biochemical studies of ABCA4 are consistent with the ATP switch model [68](figure 5). N-retinylidene-PE binds to a high affinity site on ABCA4 in the absence of ATP [64]. This binding causes a conformational change which is relayed to the NBDs as revealed by a substrate induced change in the Km for ATP hydrolysis [36, 55]. The addition of ATP even at low temperature (conditions where ATP hydrolysis does not occur) results in the dissociation of N-retinylidene-PE from detergent solubilized ABCA4. This suggests that the binding of ATP and presumably dimerization of the NBDs is sufficient to cause a conformation change in the TMD resulting in the loss in the high affinity substrate binding site and release of the substrate [64, 69]. As suggested in the ATP switch model, ATP hydrolysis and dissociation of ADP is envisioned to return ABCA4 to its initial state. The role of each of the NBDs is not understood. The model depicted in Figure 5 shows ATP binding and hydrolysis at both NBD1 and NBD2. However, photoaffinity labeling studies and ATPase measurements on mutant proteins indicate that the NBDs have distinct properties [35, 37]. Although both NBDs are critical for transport function, the role of each NBD remains to be elucidated. A detailed understanding of the mechanism of lipid transport by ABCA4 will require the development of a transport assay, determination of the role of each NBD in the transport mechanism, and ultimately a high resolution protein structure.

Figure 5
A simplified model for the transport of N-retinylidene-PE across the disc membrane by ABCA4. In the absence of ATP, N-retinylidene-PE binds to a high affinity site within the transmembrane domain (TMD) of ABCA4 altering the affinity of the nucleotide ...

6. Analysis of ABCA4 Knockout Mice

The role of ABCA4 as an N-retinylidene-PE transporter has also been proposed from studies of abca4 knockout mice [7072](figure 6). The photoreceptor cells of the abca4 deficient mice exhibit a normal appearance with well-organized outer segments. This suggests that ABCA4 is not directly involved in outer segment morphogenesis or structure. Likewise, the photoresponse of these mice is normal except for relatively small changes in the recovery of the rod photoresponse after photobleaching [70, 73]. However, significant light-dependent changes in retinoids and lipids have been observed [70, 74]. Compared to wild-type mice, abca4 knockout mice show a light-dependent elevation of all-trans retinal, protonated N-retinylidene-PE, and PE in the retina. Furthermore, abca4 knockout mice raised under constant or cyclic lighting display a high degree of autofluorescence associated with the accumulation of lipofuscin deposits in the retinal pigment epithelial (RPE) cells adjacent to the photoreceptor cells. The lipofuscin deposits in the abca4 knockout mice are associated with elevated levels of diretinyl compounds, including the diretinal pyridinium compound known as A2E and all-trans retinal dimers [70, 71, 7578]. These diretinal compounds are formed through the initial condensation of all-trans retinal with N-retinylidene-PE in the disc membranes (see figure 4). In addition to these derivatives, potentially toxic photoreactive products of A2E and retinal dimers including epoxides and oxiranes have been reported to accumulate in abca4 knockout mice [79, 80](figure 6).

Figure 6
Model for the ABCA4-mediated photoreceptor degeneration in Stargardt disease. Partial or complete loss of ABCA4 function due to disease-causing mutations (ABCA4 – star) results in the accumulation of all-trans retinal and N-retinylidene-PE (N-Ret-PE) ...

7. ABCA4 and Retinal Degenerative Diseases

7.1 Stargardt Macular Degeneration and Related Retinal Disorders

Stargardt disease is an early onset, autosomal recessive disease first described by the German ophthalmologist Karl Stargardt in 1909 [81]. It is the most common inherited macular dystrophy with a prevalence estimated to be 1 in 10,000 [82]. Characteristic features include significant loss in central vision in the first or second decade of life, progressive bilateral atrophy of the RPE and photoreceptors, yellow or white lipofuscin deposits within the macula at the level of the RPE cells associated with autofluorescence, and a delay in dark adaptaion [8387]. Elevated levels of N-retinlyidiene-PE in the retina and A2E and related diretinal fluorophores in the RPE layer have been found in tissue samples from Stargardt patents [71] . Fundus flavimaculatus, a retinal disorder with similar clinical features, is now considered to be a late onset, slow progressive form of Stargardt diseases [88, 89].

Since the identification of the ABCA4 gene over 10 years ago [29], an international effort has been undertaken to screen Stargardt patients for mutations in the ABCA4 gene [30, 90101]. To date over 500 different mutations are now known to cause Stargardt disease. These include missense, nonsense, splice-site, frameshift, and small deletion and insertion mutations. Of these, missense mutations are most common and distributed throughout the protein. A subset of missense mutations reside in NBD1 (N965S, T971N, A1038V, S1071V, E1087K, R1108C, R1129L) and NBD2 (G1961E, L1971R, G1977S, L2027F, R2038W, R2077W, R2106C, R2107H). Several of these, including N965S, T971N, E1087K, L1971R, G1977S, reside inside or close to the Walker A and B motifs [29][[90, 95, 97, 100] [92, 102].

In addition to Stargardt disease, mutations in ABCA4 are known to cause more severe retinal degenerative diseases including autosomal recessive cone-rod dystrophy and a recessive form of retinitis pigmentosa (RP) [91, 98, 102, 103]. Cone-rod dystrophy is a retinal disorder in which cone degeneration is more severe than rod degeneration as assessed by a more marked reduction in the cone b-wave of the photopic electroretinogram than the rod b-wave of the scotopic electroretinogram. Individuals with cone-rod dystrophy typically experience severe loss in visual acuity and color vision, and show evidence of significant degeneration of the macula. RP is a particularly severe retinal degenerative disease associated with night blindness, reduction in peripheral vision and progressive loss in central vision most often leading to complete blindness. RP patients also display narrowed retinal vessels, bone-spicule pigmentation and a waxy pallor of the optic disk. Over 40 different genes are known to be associated with various forms of RP. Genetic studies have linked selected mutations in ABCA4 with a subset of autosomal recessive RP known as RP19 [102]. Affected individuals show an early loss in central vision and atrophy of the macula along with the characteristic features of RP.

In addition to these disorders, mutations in ABCA4 have been reported to increase one’s risk of developing age-related macular degeneration (AMD). In an initial report individuals heterozygous with selected mutations in ABCA4 known to cause Stargardt disease were found in 16% of patients with AMD [28]. Subsequent studies, however, indicate that mutations in ABCA4, if indeed they are associated with AMD, are a minor cause of this disease [97].

