Light Dependency Threshold in Transducin Translocation in Rods

Abstract

Purpose:: Light causes massive transducin translocation from rod outer segments. A striking feature of this phenomenon is that the majority of transducin translocates only when illumination reaches a critical threshold level. To understand the molecular mechanisms underlying this threshold we compared the light dependency of transducin translocation in several mutant mice. We also analyzed the connection between the hydrophobicity of transducin α-subunit lipid modification and translocation.

Methods:: Anesthetized mice with dilated pupils were illuminated by light of various intensity from a calibrated source. The rate of rhodopsin bleaching was analyzed by difference spectroscopy. The distribution of transducin subunits was analyzed by immunostaining retina cross-sections with antibodies against transducin α and ß subunits. The relation between the nature of lipid modification and the mobility of the α subunit was established by precipitating transducin from serial tangentional sections representing the individual subcellular compartments of the rod followed by MALDI mass spec analysis of transducin N-terminal peptide.

Results:: We have found that the threshold of transducin translocation corresponds to the light intensity at which the rod response becomes completely saturated. In mutant mice, the threshold was shifted to either a lower (R9AP knockout and W70A) or higher (R9AP overexpressors) light intensity, dependent on whether the ability of the GTPase activating complex to inactivate GTP-bound transducin was decreased or increased. The threshold was not dependent on cellular signaling downstream from transducin. Finally, we showed that neither of the four naturally occurring lipid modifications assures complete translocation or retention of transducin α subunit, but the extent of its translocation is affected by the hydrophobicity of the acyl moiety.

Conclusions:: Our data suggest that transducin translocation is triggered when the cell exhausts its capacity to activate transducin GTPase and a portion of transducin remains active for a sufficient time to dissociate from membranes and to escape from the outer segment. This threshold marks the switch of the rod from the highly light-sensitive mode of operation required under limited lighting conditions to the less sensitive energy saving mode beneficial in bright light when vision is dominated by cones.

Keywords: photoreceptors • retina: distal (photoreceptors, horizontal cells, bipolar cells) • proteomics