Light-induced Raft In The Disc Membrane Of Rod Photoreceptor

Abstract

Purpose: : To characterize the light-induced raft around photoexcited-rhodopsin/transducin- complex in retinal disc membrane.

Methods: : We used single molecule imaging technique to examine the diffusivity and behavior of rhodopsin (R), transducin (G_t), and Ca²⁺-S-modulin (S) on the disc membrane of bullfrog rod photoreceptor outer segment (ROS). R was probed by Cy7-labeled Fab’ of IgG(1D4). The α subunit of G_t (G α_t) and S was directly labeled with HiLyte Fluor-750 and Cy7, respectively. Labeled proteins were reconstituted to the disc membrane, and visualized on a TIRF microscope. The effective diffusion coefficients of proteins were determined from the 2-dimensional trajectories of proteins. We also evaluate the raftophilicity, i.e. affinity to raft, of proteins and their complexes in terms of their partitioning to the detergent-resistant membrane (DRM). To disrupt the raft in the disc membrane, we used methyl-b-cyclodextrin (MCD).

Results: : Both R and photoexcited R (R*) undergo diffusion in the disc membrane with an effective diffusion coefficient (D_100ms) of ~0.3 µm²/s. The D_100ms of G_t in dark-adapted disc membrane is ~0.7 µm²/s, much higher than rhodopsin. However, G_t complexed with R* (R*/G_t-complex) in light-exposed disc membrane shows slower diffusion rate than R or R*, i.e. 0.18 µm²/s. Since MCD restores the D_100ms, raftophilic interaction seems to hinder the diffusion of the complex. GTP also releases the constraint. Artificially dimerized rhodopsin (R²) crosslinked by IgG exhibits low diffusivity and high raftophilicity. Further, the R² in DRM is partially resistant to MCD. These characters of R² are very similar to those of R*/G_t complex. Thus, we examined the stoichiometric ratio between R* and G_t in the complex residing in DRM, and found that R*:G_t=2:1. In addition, D_100ms of Ca²⁺-S-modulin was unexpectedly dependent on light, GTP and MCD-treatment of the disc membrane.

Conclusions: : Light-induced binding of G_t to R* stabilizes the dimer of rhodopsin, which can organize raft around it. This raft must be very small and transient entity. This light-induced raft may participate in recruitment of regulatory proteins to the vicinity of R* to shape up photoresponse in rod photoreceptor.

Keywords: photoreceptors • signal transduction • lipids