Identification of human P2X1 receptor-interacting proteins reveals a role of the cytoskeleton in receptor regulation.
Identification of human P2X1 receptor-interacting proteins reveals a role of the cytoskeleton in receptor regulation.
复制标题
人P2X1受体相互作用蛋白的鉴定揭示了细胞骨架在受体调节中的作用。
DOI：
10.1074/jbc.m111.253153
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发表时间：
2011-09-02
期刊：
影响因子：
--
通讯作者：
Evans RJ
中科院分区：
文献类型：
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作者：
Lalo U;Roberts JA;Evans RJ
P2X1 受体是由平滑肌和血细胞表达的 ATP 门控离子通道。羧基末端带有 His-FLAG 标签的人 P2X1 受体在 HEK293 细胞中稳定表达，并与包括肌动蛋白在内的细胞骨架蛋白共纯化。用细胞松弛素 D 破坏肌动蛋白细胞骨架可抑制 P2X1 受体电流，但对反应时程或受体表面表达没有影响。用 jasplakinolide 稳定细胞骨架对 P2X1 受体电流没有影响，但降低了受体迁移率。 P2X2 受体电流不受细胞松弛素影响，P2X1/2 受体嵌合体用于鉴定肌动蛋白敏感性的分子基础。这些研究表明，细胞内氨基末端对细胞骨架破坏的抑制作用与脂筏/胆固醇敏感性相似。用 jasplakinolide 稳定细胞骨架消除了胆固醇消耗对 P2X1 受体电流的抑制作用，表明脂筏可能通过稳定细胞骨架来调节受体。这些研究表明细胞骨架在P2X1受体调节中发挥重要作用。
P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor. Stabilization of the cytoskeleton with jasplakinolide had no effect on P2X1 receptor currents but decreased receptor mobility. P2X2 receptor currents were unaffected by cytochalasin, and P2X1/2 receptor chimeras were used to identify the molecular basis of actin sensitivity. These studies showed that the intracellular amino terminus accounts for the inhibitory effects of cytoskeletal disruption similar to that shown for lipid raft/cholesterol sensitivity. Stabilization of the cytoskeleton with jasplakinolide abolished the inhibitory effects of cholesterol depletion on P2X1 receptor currents, suggesting that lipid rafts may regulate the receptor through stabilization of the cytoskeleton. These studies show that the cytoskeleton plays an important role in P2X1 receptor regulation.