Analysis of the mechanisms underlying light adaptation and recovery following phototransduction is one of the goals outlined in the National Plan for Eye and Vision Research (www.nei.nih.gov/strategicplanning /np_retinal.asp). One of the most striking molecular events that occurs in retinal rods in response to illumination is the migration of rod G protein, transducin. In darkness, transducin concentrates in the outer segments (OS) of rods. In light, it migrates from the OS to the inner segment (IS), nuclear layer and the synaptic terminals. The significance and the mechanism responsible for this phenomenon are a subject of debate. Based on genetic studies in mice and drosophila, a number of vision scientists hypothesized that transducin movement requires active transport machinery. However, this laboratory showed that movement of transducin does not require ATP and therefore must be driven by diffusion rather than molecular motors. In this model, the light-dependence and directionality of transducin relocalization is explained by the following simple mechanism. Upon activation, rod transducin (i) dissociates into Galpha and Gbeta-gamma, (ii) the subunits become soluble and disperse throughout the cell. Dissociation of the subunits and their subsequent solubilization are the only two events necessary and sufficient for the release of transducin from the OS and subsequent relocalization. The proposed research will use this model to establish whether transducin translocation is indeed necessary for light adaptation, and whether it contributes to cell survival (protection from light damage).
Specific Aim 1 will design and test mutant forms of rod transducin alpha subunit that cannot translocate by virtue of the altered affinity to the membranes, Gbeta-gamma, and LGN, a putative binding partner in the inner compartments of the rods. These Galpha mutants will be first validated in transfected mammalian cells and then in situ using virally transformed mouse rods.
Specific Aim 2 is to express these dominant mutants in vivo (transgenic mice) in order to examine the effect on light adaptation and rod survival (retinal degeneration). A series of electrophysiological, biochemical and histological analyses will investigate light adaptation and long- term survival of the rods. This project will achieve two goals: establish the molecular mechanism of light-dependent rod transducin redistribution and understand physiologic significance of this phenomenon. This new basic knowledge will provide a conceptual framework for designing new strategies to prevent and treat retinal degenerative diseases.
Many proteins re-localize within cells in response to external stimuli. This project investigates the basic principles and significance of light-dependent intracellular movements of a heterotrimeric G protein in photoreceptor rods. The proposed experiments will determine how the ability of the G protein to relocalize is important for light adaptation and long-term photoreceptor cells. The new insights are important for understanding currently incurable blinding diseases, and is also important for broader aspects of neuronal degeneration.
