Receptor retinoid‐binding protein (IRBP) binds cone outer segments differentially in light and dark

Abstract

Abstract Purpose Vision requires the continuous exchanges of visual cycle retinoids through the interphotoreceptor matrix (IPM). IRBP, which is synthesized by the rods and cones, is the major soluble protein component of the IPM. In the matrix, IRBP protects and targets retinoids trafficking between the photoreceptors, RPE and Muller cells. Methods Organ culture of isolated Xenopus retina was used to characterize the cellular binding of recombinant full‐length IRBP protein free of fusion tags. Xenopus was selected for these studies because of their large photoreceptors which are easily detached in both light and dark‐adapted states. xIRBP was covalently bound to the fluorescent dye Alexa‐647 at 1:1 molar ratio. Labeling had no effect on the retinoid ligand‐binding properties of the protein. Xenopus retinas were detached and incubated with xIRBP‐647 for 1 hour at room temperature in light and dark. The distribution of xIRBP‐647 was studied by confocal microscopy in retinal flat whole mounts and cryosections. Results In light, xIRBP‐647 bound to cone outer segments with lighter labeling of the rod outer segments. Retinas incubated with the xIRBP‐647 in the presence of a 50 fold excess of unlabeled protein showed reduced cone and rod outer segment labeling. In dark, outer segment binding was not appreciated. Positive staining near the level of the external‐limiting membrane was noted in dark but not light. Conclusion The binding of xIRBP‐647 to cells of the isolated retina was different in light versus dark. Our data suggests that the direct interaction of IRBP with the outer segments and Muller cells plays an important role in retinoid trafficking in the the visual cycle.