Formation Of All-Trans Retinol In Mouse Rod Photoreceptors

Abstract

ABCA4 is a member of the superfamily of ATP binding cassette (ABC) proteins that is localized in outer segment disc membranes of rod and cone photoreceptor cells. Mutations in the ABCA4 gene are responsible for Stargardt macular dystrophy, cone-rod dystrophy and retinitis pigmentosa. Biochemical studies together with analysis of abca4 knockout mice implicate ABCA4 in the transport of N-retinylidene-phosphatidylethanolamine across disk membranes. This transport process facilitates the complete removal of retinal derivatives from photoreceptors following the photobleaching of rhodopsin and cone opsin as part of the visual cycle. Loss in the activity of ABCA4 leads to the production of diretinal derivatives in disc membranes which accumulate in adjacent retinal pigment epithelial (RPE) cells as lipofuscin deposits following phagocytosis of outer segments. Progressive buildup of these toxic diretinal compounds causes the degeneration of RPE and photoreceptors and a loss in vision. Recently, we have investigated the effect of C-terminal deletion, including several disease related mutations, on the structural and functional properties of ABCA4. Our studies indicate that ABCA4 contains a conserved motif near the C-terminus that is crucial for proper protein folding and functional activity of ABCA4. Individuals missing this motif due to C-terminal truncation of ABCA4 exhibit a severe form of retinal degeneration known as cone-rod dystrophy. Light detection destroys the visual pigment of vertebrate rod photoreceptors, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. Rhodopsin is regenerated through a series of reactions that begin in the rod outer segment with the release of the all-trans retinal and its reduction to all-trans retinol. All-trans retinol is then transported to the neighboring retinal pigment epithelial cells where it is used to remake 11-cis retinal. The reduction of all-trans retinal to all-trans retinol is catalyzed by retinol dehydrogenase and requires metabolic input in the form of NADPH. We have used the fluorescence of all-trans retinol to monitor its concentration in isolated mouse rod photoreceptors. After the bleaching of rhodopsin, all-trans retinol formation proceeds with a rate of~0.06 min -1 , which is faster than the rate of rhodopsin regeneration in whole animals; this would allow recycled chromophore to contribute to the 11-cis retinal used for regeneration. Inner segment metabolic pathways appear to make a significant contribution to the pool of NADPH needed for the reduction of all-trans retinal, as formation of all-trans retinol is suppressed in rod outer segments separated from the cell body. Finally, generation of all-trans retinol is suppressed in the absence of glucose, indicating a critical dependence of all-trans retinol formation on the level of metabolic activity. The recovery of sensitivity following photopigment bleaching requires the quenching of phototransduction, and the reduction of all-trans retinal is key. Retinol fluorescence increases after ...