The light responses of rods and cones have activation and recovery phases. The biochemical mechanisms underlying the activation phase, i.e., the phototransduction G-protein cascade, are now thoroughly understood. In contrast, the mechanisms responsible for the recovery phase of photorespones in situ are less well understood, although a wealth of processes have been discovered and characterized in vitro that down-regulate or modulate each of the activation steps of the cascade. For example, photoactivated rhodopsin (R*) is down-regulated by rhodopsin kinase, by arrestin binding, and by a yet-to-be isolated Ca2+-binding protein. R*-activated transducin (G*=G-alpha-GTP) is down regulated by transducin-GTPase, whose rate in turn is regulated by the binding of G* to its target, the gamma subunit of phosphodiesterase (PDE). Activated PDE (PDE*) is inactivated by transducin-GTPase, but PDE* activity is also modulated by cGMP binding to noncatalytic binding sites. The experiments proposed will test hypotheses about the kinetic roles that these and other biochemical processes play in the recovery phase of photoresponses of isolated amphibian rods, and recoveries of murine rod photoresponses in situ. Since the recoveries of rods to saturating flashes reveal that one of the underlying inactivation mechanisms has a clearly dominant time constant, experiments are proposed that will test hypotheses about the biochemical identity of the dominant mechanism. To test the hypothesis that R* lifetime is dominant, pharmacological and molecular-biological manipulations of R* phosphorylation and arrestin binding are proposed, and the kinetic effect of these manipulations predicted. To test the hypothesis that the lifetime of the G*/PDE* complex is the dominant recovery step, the concentration of PDE-gamma will be manipulated, both in isolated rods by dialysis and in situ by molecular-biological methods. Other experiments are proposed that will quantify the role of steady PDE activity in shaping the photoresponse waveform, and of the longitudinal diffusion of cGMP in the outer segment during the single-photon response. Experiments are also proposed that characterize the role in accelerating recovery of the decline in internal calcium concentration that accompanies the light response. With the increased understanding of the phototransduction cascade, many disease processes that affect its various component proteins can be studied in greater depth. The development in this work of non-invasive murine electroretinographic analyses, combined with molecular-biological (transgenic) manipulations will provide tools and models for the study of a variety of retinal diseases.
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