Together, these studies suggest that mutations in ABCA4 cause a spectrum of related retinal degenerative diseases with variable clinical severity. For the recessive disorders, Stargardt disease has the mildest phenotype, cone-rod dystrophy a moderate to severe phenotype, and RP the most severe phenotype. A model has been proposed in which the severity of the disease phenotype is inversely correlated with the residual functional activity of the mutant ABCA4 [92, 104, 105]. In this model, mutations in both alleles that result in complete loss of ABCA4 activity or produce a mutant protein with a detrimental effect on photoreceptor survival cause the RP phenotype. Compound heterozygous individuals with only residual ABCA4 function develop cone-rod dystrophy. Finally, Stargardt disease is associated with mutations that retain partial functional activity of ABCA4. Although it is likely that there is a correlation between residual ABCA4 activity and disease phenotype as suggested by this model, other factors including the effect of mutations on the trafficking of ABCA4 to photoreceptor outer segment disc, age of diagnosis, progression of the disease, and the general genetic variability of the population also contribute to the observed phenotype [106, 107] .

7.2. Effect of Disease linked Mutations on the Properties of ABCA4

The effect of a number of disease-causing mutations on ABCA4 protein expression and ATP binding and hydrolysis has been investigated by heterologous expression in HEK293 or COS7 cells [35, 38]. Mutations resulting in internal amino acid deletions and introduction of charge residues in the putative transmembrane segments were found to significantly reduce protein expression suggesting that these mutations result in a highly misfolded protein that is rapidly degraded by the cell [35]. A number of mutants including many in the NBDs expressed at protein levels similar to wild-type ABCA4, but exhibited reduced ATP binding and hydrolysis. Some mutations affect only the retinal activated ATPase activity such as the G1975D and K1978M mutations in NBD2, whereas a large number of mutants affect both the basal and retinal stimulated ATPase activity of ABCA4 as exemplified by the L541P mutation in ECD1, T971N in NBD1 and E2096K in NBD2 [35].

More recently, the effect of two disease-associated C-terminal deletion mutants on N-retinylidene-PE binding and ATP binding and hydrolysis was studied [38]. An ABCA4 mutant lacking the C-terminal 30 amino acids including a conserved VFVNFA motif and associated with cone rod dystrophy showed decreased protein expression, protein misfolding associated with retention in the endoplasmic reticulum, and complete loss in retinal and ATP binding and hydrolysis activity. In contrast, a mutant lacking the C-terminal 24 amino acids but retaining the VFVNFA motif and associated with a mild form of Stargardt disease showed only a limited decrease in protein expression and relatively normal intravesicular localization, and a relatively small reduction in retinal stimulated ATPase activity. The expression and activity of these mutants are generally consistent with the observed phenotypes of the patients and highlight the importance of the conserved VFVNFA in proper protein folding and functional activity of ABCA4 [99, 108].

The effect of several disease-associated mutations on the trafficking of ABCA4 to outer segments has been studied in transgenic Xenopus laevis [107]. A number of mutants were retained in the inner segment of the photoreceptors. This suggests that protein mislocalization as well as misfolding and diminished function can contribute to ABCA4 associated diseases.

8. Model for the Role of ABCA4 in the Visual Cycle and Retinal Degenerative Diseases

Analyses of Stargardt patients together with biochemical characterization of ABCA4 and abca4 knockout mice have implicated ABCA4 in removal of retinoids from outer segments as part of the visual or retinoid cycle [55, 69, 70]. Following photoexcitation, all-trans retinal is released from rhodopsin and partitions into the lipid bilayer of disc membranes. Most of the all-trans retinal is reduced to all-trans retinol on the cytoplasmic side of the membrane by the retinol dehydrogenase RDH8 and possibly other NADPH-dependent dehydrogenases [109, 110]. All-trans retinol is then transported from photoreceptors to the RPE cell where it is converted to all-trans retinal ester by lecithin:retinol acyl transferase (LRAT), isomerized to 11-cis retinol by RPE65-isomerase, and oxidized to 11-cis retinal by 11-cis retinol dehydrogenases (Figure 3). 11-cis retinal is then transported back to the photoreceptor outer segment where is can recombine with opsin to regenerate rhodopsin or cone opsin [109, 110]. However, a fraction of all-trans retinal generated from the photobleaching of rhodopsin or cone opsin reacts with PE to form N-retinylidene-PE. The acidic pH of the disc lumen can trap the protonated form of N-retinylidiene-PE in the lumen leaflet of the disc membranes [111]. The function of ABCA4 is envisioned to actively transport or flip trapped N-retinylidene-PE from the lumen to the cytoplasmic side of the disc membrane. On the cytoplasmic surface, N-retinylidene-PE can dissociate into all-trans retinal and PE, and all-trans retinal can then be reduced to all-trans retinol by RDH8 for the resynthesis of 11-cis retinal and the regeneration of rhodopsin via the visual cycle. In this model the function of ABCA4 is to insure that all-trans retinal and N-retinylidene-PE is completely removed from disc membranes following photobleaching of rhodopsin in rod cells and cone opsin in cone cells.

Loss or decrease in ABCA4 transport activity will result in an accumulation of N-retinylidene-PE and all-trans retinal in disc membranes as found in abca4 knockout mice and Stargardt patients [70, 71] (Figure 5, Figure 6). N-retinylidene-PE can react with all-trans retinal to form the diretinal derivative A2PE (figure 4) and related diretinal compounds in outer segments [71, 75, 76]. Rod and cone outer segments undergo a continual renewal process in which new membrane is added at the base of the outer segments while packets of aged outer segments are removed by shedding and phagocytosis by adjacent RPE cells. Through this process, the outer segment is renewed once every 10 days. Ingested phagosomes containing A2PE, all-trans retinal dimers and other retinoid compounds fuse with lysosomes to form phagolysosomes which contain enzymes that degrade the outer segments (Figure 6). A2PE is hydrolyzed to A2E, but cannot be further metabolized. As a result, A2E and related retinal compounds progressively accumulate as lipofuscin deposits in RPE cells [71, 76, 77]. Upon exposure to blue light, A2E can be converted into epoxides [79]. A2E and related epoxides can lead to loss in RPE function and cell death [75, 77, 78, 112, 113]. Since RPE cells are required for photoreceptor cell survival, the loss in RPE cells will cause the photoreceptor cell death and a loss in vision.

