Abstract
Purpose:
Rapid inactivation of the visual pigment by phosphorylation is essential for the timely termination of rod and cone photoresponses and the recovery of their photosensitivity following a bleach. However, the role of pigment dephosphorylation in that process and the nature of the pigment phosphatase are unknown. We investigated the role of putative opsin phosphatase PP2A in mammalian rods and cones.
Methods:
Expression of PP2A in mouse rods and cones was established by in situ hybridization in WT and Nrl-/- retinas, respectively. We then generated floxed PP2A catalytic subunit mice and crossed them with Cre recombinase mice to generate rod (Rho-Cre) and cone (HGRP-Cre) PP2A conditional knockout (CKO) mice. To facilitate M-cone recordings, we produced cone PP2A-CKO mice on Gnat1-/- background. We assessed the function of PP2A-deficient rods by single-cell suction recordings, and that of PP2A-deficient cones by ERG recordings. We also tested by ERG the effect of PP2A deletion on rod and cone dark adaptation in vivo.
Results:
Rod and cone photoresponse amplitude and sensitivity were comparable in Cre controls and PP2A-CKO mice. The deletion of PP2A in rods (but not cones) reduced their phototransduction amplification by 1.5-fold. Rod response recovery following dim, saturating, and 6% pigment bleach flashes was normal in isolated PP2A-deficient rods. Surprisingly, following a brighter 10% pigment bleach, the dark current and sensitivity of PP2A-deficient rods recovered ∼3 times faster than in Cre controls. This result suggests that the abnormally persistent phosphorylation of metarhodopsins in the absence of PP2A either accelerates their thermal decay following a bleach or decreases the rod desensitization induced by them. In stark contrast, the rate of rod dark adaptation following >90% bleach in vivo was substantially reduced in the absence of PP2A. M-cone phototransduction and dark adaptation were largely unaffected by the deletion of PP2A.
Conclusions:
The lack of PP2A affects the recovery of rods following a bleach both in the absence of pigment regeneration in isolated rods and with pigment regeneration in vivo. Thus, timely dark adaptation of mouse rods in vivo requires rhodopsin dephosphorylation by PP2A, likely prior to opsin recombination with chromophore. In contrast, dark adaptation of mouse M-cones either is less dependent of cone opsin dephosphorylation or is regulated by a different phosphatase.
Keywords: 508 electrophysiology: non-clinical •
648 photoreceptors