Activation-dependent Hindrance of Photoreceptor G Protein Diffusion by Lipid Microdomains

The dynamics of G protein-mediated signal transduction depend on the two-dimensional diffusion of membrane-bound G proteins and receptors, which has been suggested to be rate-limiting for vertebrate phototransduction, a highly amplified G protein-coupled signaling pathway. Using fluorescence recovery after photobleaching (FRAP), we measured the diffusion of the G protein transducin α-subunit (Gαt) and the G protein-coupled receptor rhodopsin on disk membranes of living rod photoreceptors from transgenic Xenopus laevis. Treatment with either methyl-β-cyclodextrin or filipin III to disrupt cholesterol-containing lipid microdomains dramatically accelerated diffusion of Gαt in its GTP-bound state and of the rhodopsin-Gαβγt complex but not of rhodopsin or inactive GDP-bound Gαβγ. These results imply an activity-dependent sequestration of G proteins into cholesterol-dependent lipid microdomains, which limits diffusion and exclude the majority of free rhodopsin and the free G protein heterotrimer. Our data offer a novel demonstration of lipid microdomains in the internal membranes of living sensory neurons.