Functional Association of Purinergic P2X4 Receptors with Caveolar Rafts in Undifferentiated PC12 Cells

Lipid-rafts and caveolae are the membrane microdomains, known to localize many signaling proteins including ion channels and neurotransmitter receptors. In the present study, localization of purinergic receptors with lipid-rafts in PC12-cells and the functional impact of this localization were studied. Association of P2X receptors with rafts was examined by modifying the raft structures in the cells, by depleting cholesterol. ATP induced calcium-signal was inhibited upon cholesterol depletion which was restored in cholesterol replenished cells, indicating ATP mediated responses are sensitive to cholesterol depletion and hence lipid raft disruption. Further, P2 receptor subtypes were identified. ATP mediated calcium signal in undifferentiated PC12 cells was completely blocked in the presence of 1mM EGTA in calcium free buffer, indicating involvement of ionotropic P2X-receptors. Benzoyl-ATP and

α

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β

methyl ATP, agonists of P2X7 and P2X1 respectively, did not evoke calcium response in undifferentiated cells. However CTP-citidine triphosphate, specific agonist of P2X4 receptors, induced calcium influx, which was inhibited upon cholesterol depletetion, indicating the involvement of rafts in the functioning of P2X4-receptors. The association of P2X4-receptors with rafts in intact cells was examined by immunofluorescence by double labeling the cells with anti-P2X4 and anti-thy-1/anti-caveolin antibodies. P2X4 receptors were found to be colocalized with caveolin but not with Thy-1 indicating that P2X4 receptors are localized in caveolar rafts.