The recovery of visual sensitivity during and following bright light (bleaching) exposure requires that the visual pigment within rods and cones be regenerated quickly and efficiently to terminate the persistent activation of the transduction cascade by the transient photoproducts of bleaching and opsin. Sensitivity recovery also requires that activated rhodopsin, which is normally phosphorylated and bound by arrestin following light activation, be dephosphorylated to return to the dark-adapted ground state. The reversal of these processes is slow, and their role in sensitivity recovery is poorly understood. Here we propose to use a multidisciplinary approach, including physiological and biochemical methods, on rod photoreceptors from transgenic mice to determine how rhodopsin dephosphorylation and opsin adaptation impact the overall process of dark adaptation. We will focus on elucidating the properties of two mechanisms. The first is rhodopsin dephosphorylation, a slow process that occurs normally as regenerated visual pigment is returned to its dark-adapted ground state. The second is to determine the role that free opsin plays in bleaching (opsin) adaptation. Photoreceptor sensitivity will be measured electrophysiologically (single cell and electroretinogram recordings), the rate and extent of pigment bleaching/ regeneration will be measured microspectrophotometrically, and rhodopsin phosphorylation will be measured biochemically by immunofluorescence of differentially phosphorylated rhodopsin (isoelectric focusing). There are three Specific Aims. In Aim I we will determine the cellular mechanisms that regulate rhodopsin dephosphorylation in rod photoreceptors isolated from the retinal pigment epithelium, and following exposure to bright light. These experiments will be based on our recent observation that the rate of rhodopsin dephosphorylation is regulated in mouse rods by the oxygen and lactate content in the medium bathing the retina. We will determine the relation of lactate and O2 to dephosphorylation, and whether they work singly or in concert. We will then determine whether lactate and O2 work through controlling the NAD/NADP ratio in rods, whether they act through Müller cells, or whether their effects are mediated through monocarboxylate transporters. Experiments in Aim II will determine the dependence of the rate of recovery and/or the extent of dark adaptation on lactate and O2. Experiments in Aim III will establish the phosphorylation state of free opsin responsible for bleaching adaptation, and we will determine the extent to which phosphorylated and unphosphorylated opsin activate transducin. Results of these studies will provide a molecular understanding of those mechanisms of dark adaptation that are related to rhodopsin dephosphorylation and opsin adaptation to provide insights into effective therapies for the treatment of blinding eye diseases.

Narrative The regeneration and dephosphorylation of rhodopsin are absolute requirements for maintenance of sensitivity in the mammalian retina, but the relationship between these mechanisms and sensitivity regulation is poorly understood. A molecular appreciation of these relationships is critically important, as impaired visual pigment regeneration and the resulting slowing of dark adaptation are the basis of a number of blinding eye diseases. The experiments in this proposal are designed to elucidate the cellular basis and relationship of these processes in wild-type and transgenic mice, with a long term goal to understand photoreceptor function under normal and disease related conditions.