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UBC Theses and Dissertations

The role of photoreceptor ABC transporter ABCA4 in retinal and lipid transport and Stargardt macular degeneration Quazi, Faraz


ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) transporters implicated in the clearance of retinoids from photoreceptor outer segments and associated with Stargardt macular degeneration. Stargardt patients display lipofuscin deposits and photoreceptor degeneration that invariably lead to the loss of central vision. In biochemical and knockout mice studies, ABCA4 has been implicated in the transport of N-retinylidene-PE, the Schiff base conjugate formed from all-trans retinal and phosphatidylethanolamine (PE). The principal challenge in assessing direction and transport has been impeded by the unstable hydrophobic nature of the proposed substrate. As part of this study, a novel biochemical transport assay was developed and used to show that ABCA4 actively flips N-retinylidene-PE from the lumen to the cytosolic side of membranes. This is the first mammalian ABC transporter shown to function as an importer. 11-cis retinal delivered in excess to photoreceptors is a defining cause of lipofuscin/A2E formation. Purified ABCA4 transports 11-cis retinylidene-PE. By HPLC analysis, 11-cis retinal also showed isomerization to all-trans retinal with PE present in discs. Thus, ABCA4 insures the complete removal of N-retinylidene-PE conjugates from the disc-lumen, thereby preventing accumulation of lipofuscin precursors including A2PE. Stargardt mutants which showed reduced functionality were rescued by allosteric enhancement of ATPase with dronedarone, an anti-arrhythmic drug, acting in concert with an increase in N-retinylidene-PE transport. Additionally, protein misfolding was selectively restored by 4-phenylbutyrate, a chemical chaperone. Genetic mutations in several ABCA subfamily transporters cause severe-inherited lipid disorders. The structure-function relationships underlying substrate specificity remain unclear. ABCA1 linked to Tangier disease has been implicated in the export of cholesterol and phospholipids to the apolipoproteinA-I acceptor by cell-based assays. In this study, biochemical analysis indicated that purified and reconstituted ABCA1 actively flipped phosphatidylcholine, phosphatidylserine, and sphingomyelin from the cytoplasmic to the lumenal side of membranes, while ABCA7 preferred phosphatidylserine. In contrast, ABCA4 transported PE in the reverse direction. Tangier and Stargardt mutants showed reduced lipid transport activities. This thesis demonstrates the importance of ABCA4 as a unique ABC importer, reports the first reconstitution of phospholipid flippase activity for ABCA transporters, and identifies several compounds as potential therapeutic drugs for Stargardt disease.

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