Interphotoreceptor retinoid-binding protein is the physiologically relevant carrier that removes retinol from rod photoreceptor outer segments.

The vertebrate cells responsible for vision are the rod and cone photoreceptors of the retina that convert incoming light to an electrical signal. This conversion takes place in the photoreceptor outer segments, which are full of membrane disks containing the visual pigment, and, in a physiologically important arrangement, are enveloped by the retinal pigment epithelium (RPE). The visual pigment is composed of a chromophore, 11-cis retinal, attached to an integral membrane protein, opsin. The detection of light begins with the absorption of incoming photons by the visual pigment. An absorbed photon isomerizes the chromophore moiety from 11-cis to all-trans bringing about a conformational change that initiates a cascade of reactions culminating in membrane potential change. The recovery of the cell from light involves the deactivation of the intermediates activated by light, and the reestablishment of membrane potential (1, 2). However, the isomerized chromophore, all-trans retinal, remains. For vision to be possible, it is essential that the visual pigment regenerate: that is, the all-trans retinal has to be removed, and fresh 11-cis retinal has to be provided to combine with opsin and reform the visual pigment. The reactions regenerating the pigment are known as the Visual Cycle (3–5). In the case of the rod photoreceptors, the cells responsible for vision at low light intensities, the Visual Cycle encompasses reactions in the outer segment and in the adjacent RPE cells. The first step in the Cycle is the release of all-trans retinal from photoactivated rhodopsin after hydrolysis of the Schiff base bond linking the chromophore to opsin. All-trans retinal is then reduced to all-trans retinol in a reaction catalyzed by retinol dehydrogenase (6, 7), requiring NADPH, and taking place on the cytoplasmic side of the outer segment disks. It is possible that all-trans retinal ends up inside the disks, bound via a Schiff base to phosphatidylethanolamine, in which case the phosphatidylethanolamine-all-trans-retinal compound is transported to the cytoplasmic side by the ABCR transporter (8–10) making all-trans retinal available for reduction. The all-trans retinol formed in the rod outer segment is transported to the RPE, in a process that can be facilitated by the interphotoreceptor retinoid binding protein (IRBP; (11–13)). In the RPE, retinol is converted by lecithin-retinol acyltransferase to retinyl ester (LRAT; (14, 15)), which is isomerized to 11-cis retinol (16, 17) by the RPE65 protein (18–21). 11-cis retinol is then oxidized to 11-cis retinal, and transported back to photoreceptor outer segments where it associates with opsin to reform rhodopsin. Previous work (22–25) has established that all-trans retinol can be monitored in the outer segments of living isolated rod and cone photoreceptors from its distinctive fluorescence. Here, we have taken advantage of two properties of frog rod photoreceptors to actually measure the amounts of all-trans retinol produced with quantitative biochemical and physiological methods. One, in contrast to larval salamander photoreceptors that contain two types of chromophore (based on vitamins A1 and A2) and in widely varying ratios (24), frog rods contain a single, vitamin A1-based chromophore. Two, the metabolic supply of NADPH is not limiting for the formation of all-trans retinol in the case of frog rods (23), allowing a simplified analysis and direct comparisons between biochemical and physiological data. This has further allowed us to properly characterize the removal of all-trans retinol by different lipophilic carriers. In experiments with purified rod outer segment membranes, whole retinas, and living isolated rods, we have separately measured the different steps involved in all-trans retinol formation and removal. On the basis of these measurements, we have calculated the predicted kinetics of retinol formation and removal in rod outer segments, and found that they are in close agreement with those measured directly from isolated rod photoreceptors. We also characterized the effect of different concentrations of lipophilic carriers on the removal of all-trans retinol, and established that for the physiological concentrations of carriers the rate of all-trans retinol removal is determined by the IRBP concentration. Our results strongly support a specific interaction mediating the removal of all-trans retinol by IRBP, perhaps through a receptor on the rod outer segment plasma membrane. Throughout the text, when not specifically designated as the 11-cis isomers, unqualified retinal and retinol refer to the all-trans forms.