This model is consistent with many of the characteristic features observed for individuals with Stargardt disease and mice deficient in ABCA4. These include elevated levels of all-trans retinal and protonated N-retinylidiene-PE in photoreceptor cells, and the progressive accumulation of A2E and other diretinal compounds as fluorescent lipofuscin deposits in RPE cells. In addition the accumulation of all-trans retinal in disc membranes can explain the delay in dark adaptation found in Stargardt patients and abca4 mice.

Although there is considerable support for this model in which ABCA4 functions as N-retinylidene-PE transporter, a number of issues remain to be resolved. In particular, it is necessary to directly show that ABCA4 functions to transport or flip N-retinylidene-PE across disc membranes. The mechanism by which ATP hydrolysis is coupled to the transport of N-retinylidene-PE across membranes remains to be elucidated. Importantly, the direction of substrate transport needs to be clarified. In the present model, ABCA4 is envisioned to transport N-retinylidene-PE from the lumen to the cytoplasmic leaflet of disc membranes. This is the opposite direction of transport for many well studied mammalian ABC transporters. ABC transporters are known to be regulated through protein-protein interactions and/or phosphorylation. To date there is no information on factors which regulate the activity of ABCA4. Resolution of these issues awaits the further development of functional transport assays and the generation of high resolution structures of ABCA4.

9. Therapeutic Strategies for Stargardt Macular Degeneration and Related Diseases

Insight into the genetic and molecular basis for Stargardt and related ABCA4 retinal degenerative diseases has led to investigations into possible treatments for this set of disorders. Gene therapy offers a promising approach to prevent or slow the onset of ABCA4 related diseases [114]. For recessive diseases, this typically involves the delivery of the normal gene to cells harboring the defective gene. This strategy has proven successful in animal models for a number of retinal degenerative diseases caused by mutations in genes expressed in RPE or photoreceptor cells [115117]. Most studies have employed recombinant adeno-associated viral (rAAV) vectors for gene delivery. These vectors have the advantage that they are nontoxic and relatively nonimmunogenic when administered by subretinal injections. In addition, rAAV vectors efficiently transduce non-dividing cells such as RPE and photoreceptors for long term gene expression. For example, rAAV containing the RPE65 gene delivered to RPE cells restored vision in a canine model for Leber Congenital Amaurosis (LCA), a severe early onset retinal degenerative disease associated with complete loss in vision [118]. Success in preclinical animal studies for this disease has led to recent Phase I clinical trials in which partial recovery of vision has been demonstrated for individuals affected with LCA [119121].

A potential disadvantage of rAAV for use in Stargardt disease is its limited gene packaging size of 4.7 kb. However, a recent study has shown that large genes including the 6.8 kb ABCA4 gene can be stuffed into a chimeric rAAV (2/5) vector [122]. This vector has been used to deliver the mouse Abca4 gene to photoreceptors of an abca4 knockout mouse. In another study the human ABCA4 gene has been delivered to photoreceptors of an abca4 knockout mouse using lentiviral vector [123]. In both these studies, expression of ABCA4 in photoreceptors correlated with a reduction in A2E levels in RPE cells. These initial studies suggest that gene therapy has potential application as a treatment for Stargardt and other ABCA4 associated diseases.

Another approach has been to reduce the level of 11-cis retinal and consequently all-trans retinal in photoreceptors through the application of drugs that inhibit enzymes of the visual cycle [110]. In initial studies, isotretinoin or 13-cis-retinoic acid, also known as Accutane, a widely used agent for the treatment of acne, was used to inhibit 11-cis-RDH, the enzyme responsible for the formation of 11-cis retinal in RPE cell [124]. When abca4 knockout mice were given isotretinoin, a marked reduction in the light-induced accumulation of A2E containing lipofuscin deposits was observed. Since isotretinoin is associated with side effects, other retinoid-based inhibitors have been developed and tested in animal studies. These include N-(4-hydroxyphenyl)retinamide known to reduce serum levels of vitamin A and retinylamine, an inhibitor of isomerase activity in RPE cells [125, 126]. Both these retinoid compounds have been shown to reduce A2E accumulation in animal studies pointing to the therapeutic potential for these drugs.

10. Conclusions

ABCA4 is an ABC transporter that is expressed in vertebrate rod and cone photoreceptors and localized to the rim regions of outer segment disc membranes. Mutations in ABCA4 are responsible for Stargardt macular degeneration and related retinal degenerative diseases that cause significant loss in vision. Functional properties of the purified protein together with biochemical analysis of abca4 knockout mice have implicated ABCA4 in the transport of N-retinylidene-PE across the disc membranes following photoexcitation an essential step in the complete removal of all-trans retinal and N-retinylidene-PE from photoreceptor membranes following photoexcitation. Loss in the lipid transport function of ABCA4 due to disease-causing mutations leads to the accumulation of all-trans retinal and N-retinylidene-PE in disc membranes and the formation of potentially toxic diretinal compounds. Upon phagocytosis of outer segments, the diretinal compounds including A2E progressively accumulate in RPE cells as lipofuscin deposits. A2E and related diretinal compounds compromise the function and viability of RPE cells ultimately resulting in photoreceptor degeneration and a loss in vision.

Although considerable progress has been made in understanding the role of ABCA4 in the visual cycle and the pathogenesis of ABCA4 associated diseases, a number of issues remain to be resolved. These include generation of a high resolution structure of ABCA4, development of a transport assay to measure the direction of substrate transport, identification of mechanisms that regulate the activity of ABCA4, elucidation of the molecular mechanisms underlying the coupling of transport to ATP binding and hydrolysis, and further development of genetic and drug based therapies for Stargardt and other ABCA4 associated diseases.


This work was supported by grants from the Canadian Institutes of Health Research (MT 5822), National Eye Institute (EY02422) and a grant from the Macula Vision Research Foundation.


Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


1. Paulusma CC, Oude Elferink RP. Diseases of intramembranous lipid transport. FEBS Lett. 2006;580:5500–5509. [PubMed]
2. Higgins CF. ABC transporters: from microorganisms to man. Annu Rev Cell Biol. 1992;8:67–113. [PubMed]
3. Dean M, Allikmets R. Complete characterization of the human ABC gene family. J Bioenerg Biomembr. 2001;33:475–479. [PubMed]
4. Borst P, Zelcer N, van Helvoort A. ABC transporters in lipid transport. Biochim Biophys Acta. 2000;1486:128–144. [PubMed]
5. Kaminski WE, Piehler A, Wenzel JJ. ABC A-subfamily transporters: structure, function and disease. Biochim Biophys Acta. 2006;1762:510–524. [PubMed]
6. Mosser J, Douar AM, Sarde CO, Kioschis P, Feil R, Moser H, Poustka AM, Mandel JL, Aubourg P. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature. 1993;361:726–730. [PubMed]
7. Berge KE, Tian H, Graf GA, Yu L, Grishin NV, Schultz J, Kwiterovich P, Shan B, Barnes R, Hobbs HH. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters. Science. 2000;290:1771–1775. [PubMed]
8. Brooks-Wilson A, Marcil M, Clee SM, Zhang LH, Roomp K, van Dam M, Yu L, Brewer C, Collins JA, Molhuizen HO, Loubser O, Ouelette BF, Fichter K, Ashbourne-Excoffon KJ, Sensen CW, Scherer S, Mott S, Denis M, Martindale D, Frohlich J, Morgan K, Koop B, Pimstone S, Kastelein JJ, Hayden MR, et al. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency. Nat Genet. 1999;22:336–345. [PubMed]
9. de Vree JM, Jacquemin E, Sturm E, Cresteil D, Bosma PJ, Aten J, Deleuze JF, Desrochers M, Burdelski M, Bernard O, Oude Elferink RP, Hadchouel M. Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proc Natl Acad Sci U S A. 1998;95:282–287. [PubMed]
10. Jacquemin E, Cresteil D, Manouvrier S, Boute O, Hadchouel M. Heterozygous non-sense mutation of the MDR3 gene in familial intrahepatic cholestasis of pregnancy. Lancet. 1999;353:210–211. [PubMed]
11. Dean M, Annilo T. Evolution of the ATP-binding cassette (ABC) transporter superfamily in vertebrates. Annu Rev Genomics Hum Genet. 2005;6:123–142. [PubMed]
12. Chen ZQ, Annilo T, Shulenin S, Dean M. Three ATP-binding cassette transporter genes, Abca14, Abca15, and Abca16, form a cluster on mouse Chromosome 7F3. Mamm Genome. 2004;15:335–343. [PubMed]
13. Peelman F, Labeur C, Vanloo B, Roosbeek S, Devaud C, Duverger N, Denefle P, Rosier M, Vandekerckhove J, Rosseneu M. Characterization of the ABCA transporter subfamily: identification of prokaryotic and eukaryotic members, phylogeny and topology. J Mol Biol. 2003;325:259–274. [PubMed]
14. Illing M, Molday LL, Molday RS. The 220-kDa rim protein of retinal rod outer segments is a member of the ABC transporter superfamily. J Biol Chem. 1997;272:10303–10310. [PubMed]
15. Bungert S, Molday LL, Molday RS. Membrane topology of the ATP binding cassette transporter ABCR and its relationship to ABC1 and related ABCA transporters: identification of N- linked glycosylation sites. J Biol Chem. 2001;276:23539–23546. [PubMed]
16. Oram JF. ATP-binding cassette transporter A1 and cholesterol trafficking. Curr Opin Lipidol. 2002;13:373–381. [PubMed]
17. Bodzioch M, Orso E, Klucken J, Langmann T, Bottcher A, Diederich W, Drobnik W, Barlage S, Buchler C, Porsch-Ozcurumez M, Kaminski WE, Hahmann HW, Oette K, Rothe G, Aslanidis C, Lackner KJ, Schmitz G. The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease. Nat Genet. 1999;22:347–351. [PubMed]
18. Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette JC, Deleuze JF, Brewer HB, Duverger N, Denefle P, Assmann G. Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1. Nat Genet. 1999;22:352–355. [PubMed]
19. Oram JF. ATP-binding cassette transporter A1 and cholesterol trafficking. Curr Opin Lipidol. 2002;13:373–381. [PubMed]
20. Ban N, Matsumura Y, Sakai H, Takanezawa Y, Sasaki M, Arai H, Inagaki N. ABCA3 as a lipid transporter in pulmonary surfactant biogenesis. J Biol Chem. 2007;282:9628–9634. [PubMed]
21. Shulenin S, Nogee LM, Annilo T, Wert SE, Whitsett JA, Dean M. ABCA3 gene mutations in newborns with fatal surfactant deficiency. N Engl J Med. 2004;350:1296–1303. [PubMed]
22. Yamano G, Funahashi H, Kawanami O, Zhao LX, Ban N, Uchida Y, Morohoshi T, Ogawa J, Shioda S, Inagaki N. ABCA3 is a lamellar body membrane protein in human lung alveolar type II cells. FEBS Lett. 2001;508:221–225. [PubMed]
23. Lefevre C, Audebert S, Jobard F, Bouadjar B, Lakhdar H, Boughdene-Stambouli O, Blanchet-Bardon C, Heilig R, Foglio M, Weissenbach J, Lathrop M, Prud'homme JF, Fischer J. Mutations in the transporter ABCA12 are associated with lamellar ichthyosis type 2. Hum Mol Genet. 2003;12:2369–2378. [PubMed]
24. Akiyama M, Sugiyama-Nakagiri Y, Sakai K, McMillan JR, Goto M, Arita K, Tsuji-Abe Y, Tabata N, Matsuoka K, Sasaki R, Sawamura D, Shimizu H. Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer. J Clin Invest. 2005;115:1777–1784. [PMC free article] [PubMed]
25. Annilo T, Shulenin S, Chen ZQ, Arnould I, Prades C, Lemoine C, Maintoux-Larois C, Devaud C, Dean M, Denefle P, Rosier M. Identification and characterization of a novel ABCA subfamily member, ABCA12, located in the lamellar ichthyosis region on 2q34. Cytogenet Genome Res. 2002;98:169–176. [PubMed]
26. Azarian SM, Travis GH. The photoreceptor rim protein is an ABC transporter encoded by the gene for recessive Stargardt's disease (ABCR) FEBS Lett. 1997;409:247–252. [PubMed]
