Rod outer segment retinol formation is independent of Abca4, arrestin, rhodopsin kinase, and rhodopsin palmitylation.

Rhodopsin, the primary light detector of vertebrate rod photoreceptor cells, is composed of a chromophore, 11-cis retinal, which is covalently bound to the apoprotein opsin.1,2 Photon absorption isomerizes the chromophore to all-trans, leading to the generation of an active intermediate, metarhodopsin II, that initiates a cascade of reactions culminating in a change in membrane potential and hence the conversion of light to an electrical signal.3,4 The cell recovery from light stimulation includes the deactivation of metarhodopsin II and of the other intermediates of the reaction cascade; metarhodopsin II is deactivated through its phosphorylation by rhodopsin kinase followed by the binding of arrestin. Necessary for recovery is also the regeneration of rhodopsin that was destroyed by the photoisomerization of its chromophore moiety. The regeneration is achieved through the removal of the all-trans chromophore and the binding of opsin with fresh 11-cis retinal, which is accomplished through a series of reactions called the visual cycle.5–7 The removal of the all-trans chromophore encompasses several steps, beginning with the hydrolysis of the Schiff base bond linking it to opsin, followed by its release from photoactivated rhodopsin as all-trans retinal. The all-trans retinal is subsequently reduced to all-trans retinol by retinol dehydrogenase,8,9 and transferred from the rod outer segment to the adjacent retinal pigment epithelial cells by interphotoreceptor retinoid binding protein (IRBP).10,11 In the retinal pigment epithelium, all-trans retinol is converted to 11-cis retinal,12 which can then be used for the regeneration of rhodopsin. The formation of all-trans retinol achieves the removal of the all-trans retinal released from the photoactivated rhodopsin. all-trans Retinal can form potentially toxic bisretinoid adducts with components of the rod outer segment13 and is also a photosensitizer.14,15 Because all-trans retinal is toxic and is also the substrate for the visual cycle, its processing in the rod outer segment has attracted considerable experimental attention. It has been shown that the binding of arrestin to photoactivated phosphorylated rhodopsin slows down the release of all-trans retinal.16,17 After the release, all-trans retinal can remain noncovalently bound to opsin, to a site formed partly by the two palmitate groups attached to the opsin cysteines 322 and 323.18 The all-trans retinal can also be sequestered inside the discs, bound via a Schiff base to phosphatidylethanolamine, and remain inaccessible to retinol dehydrogenase. The Abca4 transporter protein, present at the rim of the discs, is proposed to transport the phosphatidylethanolamine–all-trans retinal complex to the cytosolic side thereby making all-trans retinal available for reduction.19–21 Defects in the proteins involved in these reactions could result in increased levels of all-trans retinal consequent to light detection, restrict the amounts of available 11-cis retinal, or both. Importantly, such defects have been linked to serious visual deficiencies: Mutations in Abca4 are responsible for Stargardt disease, a form of childhood macular degeneration,22,23 and mutations in arrestin and rhodopsin kinase lead to Oguchi disease, a form of congenital stationary night blindness.24,25 To probe the relevance of these different factors in the clearance of all-trans retinal through its reduction to all-trans retinol, we measured the kinetics of all-trans retinol formation in the outer segments of rod photoreceptors separated from the pigment epithelium. In such a preparation, the all-trans retinol formed after exposure to light cannot be transported away and accumulates in the outer segment membranes. This process allows the examination of the steps involving all-trans retinal and resulting in all-trans retinol formation. We used retinas from wild-type and four types of genetically modified mice, lacking rhodopsin kinase (Rhok−/−), arrestin (Sag−/−), the Abca4 transporter (Abca4−/−), or the palmitate groups of rhodopsin (Palm−/−). That is, they have biochemical defects that can influence either the release of all-trans retinal from photoactivated rhodopsin or its access to retinol dehydrogenase. We measured the formation of all-trans retinol by quantitative HPLC of retinoid extracts and from its fluorescence. For comparison, we separately measured the release of all-trans retinal from photoactivated rhodopsin in purified rod outer segment membranes. We found no substantial differences in the kinetics and levels of all-trans retinol formation between wild-type and the four types of genetically modified mice. The formation of all-trans retinol, however, is much slower than the release of all-trans retinal. It should be emphasized that the conclusions in this article are based solely on the data obtained from isolated retina and cell preparations and not from an intact eye. In the rest of the text, retinal and retinol refer to the all-trans isomers.