Interphotoreceptor retinoid-binding protein as the physiologically relevant carrier of 11-cis-retinol in the cone visual cycle

Abstract

Cones function in constant light and are responsible for mediating daytime human vision. Like rods, cones use the photosensitive molecule 11-cis-retinal to detect light, and in constant illumination, a continuous supply of 11-cis-retinal is needed. A retina visual cycle is thought to provide a privileged supply of 11-cis-retinal to cones by using 11-cis-retinol generated in Müller cells. In the cycle, 11-cis-retinol is transported from Müller cells to cone inner segments, where it is oxidized to 11-cis-retinal. This oxidation step is only performed in cones, thus rendering the cycle cone-specific. Interphotoreceptor retinoid-binding protein (IRBP) is a retinoid-binding protein in the subretinal space that binds 11-cis-retinol endogenously. Cones in Irbp(-/-) mice are retinoid-deficient under photopic conditions, and it is possible that 11-cis-retinol supplies are disrupted in the absence of IRBP. We tested the hypothesis that IRBP facilitates the delivery of 11-cis-retinol to cones by preserving the isomeric state of 11-cis-retinol in light. With electrophysiology, we show that the cone-like photoreceptors of Nrl(-/-) mice use the cone visual cycle similarly to wild-type cones. Then, using oxidation assays in isolated Nrl(-/-)Rpe65(-/-) retinas, we show that IRBP delivers 11-cis-retinol for oxidation in cones and improves the efficiency of the oxidation reaction. Finally, we show that IRBP protects the isomeric state of 11-cis-retinol in the presence of light. Together, these findings suggest that IRBP plays an important role in the delivery of 11-cis-retinol to cones and can facilitate cone function in the presence of light.

Figure 1.

The retinas of mice lacking NRL have a functional cone visual cycle. A, Cone trans-retinal ERG responses from dark-adapted Nrl^−/− retina (left) and bleached retinas followed by different recovery times in darkness. The red traces represent the light responses elicited by 276,511 photons · μm⁻² at 500 nm. B, Kinetics of cone sensitivity recovery. Retinas were exposed to brief bleaching light at time 0. It took 4 h for Nrl^−/− cones to recover their sensitivity to the plateau at ∼50% of its dark-adapted value (black trace, open squares), while WT cones recovered their sensitivity after an identical bleach to the same level within 10 min (blue trace, filled squares). Data were fitted by a single exponential function with τ = 84 min for Nrl^−/− cones and τ = 4.5 min for WT cones. To determine the effects of 11-cis-retinol on the recovery of cone sensitivity, retinas were treated with 100 μm 11-cis-retinol in 0.1% ethanol and incubated at room temperature for 30 min. Exogenous 11-cis-retinol promoted rapid cone dark adaptation (red, open triangle). Sensitivity measurements were normalized to the corresponding dark-adapted value. Error bars indicate SEM; n = 3. C, Post-bleach recovery of Nrl^−/− cone sensitivity was accelerated by exogenous 11-cis-retinol. Cone sensitivity reached the maximum level within 30 min. The red traces represent the photo responses elicited by 276,511 photons · μm⁻² at 500 nm.

Figure 2.

11-cis-Retinol is oxidized in Nrl^−/−Rpe65^−/− retinas. A, HPLC traces of retinoids extracted from six Nrl^−/−Rpe65^−/− (P17) retinas. After incubating (60 min) with IRBP (24 μm) alone, 11-cis-retinal (11-cRAL) was essentially absent. After treating with IRBP (24 μm) and 11-cis-retinol (11-cROL) (24 μm), 11-cis-retinal was generated in the retinas. B, 11-cis-Retinal production in isolated retinas from Nrl^−/−Rpe65^−/− mice of increasing age. The decline in 11-cis-retinal production corresponds to the loss of photoreceptors seen in Nrl^−/−Rpe65^−/− mice, suggesting that 11-cis-retinal production occurred in the photoreceptors. Data points represent mean values of 11-cis-retinal per retina obtained from three samples with four retinas used per sample.

Figure 3.

11-cis-Retinol is the preferred substrate for oxidation in the retina. To determine whether 11-cis-retinol is the preferred substrate for oxidation in the retina, isolated retinas from Nrl^−/−Rpe65^−/− mice were treated with IRBP (24 μm) and either all-trans-retinol (n = 4) or 11-cis-retinol (n = 4). After 60 min, retinoids were extracted and levels of all-trans-retinal or 11-cis-retinal were measured by HPLC. The use of IRBP to deliver all-trans-retinol did not result in an increase in all-trans-retinal (p = 0.52). However, the use of IRBP to deliver 11-cis-retinol resulted in a significant increase in 11-cis-retinal (p = 0.001).

Figure 4.

IRBP improves the efficiency of 11-cis-retinal production in Nrl^−/−Rpe65^−/− retinas. Isolated retinas were treated with 11-cis-retinol (5 μm) using either BSA or IRBP (15 μm) as carriers. The use of IRBP resulted in higher percentages of 11-cis-retinal (n = 4; p = 0.001) and lower amounts of side-products, such as all-trans-retinol (n = 4; p = 0.01), after 60 min at 37°C. Thus, IRBP helps preserve the isomeric state of 11-cis-retinol and improves the efficiency of 11-cis-retinal production.

Figure 5.

IRBP protects the isomeric state of 11-cis-retinol. 11-cis-Retinol (10 μm) was exposed to light in the presence or absence of IRBP. In control conditions (no carrier), the addition of light caused a significant reduction in the levels of 11-cis-retinol extracted (n = 4; p = 0.008). In the presence of IRBP (15 μm), exposure to light had no effect on 11-cis-retinol levels (n = 4; p = 0.59). Thus, IRBP protects 11-cis-retinol from photodegradation. Data points represent means ± 1 SD.