27. Papermaster DS, Schneider BG, Zorn MA, Kraehenbuhl JP. Immunocytochemical localization of a large intrinsic membrane protein to the incisures and margins of frog rod outer segment disks. J Cell Biol. 1978;78:415–425. [PMC free article] [PubMed]
28. Allikmets R, Shroyer NF, Singh N, Seddon JM, Lewis RA, Bernstein PS, Peiffer A, Zabriskie NA, Li Y, Hutchinson A, Dean M, Lupski JR, Leppert M. Mutation of the Stargardt disease gene (ABCR) in age-related macular degeneration. Science. 1997;277:1805–1807. [PubMed]
29. Allikmets R, Singh N, Sun H, Shroyer NF, Hutchinson A, Chidambaram A, Gerrard B, Baird L, Stauffer D, Peiffer A, Rattner A, Smallwood P, Li Y, Anderson KL, Lewis RA, Nathans J, Leppert M, Dean M, Lupski JR. A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy [see comments] Nat Genet. 1997;15:236–246. [PubMed]
30. Nasonkin I, Illing M, Koehler MR, Schmid M, Molday RS, Weber BH. Mapping of the rod photoreceptor ABC transporter (ABCR) to 1p21-p22.1 and identification of novel mutations in Stargardt's disease. Hum Genet. 1998;102:21–26. [PubMed]
31. Sun H, Nathans J. Stargardt's ABCR is localized to the disc membrane of retinal rod outer segments [letter] Nat Genet. 1997;17:15–16. [PubMed]
32. Molday LL, Rabin AR, Molday RS. ABCR expression in foveal cone photoreceptors and its role in Stargardt macular dystrophy. Nat Genet. 2000;25:257–258. [PubMed]
33. Papermaster DS, Reilly P, Schneider BG. Cone lamellae and red and green rod outer segment disks contain a large intrinsic membrane protein on their margins: an ultrastructural immunocytochemical study of frog retinas. Vision Res. 1982;22:1417–1428. [PubMed]
34. Bhongsatiern J, Ohtsuki S, Tachikawa M, Hori S, Terasaki T. Retinal-specific ATP-binding cassette transporter (ABCR/ABCA4) is expressed at the choroid plexus in rat brain. J Neurochem. 2005;92:1277–1280. [PubMed]
35. Sun H, Smallwood PM, Nathans J. Biochemical defects in ABCR protein variants associated with human retinopathies. Nat Genet. 2000;26:242–246. [PubMed]
36. Ahn J, Wong JT, Molday RS. The effect of lipid environment and retinoids on the ATPase activity of ABCR, the photoreceptor ABC transporter responsible for Stargardt macular dystrophy. J Biol Chem. 2000;275:20399–20405. [PubMed]
37. Ahn J, Beharry S, Molday LL, Molday RS. Functional interaction between the two halves of the photoreceptor-specific ATP binding cassette protein ABCR (ABCA4). Evidence for a non-exchangeable ADP in the first nucleotide binding domain. J Biol Chem. 2003;278:39600–39608. [PubMed]
38. Zhong M, Molday LL, Molday RS. Role of the C-terminus of the photoreceptor ABCA4 transporter in protein folding, function and retinal degenerative diseases. J Biol Chem. 2008 [PMC free article] [PubMed]
39. Goldberg AF, Moritz OL, Molday RS. Heterologous expression of photoreceptor peripherin/rds and Rom-1 in COS-1 cells: assembly, interactions, and localization of multisubunit complexes. Biochemistry. 1995;34:14213–14219. [PubMed]
40. Moritz OL, Molday RS. Molecular cloning, membrane topology, and localization of bovine rom-1 in rod and cone photoreceptor cells. Invest Ophthalmol Vis Sci. 1996;37:352–362. [PubMed]
41. Molday RS, Hicks D, Molday L. Peripherin. A rim-specific membrane protein of rod outer segment discs. Invest Ophthalmol Vis Sci. 1987;28:50–61. [PubMed]
42. Fitzgerald ML, Morris AL, Rhee JS, Andersson LP, Mendez AJ, Freeman MW. Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I. J Biol Chem. 2002;277:33178–33187. [PubMed]
43. Chroni A, Liu T, Fitzgerald ML, Freeman MW, Zannis VI. Cross-linking and lipid efflux properties of apoA-I mutants suggest direct association between apoA-I helices and ABCA1. Biochemistry. 2004;43:2126–2139. [PubMed]
44. Fitzgerald ML, Okuhira K, Short GF, 3rd, Manning JJ, Bell SA, Freeman MW. ATP-binding cassette transporter A1 contains a novel C-terminal VFVNFA motif that is required for its cholesterol efflux and ApoA-I binding activities. J Biol Chem. 2004;279:48477–48485. [PubMed]
45. Vetter IR, Wittinghofer A. Nucleoside triphosphate-binding proteins: different scaffolds to achieve phosphoryl transfer. Q Rev Biophys. 1999;32:1–56. [PubMed]
46. Linton KJ, Higgins CF. Structure and function of ABC transporters: the ATP switch provides flexible control. Pflugers Arch. 2007;453:555–567. [PubMed]
47. Kim IW, Peng XH, Sauna ZE, FitzGerald PC, Xia D, Muller M, Nandigama K, Ambudkar SV. The conserved tyrosine residues 401 and 1044 in ATP sites of human P-glycoprotein are critical for ATP binding and hydrolysis: evidence for a conserved subdomain, the A-loop in the ATP-binding cassette. Biochemistry. 2006;45:7605–7616. [PubMed]
48. Oswald C, Holland IB, Schmitt L. The motor domains of ABC-transporters. What can structures tell us? Naunyn Schmiedebergs Arch Pharmacol. 2006;372:385–399. [PubMed]
49. Schmitt L, Benabdelhak H, Blight MA, Holland IB, Stubbs MT. Crystal structure of the nucleotide-binding domain of the ABC-transporter haemolysin B: identification of a variable region within ABC helical domains. J Mol Biol. 2003;330:333–342. [PubMed]
50. Jones PM, George AM. Subunit interactions in ABC transporters: towards a functional architecture. FEMS Microbiol Lett. 1999;179:187–202. [PubMed]
51. Nikaido H. How are the ABC transporters energized? Proc Natl Acad Sci U S A. 2002;99:9609–9610. [PubMed]
