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 Galphaq and Homer. Membranes isolated from agonist-stimulated astrocytes showed an enhanced recruitment of phospholipase C (PLCbeta1), IP3R2 and protein kinase C (PKC-alpha) into lipid raft fractions. IP3R2, TRPC1 and Homer co-immunoprecipitated, suggesting protein-protein interactions. Disruption of rafts by cholesterol depletion using methyl-beta-cyclodextrin (beta-MCD) altered the distribution of caveolin-1 and GM1 to non-raft fractions with higher densities. beta-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.
Weerth
Susanna H
SH
Section for Cell Biology and Signal Transduction, National Institute of Child Health and Human Development, National Institute of Health, Bethesda, MD 20892-4480, USA.
Holtzclaw
Lynne A
LA
Russell
James T
JT
D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D020013:Calcium Signaling; D051242:Caveolin 1 / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism* / Q000502:physiology; D009457:Neuroglia / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D051381:Rats; D012995:Solubility
D051242:Caveolin 1; D053496:Inositol 1,4,5-Trisphosphate Receptors; D047908:Intracellular Signaling Peptides and Proteins; D017830:Octoxynol; D002784:Cholesterol; D002118:Calcium