FIGURE 5.

Deviation of observed lateral diffusion from predictions for membrane homogeneity and derived model of membrane heterogeneity. A, observed diffusion coefficients versus predictions for homogeneous lipid. The bar graph show the ratio of diffusion coefficients between our observation and the theoretical prediction based on homogenous lipid membrane calculated as described under “Experimental Procedures.” The treatments (additions to oxygenated Ringer's solution with 10 mm glucose, except as noted) for each sample were: Gα_t, cholesterol added (α-toxin/MβCD/cholesterol); Gα_t (no additions or α-toxin/ATP/GTPγS); Rho₂-Gαβγ_t (2-DG/sodium azide, no glucose); Gαβγ_t (hydroxylamine); Rho₂ (rhodospin-EGFP transgenics; no additions); Gα_t, cholesterol depleted (α-toxin/MβCD); Rho₂-Gαβγ_t, cholesterol depleted (α-toxin/MβCD/2-DG/sodium azide, no glucose); Gαβγ_t, cholesterol depleted (α-toxin/MβCD/hydroxylamine); and Rho₂, cholesterol depleted (rhodospin-EGFP transgenics, α-toxin/MβCD). Unless otherwise indicated, all measurements used Gα_t-EGFP transgenic tadpoles. B, proposed model of disk membrane heterogeneity and diffusion. Based on our findings, the following sequence of events is proposed. In the dark, and immediately after photoexcitation of rhodopsin, both rhodopsin and GDP-Gαβγ_t diffuse freely in a bulk lipid phase with relatively low effective viscosity. Upon binding of GDP-bound Gαβγ_t to light-activated rhodopsin (R^*), the complex moves from bulk lipid into microdomains (or microdomains assemble around the complex), which may facilitate the conformational changes necessary for GDP-GTP exchange and dissociation of activated GTP-Gα_t from R^*. GTP-Gα_t remains associated with microdomains while in the active state.