Decrease in Amplification of Phototransduction Correlated with Light-dependent Transducin Translocation in Mouse Rod Photoreceptors

Abstract

Abstract: : Purpose: Our laboratories previously reported a mechanism of light adaptation based upon the light-dependent redistribution of transducin subunits (Gα_t and Gß₁γ₁) from rod outer segments (ROS) to other parts of the photoreceptor cell, accompanied by a decrease in rod photoresponse amplification in living rats. Here we investigate whether this mechanism occurs in the mouse rod. Methods: Wild type C57BL6 mice were dark-adapted 15 hours prior to all experiments. The technique of serial tangential sectioning with Western blotting, previously described for quantification of protein redistributions in rat rods, and ERG a-wave recordings were conducted and analyzed with published protocols (Sokolov et al, Neuron 2002). Difference spectroscopy of sonicated retinal extracts with or without the addition of 11-cis retinal was used to determine rhodopsin bleach levels at the time of termination of the ERG recording. For light conditioning experiments the mice were anesthetized, the pupils dilated, and the eyes exposed to a light intensity level of 100 cd m² for a period of 30 minutes. Results: The transducin translocation mechanism of light adaptation observed in the rat retina also exists in mouse rod photoreceptors. The ROS of dark-adapted mouse retinas contain ~88% of the total cellular content of Gα_t and ~66% of the total content of Gß₁γ₁, slightly lower than the amounts measured in rat, 91% and 86% for the Gα_t and Gß₁γ₁ subunits, respectively. After illumination ~23% of the total Gα_t and ~12% of total Gß₁γ₁ remains in OS of mouse rods. ERG measurements show that 15 minutes after the 100 cd m² conditioning light is extinguished, the rod circulating current has recovered to about 40% of its dark-adapted level, and the amplification of the a-wave (adjusted for the level of rhodopsin bleaching) is reduced about 4 fold. This is comparable to the 5-fold decrease observed in the rat and consistent with the ~four fold reduction in the Gα_t content of illuminated ROS. Conclusions: This work lays the foundation for ongoing studies of light-dependent protein translocation utilizing transgenic mouse models. Two of these models, in which the genes for Gα_t and arrestin have been deleted, are currently under investigation in our laboratory. This work is supported by a Knights Templar Pediatric Ophthalmology Award (KJS and MS), and NIH grants EY10336 (VYA) and EY02660 (ENP) and by RPB (VYA and ENP).

Keywords: photoreceptors • retina: distal(photoreceptors, horizontal cell • electrophysiology: non-clinical