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D. H. Rosenzweig, K. S. Nair, Q. Wang, J. Wei, G. G. Garwin, J. B. Hurley, V. Z. Slepak; The Role of Subunit Dissociation in Light-Induced Transducin Migration in Rods and Cones. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1114.
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It is believed that one of the mechanisms involved in rod light adaptation is the light-dependent re-localization of transducin (Gt) between the rod outer segments (OS) and inner compartments. Here we examined the molecular mechanism responsible for rod trasducin migration and investigated why transducin does not migrate in cones.
Wild-type and Nrl-/- mice, which have cone-like photoreceptors were used in this study. Explanted retinas were permeabilized with Staphylococcal alpha toxin to allow the free diffusion of small molecules between the bathing medium and cytosol. Retinas were depleted of ATP by incubating them in glucose-free DMEM supplemented with KCN and deoxyglucose. Localization of rod and cone transducin subunits in the cells was examined by immunofluorescence microscopy. Cone opsin activation was measured by HPLC analysis of retinoid levels. Biochemical analysis of transducin status involved cell fractionation, trypsinolysis and chromatography followed by western blot.
Light-induced migration of rod Gt occurs in ATP-depleted rods with roughly the same kinetics and to the same extent as in live animals. GTP (or GTPγS) is required for rod Gt translocation in light. Translocation also occurs without GTP in the dark in the presence of a synthetic peptide mSIRK, which causes G protein subunit dissociation. Cone transducin does not migrate upon activation of as much as 80% of cone pigment in Nrl-/- retinas. Light-induced GTPγS binding to cone Gt results in migration of only 10% of cone Gαt. However, application of GTPγS together with mSIRK causes up to 60% relocalization of cone Gαt. Accordingly, while rod Gt readily dissociates from the membranes in light plus GTP, cone G protein requires simultaneous application of saturating light, GTPγS and mSIRK.
Subunit dissociation is sufficient to cause redistribution of rod transducin. When the subunits are stimulated to dissociate, either by light or by a synthetic peptide, they detach from the OS membranes and diffuse throughout the cytosol in all compartments of the photoreceptor. Thus, light-regulated migration of rod transducin is independent of molecular motors or other processes directly requiring metabolic energy. Activated cone Gt does not diffuse to other compartments evidently because its subunits do not readily dissociate upon activation and remain anchored in the OS membranes.
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