Retinal stimulates ATP hydrolysis by purified and reconstituted ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt disease

Many substrates for P-glycoprotein, an ABC transporter that mediates multid rug resistance in mammalian cells, have been shown to stimulate its ATPase activity in vitro. In the present study, we used this property as a criteri on to search for natural and artificial substrates and/or allosteric regula tors of ABCR, the rod photoreceptor-specific ABC transporter responsible fo r Stargardt disease, an early onset macular degeneration. ABCR was immunoaf finity purified to apparent homogeneity from bovine rod outer segments and reconstituted into liposomes, All-trans-retinal, a candidate ligand, stimul ates the ATPase activity of ABCR 3-4-fold, with a half-maximal effect at 10 -15 mu M. 11-cis- and 13-cis-retinal show similar activity. All-trans-retin al stimulates the ATPase activity of ABCR with Michaelis-Menten behavior in dicative of simple noncooperative binding that is associated with a rate-li miting enzyme-substrate intermediate in the pathway of ATP hydrolysis. Amon g 37 structurally diverse non-retinoid compounds, including nine previously characterized substrates or sensitizers of P-glycoprotein, only four show significant ATPase stimulation when tested at 20 mu M. The dose-response cu rves of these four compounds are indicative of multiple binding sites and/o r modes of interaction with ABCR, Two of these compounds, amiodarone and di gitonin, can act synergistically with all-trans retinal, implying that they interact with a site or sites on ABCR different from the one with which al l-trans-retinal interacts, Unlike retinal, amiodarone appears to interact w ith both free and ATP-bound ABCR, Together with clinical observations on St argardt disease and the localization of ABCR to rod outer segment disc memb ranes, these data suggest that retinoids, and most likely retinal, are the natural substrates for transport by ABCR in rod outer segments. These obser vations have significant implications for understanding the visual cycle an d the pathogenesis of Stargardt disease and for the identification of compo unds that could modify the natural history of Stargardt disease or other re tinopathies associated with impaired ABCR function.