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Alexander V Kolesnikov, Tivadar Orban, Krzysztof Palczewski, Vladimir J Kefalov; Dephosphorylation of visual pigments by PP2A is required for timely dark adaptation of rods and cones. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3575.
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© ARVO (1962-2015); The Authors (2016-present)
Rapid inactivation of the visual pigment by phosphorylation is essential for the timely termination of rod and cone photoresponses and the recovery of photoreceptor sensitivity following a bleach. However, the role of pigment dephosphorylation in dark adaptation of rods and cones and the identity of the phosphatase are still debated. We investigated the putative role of pigment phosphatase 2A (PP2A) in mammalian rods and cones.
Expression of PP2A in mouse rods and cones was revealed by in situ mRNA hybridization in WT and Nrl-/- retinas, respectively. We then generated mice with the floxed gene of the a-catalytic subunit of PP2A and crossed them with strains expressing Cre-recombinase to generate rod (Rho-Cre) and cone (HGRP-Cre) PP2A conditional knockout (CKO) lines. To facilitate M-cone recordings, we derived cone PP2A-CKO animals on a Gnat1-/- background. The lack of PP2A in photoreceptors was confirmed by immunohistochemistry. We assessed the function of PP2A-deficient rods by single-cell suction recordings, and that of PP2A-deficient cones by transretinal ERG recordings. The effect of PP2A deletion on rod and cone dark adaptation was determined by in vivo ERG. We measured the kinetics of rhodopsin phosphorylation and dephosphorylation by mass-spectrometry. Finally, we quantified dark and postbleach levels of 11-cis-retinal and other visual cycle retinoids in the eye by HPLC.
Rod and cone photoresponse amplitude and sensitivity were comparable in dark-adapted Cre controls and PP2A-CKO mice. The response recovery following dim to saturating test flashes was also normal in isolated PP2A-deficient rods. However, following >90% pigment bleach the dark adaptation of PP2A-deficient rods was substantially delayed, and the recovery of their sensitivity in vivo was reduced compared to the Cre controls. The slower rod dark adaptation in the absence of PP2A correlated with a substantially delayed dephosphorylation of rhodopsin and recycling of 11-cis-retinal chromophore in the visual cycle. Similarly, even though phototransduction was unaffected by the deletion of PP2A in M-cones, their dark adaptation was greatly retarded in vivo.
The timely dark adaptation of mouse rods and cones requires dephosphorylation of their visual pigments by PP2A. Thus, PP2A is the dominant phosphatase which resets the ground state of visual pigments following their photoactivation.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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