The long term objective of the proposed research is to understand the molecular chain of events that link the absorption of light by rhodopsin with the permeability changes of the receptor organelle of the vertebrate photoreceptor for dim light, the rod outer segment (ROS). A better understanding of these molecular events is necessary to plan a rational attack on diseases that affect the ROS. The three specific aims are: 1- To determine if cyclic GMP hydrolysis per se hyperpolarizes and is thus sufficient for transduction, it is proposed a) to monitor membrane potential while injecting active purified phosphodiesterase (PDE) into ROS of the isolated toad retina during dim adapting lights designed to counteract masking effects of cyclase, b) to analyze kinetics of responses before and after PDE injections to determine if cyclic GMP affects the initial hyperpolarizing phase of the receptor potential, and c) to inject activated transducin to determine if the action of transducin is different from PDE which would suggest a branch point in the transduction mechanism before PDE activation. 2- to determine if active PDE has an effect equivalent with light adaptation by analyzing the kinetics of responses to dim flashes during continuous dim adapting lights and after injection of activated PDE in darkness. 3- to determine how cyclic GMP and the hydrolysis of cyclic GMP exert their effects by analyzing responses to injections into ROS of purified protein kinases, antibodies to protein kinases and protein phosphatases. Injections of purified proteins will be made by pressure through the recording pipette into ROS of the isolated toad or other amphibian retinas while analyzing the membrane potential.
NIH ExPorter
NIH ExPorter