52. Ambudkar SV, Lelong IH, Zhang J, Cardarelli CO, Gottesman MM, Pastan I. Partial purification and reconstitution of the human multidrug-resistance pump: characterization of the drug-stimulatable ATP hydrolysis. Proc Natl Acad Sci U S A. 1992;89:8472–8476. [PubMed]
53. Urbatsch IL, al-Shawi MK, Senior AE. Characterization of the ATPase activity of purified Chinese hamster P-glycoprotein. Biochemistry. 1994;33:7069–7076. [PubMed]
54. Shapiro AB, Ling V. ATPase activity of purified and reconstituted P-glycoprotein from Chinese hamster ovary cells. J Biol Chem. 1994;269:3745–3754. [PubMed]
55. Sun H, Molday RS, Nathans J. Retinal stimulates ATP hydrolysis by purified and reconstituted ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt disease. J Biol Chem. 1999;274:8269–8281. [PubMed]
56. Biswas EE. Nucleotide binding domain 1 of the human retinal ABC transporter functions as a general ribonucleotidase. Biochemistry. 2001;40:8181–8187. [PubMed]
57. Biswas EE, Biswas SB. The C-terminal nucleotide binding domain of the human retinal ABCR protein is an adenosine triphosphatase. Biochemistry. 2000;39:15879–15886. [PubMed]
58. Poincelot RP, Millar PG, Kimbel RL, Jr, Abrahamson EW. Lipid to protein chromophore transfer in the photolysis of visual pigments. Nature. 1969;221:256–257. [PubMed]
59. Anderson RE, Maude MB. Phospholipids of bovine outer segments. Biochemistry. 1970;9:3624–3628. [PubMed]
60. Gottesman MM, Hrycyna CA, Schoenlein PV, Germann UA, Pastan I. Genetic analysis of the multidrug transporter. Annu Rev Genet. 1995;29:607–649. [PubMed]
61. Zhou Z, White KA, Polissi A, Georgopoulos C, Raetz CR. Function of Escherichia coli MsbA, an essential ABC family transporter, in lipid A and phospholipid biosynthesis. J Biol Chem. 1998;273:12466–12475. [PubMed]
62. van Helvoort A, Smith AJ, Sprong H, Fritzsche I, Schinkel AH, Borst P, van Meer G. MDR1 P-glycoprotein is a lipid translocase of broad specificity, while MDR3 P-glycoprotein specifically translocates phosphatidylcholine. Cell. 1996;87:507–517. [PubMed]
63. Ruetz S, Gros P. Phosphatidylcholine translocase: a physiological role for the mdr2 gene. Cell. 1994;77:1071–1081. [PubMed]
64. Beharry S, Zhong M, Molday RS. N-retinylidene-phosphatidylethanolamine is the preferred retinoid substrate for the photoreceptor-specific ABC transporter ABCA4 (ABCR) J Biol Chem. 2004;279:53972–53979. [PubMed]
65. Linton KJ. Structure and function of ABC transporters. Physiology (Bethesda) 2007;22:122–130. [PubMed]
66. Locher KP. Review. Structure and mechanism of ATP-binding cassette transporters. Philos Trans R Soc Lond B Biol Sci. 2009;364:239–245. [PMC free article] [PubMed]
67. Hollenstein K, Dawson RJ, Locher KP. Structure and mechanism of ABC transporter proteins. Curr Opin Struct Biol. 2007;17:412–418. [PubMed]
68. Higgins CF, Linton KJ. The ATP switch model for ABC transporters. Nat Struct Mol Biol. 2004;11:918–926. [PubMed]
69. Molday RS. ATP-binding cassette transporter ABCA4: molecular properties and role in vision and macular degeneration. J Bioenerg Biomembr. 2007;39:507–517. [PubMed]
70. Weng J, Mata NL, Azarian SM, Tzekov RT, Birch DG. Travis, Insights into the function of rim protein in photoreceptors and etiology of Stargardt's Disease from the phenotype in abcr knockout mice. Cell. 1999;98:13–23. [PubMed]
71. Mata NL, Weng J, Travis GH. Biosynthesis of a major lipofuscin fluorophore in mice and humans with ABCR-mediated retinal and macular degeneration. Proc Natl Acad Sci U S A. 2000;97:7154–7159. [PubMed]
72. Mata NL, Tzekov RT, Liu X, Weng J, Birch DG, Travis GH. Delayed dark-adaptation and lipofuscin accumulation in abcr+/− mice: implications for involvement of ABCR in age-related macular degeneration. Invest Ophthalmol Vis Sci. 2001;42:1685–1690. [PubMed]
73. Pawar AS, Qtaishat NM, Little DM, Pepperberg DR. Recovery of rod photoresponses in ABCR-deficient mice. Invest Ophthalmol Vis Sci. 2008;49:2743–2755. [PMC free article] [PubMed]
74. Maeda A, Maeda T, Golczak M, Palczewski K. Retinopathy in mice induced by disrupted all-trans-retinal clearance. J Biol Chem. 2008;283:26684–26693. [PMC free article] [PubMed]
75. Eldred GE, Lasky MR. Retinal age pigments generated by self-assembling lysosomotropic detergents. Nature. 1993;361:724–726. [PubMed]
76. Ben-Shabat S, Parish CA, Vollmer HR, Itagaki Y, Fishkin N, Nakanishi K, Sparrow JR. Biosynthetic studies of A2E, a major fluorophore of retinal pigment epithelial lipofuscin. J Biol Chem. 2002;277:7183–7190. [PubMed]
77. Sparrow JR, Boulton M. RPE lipofuscin and its role in retinal pathobiology. Exp Eye Res. 2005;80:595–606. [PubMed]
78. Sparrow JR, Nakanishi K, Parish CA. The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells. Invest Ophthalmol Vis Sci. 2000;41:1981–1989. [PubMed]
79. Radu RA, Mata NL, Bagla A, Travis GH. Light exposure stimulates formation of A2E oxiranes in a mouse model of Stargardt's macular degeneration. Proc Natl Acad Sci U S A. 2004;101:5928–5933. [PubMed]
80. Kim SR, Jang YP, Jockusch S, Fishkin NE, Turro NJ, Sparrow JR. The all-trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model. Proc Natl Acad Sci U S A. 2007;104:19273–19278. [PubMed]
81. Stargardt K. Uber familiare, progressive degeenration under makulagegend des augen. Albrecht von Graefes Arch Ophthalmol. 1909;71:534–550.
