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K.J. Strissel, M. Sokolov, S.M. Hanson, V.Y. Arshavsky; QUANTIFICATION OF LIGHT–DEPENDENT ARRESTIN TRANSLOCATION IN MOUSE ROD PHOTORECEPTORS . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3451.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Arrestin is one of two signaling proteins known to undergo massive translocation between cellular compartments in rod photoreceptors upon illumination. In order to study the molecular mechanisms and physiological significance of this phenomenon precise measurements of the quantities and parameters of the light–dependent translocation of arrestin are needed. Methods: Mice were dark–adapted 15 hours prior to all experiments. Dissections were performed in chilled Mouse Ringer’s on ice. For quantification of arrestin in rods, aliquots of whole retina extracts from dark–adapted mice were analyzed to determine the rhodopsin concentration by difference spectroscopy and for their arrestin content using quantitative Western blotting with recombinant mouse arrestin as the standard. For analyzing the light–dependency of arrestin translocation, anesthetized mice with dilated pupils were subjected to 30 minutes of illumination at various light intensity levels using a calibrated light source. At the end of the illumination the retinas were either prepared for determination of rhodopsin bleaching by difference spectroscopy after regeneration with 11–cis retinal or were flat–mounted and immediately frozen for serial tangential sectioning followed by Western blotting for the analysis of subcellular protein distribution. Results: These studies have revealed that arrestin is present in murine rods at a molar ratio of 1.51 ± 0.11 with rhodopsin (SEM, n=3), a quantity much higher than previously reported. The dark–adapted mouse rod outer segment contains very little arrestin, less than we could reliably measure. Arrestin translocation is initiated by moderate light intensities, but requires nearly total rhodopsin bleaching for completion. For example, the bleaching of 40% rhodopsin causes the translocation of 55.2% ± 4.3% (SEM, n=3) arrestin to rod outer segments. At the highest light intensity levels tested (bleaching >90% rhodopsin) approximately 90% of the total arrestin pool is translocated to the outer segments. Conclusions: Arrestin is present in the rod cell at a level sufficient to quench nearly all photolysed rhodopsin molecules in the outer segment and nearly the entire arrestin pool is translocated to the outer segments at saturating illumination. We suggest that the major role of arrestin translocation is to provide a means for photoresponse termination at any light level that a photoreceptor could encounter in nature. Supported by: NIH grant EY10336 (VYA), RPB (VYA), and NIH grant GM0762 (SMH)
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