Signaling proteins in raft-like microdomains are essential for Ca2+ wave propagation in glial cells

The hypothesis that calcium signaling proteins segregate into lipid raft-like microdomains was tested in isolated membranes of rat oligodendrocyte progenitor (OP) cells and astrocytes using Triton X-100 solubilization and density gradient centrifugation. Western blot analysis of gradient fractions showed co-localization of caveolin-1 with proteins involved in the Ca2+ signaling cascade. These included agonist receptors, P2Y1, and M1, TRPC1, IP3R2, ryanodine receptor, as well as the G protein Gαq and Homer. Membranes isolated from agonist-stimulated astrocytes showed an enhanced recruitment of phospholipase C (PLCβ1), IP3R2 and protein kinase C (PKC-α) into lipid raft fractions. IP3R2, TRPC1 and Homer co-immunoprecipitated, suggesting protein–protein interactions. Disruption of rafts by cholesterol depletion using methyl-β-cyclodextrin (β-MCD) altered the distribution of caveolin-1 and GM1 to non-raft fractions with higher densities. β-MCD-induced disruption of rafts inhibited agonist-evoked Ca2+ wave propagation in astrocytes and attenuated wave speeds. These results indicate that in glial cells, Ca2+ signaling proteins might exist in organized membrane microdomains, and these complexes may include proteins from different cellular membrane systems. Such an organization is essential for Ca2+ wave propagation.