82. Blacharski PA. Fundu flavimaculatus. In: Newsome DA, editor. Retinal dystrophies and degenerations. New York: Raven Press; 1988. pp. 135–159.
83. Weleber RG. Stargardt's macular dystrophy. Arch Ophthalmol. 1994;112:752–754. [PubMed]
84. Fishman GA, Farbman JS, Alexander KR. Delayed rod dark adaptation in patients with Stargardt's disease. Ophthalmology. 1991;98:957–962. [PubMed]
85. Lois N, Halfyard AS, Bird AC, Holder GE, Fitzke FW. Fundus autofluorescence in Stargardt macular dystrophy-fundus flavimaculatus. Am J Ophthalmol. 2004;138:55–63. [PubMed]
86. Delori FC, Staurenghi G, Arend O, Dorey CK, Goger DG, Weiter JJ. In vivo measurement of lipofuscin in Stargardt's disease--Fundus flavimaculatus. Invest Ophthalmol Vis Sci. 1995;36:2327–2331. [PubMed]
87. Cideciyan AV, Aleman TS, Swider M, Schwartz SB, Steinberg JD, Brucker AJ, Maguire AM, Bennett J, Stone EM, Jacobson SG. Mutations in ABCA4 result in accumulation of lipofuscin before slowing of the retinoid cycle: a reappraisal of the human disease sequence. Hum Mol Genet. 2004;13:525–534. [PubMed]
88. Noble KG, Carr RE. Stargardt's disease and fundus flavimaculatus. Arch Ophthalmol. 1979;97:1281–1285. [PubMed]
89. Gelisken O, De Laey JJ. A clinical review of Stargardt's disease and/or fundus flavimaculatus with follow-up. Int Ophthalmol. 1985;8:225–235. [PubMed]
90. Lewis RA, Shroyer NF, Singh N, Allikmets R, Hutchinson A, Li Y, Lupski JR, Leppert M, Dean M. Genotype/Phenotype analysis of a photoreceptor-specific ATP-binding cassette transporter gene, ABCR, in Stargardt disease. Am J Hum Genet. 1999;64:422–434. [PubMed]
91. Maugeri A, Klevering BJ, Rohrschneider K, Blankenagel A, Brunner HG, Deutman AF, Hoyng CB, Cremers FP. Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-Rod dystrophy. Am J Hum Genet. 2000;67:960–966. [PubMed]
92. Maugeri A, van Driel MA, van de Pol DJ, Klevering BJ, van Haren FJ, Tijmes N, Bergen AA, Rohrschneider K, Blankenagel A, Pinckers AJ, Dahl N, Brunner HG, Deutman AF, Hoyng CB, Cremers FP. The 2588G-->C mutation in the ABCR gene is a mild frequent founder mutation in the Western European population and allows the classification of ABCR mutations in patients with Stargardt disease. Am J Hum Genet. 1999;64:1024–1035. [PubMed]
93. Rivera A, White K, Stohr H, Steiner K, Hemmrich N, Grimm T, Jurklies B, Lorenz B, Scholl HP, Apfelstedt-Sylla E, Weber BH. A comprehensive survey of sequence variation in the ABCA4 (ABCR) gene in Stargardt disease and age-related macular degeneration. Am J Hum Genet. 2000;67:800–813. [PubMed]
94. Rozet JM, Gerber S, Souied E, Ducroq D, Perrault I, Ghazi I, Soubrane G, Coscas G, Dufier JL, Kaplan J. The ABCR gene: a major disease gene in macular and peripheral retinal degenerations with onset from early childhood to the elderly. Mol Genet Metab. 1999;68:310–315. [PubMed]
95. Rozet JM, Gerber S, Souied E, Perrault I, Chatelin S, Ghazi I, Leowski C, Dufier JL, Munnich A, Kaplan J. Spectrum of ABCR gene mutations in autosomal recessive macular dystrophies. Eur J Hum Genet. 1998;6:291–295. [PubMed]
96. Webster AR, Heon E, Lotery AJ, Vandenburgh K, Casavant TL, Oh KT, Beck G, Fishman GA, Lam BL, Levin A, Levin A, Heckenlively JR, Jacobson SG, Weleber RG, Sheffield VC, Stone EM. An analysis of allelic variation in the ABCA4 gene. Invest Ophthalmol Vis Sci. 2001;42:1179–1189. [PubMed]
97. Stone EM, Webster AR, Vandenburgh K, Streb LM, Hockey RR, Lotery AJ, Sheffield VC. Allelic variation in ABCR associated with Stargardt disease but not age-related macular degeneration. Nat Genet. 1998;20:328–329. [PubMed]
98. Briggs CE, Rucinski D, Rosenfeld PJ, Hirose T, Berson EL, Dryja TP. Mutations in ABCR (ABCA4) in patients with Stargardt macular degeneration or cone-rod degeneration. Invest Ophthalmol Vis Sci. 2001;42:2229–2236. [PubMed]
99. Stenirri S, Battistella S, Fermo I, Manitto MP, Martina E, Brancato R, Ferrari M, Cremonesi L. De novo deletion removes a conserved motif in the C-terminus of ABCA4 and results in cone-rod dystrophy. Clin Chem Lab Med. 2006;44:533–537. [PubMed]
100. Fishman GA, Stone EM, Grover S, Derlacki DJ, Haines HL, Hockey RR. Variation of clinical expression in patients with Stargardt dystrophy and sequence variations in the ABCR gene. Arch Ophthalmol. 1999;117:504–510. [PubMed]
101. Allikmets R. Simple and complex ABCR: genetic predisposition to retinal disease. Am J Hum Genet. 2000;67:793–799. [PubMed]
102. Cremers FP, van de Pol DJ, van Driel M, den Hollander AI, van Haren FJ, Knoers NV, Tijmes N, Bergen AA, Rohrschneider K, Blankenagel A, Pinckers AJ, Deutman AF, Hoyng CB. Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt's disease gene ABCR. Hum Mol Genet. 1998;7:355–362. [PubMed]
103. Martinez-Mir A, Paloma E, Allikmets R, Ayuso C, del Rio T, Dean M, Vilageliu L, Gonzalez-Duarte R, Balcells S. Retinitis pigmentosa caused by a homozygous mutation in the Stargardt disease gene ABCR. Nat Genet. 1998;18:11–12. [PubMed]
104. van Driel MA, Maugeri A, Klevering BJ, Hoyng CB, Cremers FP. ABCR unites what ophthalmologists divide(s) Ophthalmic Genet. 1998;19:117–122. [PubMed]
105. Shroyer NF, Lewis RA, Allikmets R, Singh N, Dean M, Leppert M, Lupski JR. The rod photoreceptor ATP-binding cassette transporter gene, ABCR, and retinal disease: from monogenic to multifactorial. Vision Res. 1999;39:2537–2544. [PubMed]
