Abstract
Purpose:
Exposure of retinal rods to light leads to photoisomerization of the rhodopsin chromophore, resulting in activation of the visual transduction cascade. This activation is terminated by the phosphorylation of a cluster of serine and threonine residues located at the carboxyl terminus of rhodopsin and by subsequent binding of arrestin. Conventionally, it is thought that recovery of sensitivity following bleaching requires, among other factors, dephosphorylation of rhodopsin and unbinding of arrestin. The purpose of our study was to test this view by determining the extent to which rhodopsin dephosphorylation affects rhodopsin regeneration and recovery of dark-adapted sensitivity.
Methods:
Flash sensitivity, rhodopsin concentration, and rhodopsin phosphorylation were compared before and following exposure to bright light that bleached a large fraction of rhodopsin, and during rhodopsin regeneration fueled by exposure to exogenous 11-cis retinal. All measurements were made in rods of transgenic mice lacking cone transducin (Gnat2-/-) to isolate rod responses. Sensitivity was determined via transretinal ERG. Rhodopsin concentration was determined by microspectrophotometry on intact rods in isolated retina. The extent of rhodopsin phosphorylation at its 6 phosphorylation sites was determined by isoelectric focusing.
Results:
Bleaching 50% of the rhodopsin in retinal rods isolated from the retinal pigment epithelium resulted in persistent desensitization as well as persistent phosphorylation at all 6 phosphorylation sites on rhodopsin. Little dephosphorylation of rhodopsin was observed in darkness during three hours subsequent to bleaching or following total pigment regeneration. Surprisingly, near complete recovery of sensitivity was observed in spite of persistent phosphorylation in a large fraction of the regenerated visual pigment, The kinetics of dim flash responses recovered to previous dark-adapted levels.
Conclusions:
Our results demonstrate that, despite the presence of substantial amounts of regenerated phosphorylated rhodopsin, the flash sensitivity recovers, almost completely. Furthermore, dim flash kinetics were fully restored, indicating that the basal PDE and cyclase activity were at dark-adapted rates.
Keywords: 675 receptors: pharmacology/physiology •
687 regeneration •
648 photoreceptors