RGR opsin and RDH10 in Müller Cells Affect Light Dependent Synthesis of Chromophore Precursor for Cone Pigment Regeneration

Abstract

Purpose : The rates of rhodopsin and cone-opsin bleaching during light exposure greatly outstrip chromophore (11cRAL) synthesis by the RPE. A second visual cycle in Müller cells is thought to convert vitamin A (atROL) released by photoreceptors into the chromophore precursor, 11cROL. Cones, but not rods, utilize 11cROL to make 11cRAL for visual pigment regeneration. The Müller-cell visual cycle thereby specifically regenerates cones. The proteins responsible for this conversion in rod-dominant species are unknown.

Methods : RGR opsin and RDH10 were expressed in cultured HEK-293T cells and assayed for conversion of atROL to 11cROL and 11cRAL under different light conditions. Co-immunoprecipitation experiments were performed with RGR opsin and several retinol dehydrogenases expressed in HEK cells. Microsomal proteins prepared from the retinas of Rgr+/+ (129/Sv) and Rgr-/- mutant mice were assayed for light-dependent retinoid processing activity.

Results : (1) HEK cells expressing RGR opsin and RDH10 exposed to light exhibited dramatic conversion of atROL to 11cROL with minimal formation of 11cRAL. No formation of 11cROL or 11cRAL was seen by cells kept in the dark. Also, no formation of 11cROL or 11cRAL was seen by cells expressing RGR or RDH10 alone, regardless of light exposure. The action spectrum for 11cROL production corresponded to the absorption spectrum of RGR opsin (λ_max = 470 nm). (2) Light-dependent conversion of atROL to 11cROL was not observed by cells expressing RGR plus RDH5, RDH8, RDH11 or RDH14; only RDH10 cooperated with RGR opsin. (3) Despite the specificity of the RGR—RDH10 interaction, RGR opsin reciprocally co-immunoprecipitated with RDH5, RDH10 and RDH14. (4) Squid retinochrome, a distant ortholog of RGR opsin, also cooperated with RDH10 to convert atROL to 11cROL during light exposure. (5) Microsomal proteins prepared from Rgr+/+ (129/Sv) mouse retinas exhibited light-dependent conversion of atROL to 11cROL with minimal formation of 11cRAL. In contrast, microsomes from Rgr-/- mouse retinas exhibited several-fold slower conversion of atROL to 11cROL in the presence of light.

Conclusions : RGR opsin and RDH10 in Müller cells exhibit a novel redox photoisomerase activity that converts atROL to 11cROL in the presence of light. The activity of these proteins is likely responsible for 11cROL synthesis and the regeneration of cone visual pigments by Müller cells.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.