首页> 外文会议>6th world congress of biomechanics (WCB 2010) >Functional Association of Purinergic P2X4 Receptors with Caveolar Rafts in Undifferentiated PC12 Cells
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Functional Association of Purinergic P2X4 Receptors with Caveolar Rafts in Undifferentiated PC12 Cells

机译:嘌呤能P2X4受体与未分化的PC12细胞中的小窝筏的功能关联。

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Lipid-rafts and caveolae are the membrane micro-domains, 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 lmM EGTA in calcium free buffer, indicating involvement of ionotropic P2X-receptors. Benzoyl-ATP and a,p" 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 im-munofluorescence 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.
机译:脂筏和小窝是膜微区,已知可以定位许多信号蛋白,包括离子通道和神经递质受体。在本研究中,研究了嘌呤能受体在脂质筏中在PC12细胞中的定位以及这种定位的功能影响。 P2X受体与筏的关联是通过消耗胆固醇来修饰细胞中的筏结构来检查的。 ATP诱导的钙信号在胆固醇耗尽时受到抑制,胆固醇在补充胆固醇的细胞中得以恢复,这表明ATP介导的反应对胆固醇耗尽并因此对脂质筏破坏敏感。此外,鉴定了P2受体亚型。在无钙缓冲液中存在lmM EGTA的情况下,未分化的PC12细胞中ATP介导的钙信号被完全阻断,表明离子型P2X受体参与其中。 P2X7和P2X1的激动剂分别为苯甲酰ATP和a,p“甲基ATP,它们在未分化的细胞中不会引起钙反应。但是,P2X4受体的特异性激动剂CTP-胞苷三磷酸诱导钙流入,胆固醇被消耗后被抑制,免疫荧光法通过对细胞进行抗-P2X4和抗-thy-1 /抗-caveolin抗体的双重标记,通过免疫荧光检查了完整细胞中P2X4-受体与筏的相关性。发现P2X4受体与小窝蛋白共定位,但与Thy-1不共存,这表明P2X4受体位于小窝筏中。

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