All- Trans Retinol in Rod Photoreceptor Outer Segments Moves Unrestrictedly by Passive Diffusion

Abstract

The visual pigment protein of vertebrate rod photoreceptors, rhodopsin, contains an 11- cis retinyl moiety that is isomerized to all- trans upon light absorption. Subsequently, all- trans retinal is released from the protein and reduced to all- trans retinol, the first step in the recycling of rhodopsin’s chromophore group through the series of reactions that constitute the visual cycle. The concentration of all- trans retinol in photoreceptor outer segments can be monitored from its fluorescence. We have used two-photon excitation (720 nm) of retinol fluorescence and fluorescence recovery after photobleaching to characterize the mobility of all- trans retinol in frog photoreceptor outer segments. Retinol produced after rhodopsin bleaching moved laterally in the disk membrane bilayer with an apparent diffusion coefficient of 2.5 ± 0.3 μm 2 s −1. The diffusion coefficient of exogenously added retinol was 3.2 ± 0.5 μm 2 s −1. These diffusion coefficients are in close agreement with those reported for lipids, suggesting that retinol is not tightly bound to protein sites that would be diffusing much more slowly in the plane of the membrane. In agreement with this interpretation, a fluorescent-labeled C-16 fatty acid diffused laterally with a similar diffusion coefficient, 2.2 ± 0.2 μm 2 s −1. Retinol also moved along the length of the rod outer segment, with an apparent diffusion coefficient of 0.07 ± 0.01 μm 2 s −1, again suggesting that it is not tightly bound to proteins that would confine it to the disks. The axial diffusion coefficient of exogenously added retinol was 0.05 ± 0.01 μm 2 s −1. In agreement with passive diffusion, the rate of axial movement was inversely proportional to the square of the length of the rod outer segment. Diffusion of retinol on the plasma membrane of the outer segment can readily account for the measured value of the axial diffusion coefficient, as the plasma membrane comprises ∼1% of the total outer-segment membrane. The values of both the lateral and axial diffusion coefficients are consistent with most of the all- trans retinol in the outer segments moving unrestricted and not being bound to carrier proteins. Therefore, and in contrast to other steps of the visual cycle, there does not appear to be any specialized processing for all- trans retinol within the rod outer segment.