106. Cideciyan AV, Swider M, Aleman TS, Tsybovsky Y, Schwartz SB, Windsor EA, Roman AJ, Sumaroka A, Steinberg JD, Jacobson SG, Stone EM, Palczewski K. ABCA4 Disease Progression and a Proposed Strategy for Gene Therapy. Hum Mol Genet. 2008 [PMC free article] [PubMed]
107. Wiszniewski W, Zaremba CM, Yatsenko AN, Jamrich M, Wensel TG, Lewis RA, Lupski JR. ABCA4 mutations causing mislocalization are found frequently in patients with severe retinal dystrophies. Hum Mol Genet. 2005;14:2769–2778. [PubMed]
108. Fumagalli A, Ferrari M, Soriani N, Gessi A, Foglieni B, Martina E, Manitto MP, Brancato R, Dean M, Allikmets R, Cremonesi L. Mutational scanning of the ABCR gene with double-gradient denaturing-gradient gel electrophoresis (DG-DGGE) in Italian Stargardt disease patients. Hum Genet. 2001;109:326–338. [PubMed]
109. Saari JC. Biochemistry of visual pigment regeneration: the Friedenwald lecture. Invest Ophthalmol Vis Sci. 2000;41:337–348. [PubMed]
110. Travis GH, Golczak M, Moise AR, Palczewski K. Diseases caused by defects in the visual cycle: retinoids as potential therapeutic agents. Annu Rev Pharmacol Toxicol. 2007;47:469–512. [PMC free article] [PubMed]
111. Chen C, Jiang Y, Koutalos Y. Dynamic behavior of rod photoreceptor disks. Biophys J. 2002;83:1403–1412. [PubMed]
112. Holz FG, Schutt F, Kopitz J, Eldred GE, Kruse FE, Volcker HE, Cantz M. Inhibition of lysosomal degradative functions in RPE cells by a retinoid component of lipofuscin. Invest Ophthalmol Vis Sci. 1999;40:737–743. [PubMed]
113. Zhou J, Jang YP, Kim SR, Sparrow JR. Complement activation by photooxidation products of A2E, a lipofuscin constituent of the retinal pigment epithelium. Proc Natl Acad Sci U S A. 2006;103:16182–16187. [PubMed]
114. Dinculescu A, Glushakova L, Min SH, Hauswirth WW. Adeno-associated virus-vectored gene therapy for retinal disease. Hum Gene Ther. 2005;16:649–663. [PubMed]
115. Pang JJ, Chang B, Kumar A, Nusinowitz S, Noorwez SM, Li J, Rani A, Foster TC, Chiodo VA, Doyle T, Li H, Malhotra R, Teusner JT, McDowell JH, Min SH, Li Q, Kaushal S, Hauswirth WW. Gene therapy restores vision-dependent behavior as well as retinal structure and function in a mouse model of RPE65 Leber congenital amaurosis. Mol Ther. 2006;13:565–572. [PubMed]
116. Min SH, Molday LL, Seeliger MW, Dinculescu A, Timmers AM, Janssen A, Tonagel F, Tanimoto N, Weber BH, Molday RS, Hauswirth WW. Prolonged recovery of retinal structure/function after gene therapy in an Rs1h-deficient mouse model of x-linked juvenile retinoschisis. Mol Ther. 2005;12:644–651. [PubMed]
117. Kootstra NA, Verma IM. Gene therapy with viral vectors. Annu Rev Pharmacol Toxicol. 2003;43:413–439. [PubMed]
118. Acland GM, Aguirre GD, Ray J, Zhang Q, Aleman TS, Cideciyan AV, Pearce-Kelling SE, Anand V, Zeng Y, Maguire AM, Jacobson SG, Hauswirth WW, Bennett J. Gene therapy restores vision in a canine model of childhood blindness. Nat Genet. 2001;28:92–95. [PubMed]
119. Cideciyan AV, Aleman TS, Boye SL, Schwartz SB, Kaushal S, Roman AJ, Pang JJ, Sumaroka A, Windsor EA, Wilson JM, Flotte TR, Fishman GA, Heon E, Stone EM, Byrne BJ, Jacobson SG, Hauswirth WW. Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics. Proc Natl Acad Sci U S A. 2008;105:15112–15117. [PubMed]
120. Maguire AM, Simonelli F, Pierce EA, Pugh EN, Jr, Mingozzi F, Bennicelli J, Banfi S, Marshall KA, Testa F, Surace EM, Rossi S, Lyubarsky A, Arruda VR, Konkle B, Stone E, Sun J, Jacobs J, Dell'Osso L, Hertle R, Ma JX, Redmond TM, Zhu X, Hauck B, Zelenaia O, Shindler KS, Maguire MG, Wright JF, Volpe NJ, McDonnell JW, Auricchio A, High KA, Bennett J. Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med. 2008;358:2240–2248. [PMC free article] [PubMed]
121. Bainbridge JW, Smith AJ, Barker SS, Robbie S, Henderson R, Balaggan K, Viswanathan A, Holder GE, Stockman A, Tyler N, Petersen-Jones S, Bhattacharya SS, Thrasher AJ, Fitzke FW, Carter BJ, Rubin GS, Moore AT, Ali RR. Effect of gene therapy on visual function in Leber's congenital amaurosis. N Engl J Med. 2008;358:2231–2239. [PubMed]
122. Allocca M, Doria M, Petrillo M, Colella P, Garcia-Hoyos M, Gibbs D, Kim SR, Maguire A, Rex TS, Di Vicino U, Cutillo L, Sparrow JR, Williams DS, Bennett J, Auricchio A. Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice. J Clin Invest. 2008;118:1955–1964. [PubMed]
123. Kong J, Kim SR, Binley K, Pata I, Doi K, Mannik J, Zernant-Rajang J, Kan O, Iqball S, Naylor S, Sparrow JR, Gouras P, Allikmets R. Correction of the disease phenotype in the mouse model of Stargardt disease by lentiviral gene therapy. Gene Ther. 2008;15:1311–1320. [PMC free article] [PubMed]
124. Radu RA, Mata NL, Nusinowitz S, Liu X, Sieving PA, Travis GH. Treatment with isotretinoin inhibits lipofuscin accumulation in a mouse model of recessive Stargardt's macular degeneration. Proc Natl Acad Sci U S A. 2003;100:4742–4747. [PubMed]
125. Radu RA, Han Y, Bui TV, Nusinowitz S, Bok D, Lichter J, Widder K, Travis GH, Mata NL. Reductions in serum vitamin A arrest accumulation of toxic retinal fluorophores: a potential therapy for treatment of lipofuscin-based retinal diseases. Invest Ophthalmol Vis Sci. 2005;46:4393–4401. [PubMed]
126. Golczak M, Kuksa V, Maeda T, Moise AR, Palczewski K. Positively charged retinoids are potent and selective inhibitors of the trans-cis isomerization in the retinoid (visual) cycle. Proc Natl Acad Sci U S A. 2005;102:8162–8167. [PubMed]