P2X3 receptor localizes into lipid rafts in neuronal cells.
P2X receptors are a family of seven (P2X(1-7)) cation channels gated by extracellular ATP, widely expressed in neurons and nonneuronal cells. Lipid rafts are cholesterol/sphingolipid-rich membrane domains, involved in many cellular processes, including transmembrane receptor signaling, vesicle traffic, and protein sorting. We provide direct biochemical evidence that P2X3 receptor localizes into lipid rafts, in primary cultures of cerebellar granule neurons as well as in brain and dorsal root ganglia extracts. We show that P2X3 exhibits all the characteristics distinctive of a protein associated with lipid rafts. These characteristics include resistance to detergent extraction at 4 degrees C, solubility after extraction of cholesterol from membranes with either saponin or methyl-beta-cyclodextrin, and partitioning to low buoyant density fractions after sucrose gradient centrifugation in both detergent-containing and detergent-free conditions. Furthermore, P2X3 localizes in raft-containing fractions in transiently transfected SH-SY5Y neuroblastoma cells. The present finding contributes to the characterization of the functional localization of P2X3 in neurons and provides a novel potential mechanism for correct targeting and dynamic activation of this receptor.
Vacca
Fabrizio
F
Fondazione Santa Lucia, Rome, Italy.
Amadio
Susanna
S
Sancesario
Giuseppe
G
Bernardi
Giorgio
G
Volonté
Cinzia
C
D000199:Actins / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western / Q000379:methods; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D003902:Detergents / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D005727:Ganglia, Spinal / Q000166:cytology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009447:Neuroblastoma; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058477:Receptors, Purinergic P2X3; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D014162:Transfection
D000199:Actins; D051242:Caveolin 1; D022461:Caveolins; D003902:Detergents; D005904:Glial Fibrillary Acidic Protein; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C546407:P2RX3 protein, human; D018048:Receptors, Purinergic P2; D058477:Receptors, Purinergic P2X3; C106354:flotillins