Phototransduction is the process by which light is converted into electrical signals in the rod and cone cells of the retina. The process may be conceptually divided into two phases: activation, the sequence of events leading from light absorption to the diminution of the current flowing into rods and cones in the dark, and inactivation, the events that restore the dark resting state. In the past few years a molecular theory of phototransduction, the cyclic GMP cascade theory, has been formulated based upon the combined evidence of electrophysiology and biochemistry. According to this theory, the protein rhodopsin absorbs a photon of light, and serially activates many G-proteins by catalyzing the binding of the nucleotide GTP to them; activated G-protein in turns activates phosphodiesterase, an enzyme that catalyzes the hydrolysis of the internal messenger, cyclic GMP; a decline in the concentration of free cGMP in the rod cytoplasm causes the closure of the cGMP-gated cation channel in the rod plasma membrane, and diminution in the dark current. Although this molecular theory provides a good qualitative account of activation, a number of key quantitative issues concerning the activation process remain unresolved; moreover, many substantive issues remain unresolved about the molecular mechanisms of inactivation. The proposed research will combine electrophysiological investigations of isolated amphibian rods, and biochemical assays of rod membranes of the same species, and will address several of these unresolved quantitative problems in activation, including (1) the catalytic rate of G-protein activation by individual rhodopsin molecules; (2) the stoichiometric relationship between G-protein activation and phosphodiesterase activation; (3) the enzymatic power of the activated phosphodiesterase in situ; (4) the quantitative relationship between the in situ activity of phosphodiesterase and the fraction of the cGMP-activated channels that remain open. The proposed research will also investigate the nature and rates of the mechanisms that inactivate rhodopsin, G-protein, and phosphodiesterase in situ. Theoretical analyses will try to link the biochemical measurements and the electrophysiological data in a biophysically rigorous model of the cGMP cascade theory.
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