P2X7-mediated ATP secretion is accompanied by depletion of cytosolic ATP
P2X7-mediated ATP secretion is accompanied by depletion of cytosolic ATP
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DOI：
10.1007/s11302-019-09654-5
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发表时间：
2019-06-01
影响因子：
3.5
通讯作者：
Haag, Friedrich
中科院分区：
文献类型：
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作者：
Johnsen, Bjarne;Kaschubowski, Klaus Eric;Haag, Friedrich
ATP及其代谢产物是作用于嘌呤能P2和P1受体的重要细胞外信号递质。大多数细胞可以主动分泌ATP以响应各种外部刺激，如P2X7受体的门控。我们使用Yac-1小鼠淋巴瘤细胞研究p2x7介导的ATP释放。这些细胞共同表达P2X7和adp核糖基转移酶ARTC2，允许P2X7通过NAD（+）依赖性adp核糖基化而不需要添加外源ATP。Yac-1细胞在NAD（+）刺激后几分钟内释放ATP到细胞外空间。通过抑制p2x7特异性纳米体13A7的预孵育可以阻断这一过程。P2X7门控作用3h后，活细胞内ATP水平显著降低，但20h后恢复正常。p2x7介导的ATP释放依赖于胞质钙的升高和细胞内钾的消耗，但不被泛连接蛋白或连接蛋白抑制剂阻断。我们使用基因编码的基于fret的ATP传感器靶向细胞质，在共表达P2X7和ARTC2的3T3成纤维细胞和Yac-1细胞中成像P2X7介导的ATP分布变化。在对NAD（+）的反应中，我们观察到细胞质中ATP的明显耗竭。这项研究证明了ATP传感器作为研究其他细胞类型在其他条件下调节ATP释放的工具的潜力。
ATP and its metabolites are important extracellular signal transmitters acting on purinergic P2 and P1 receptors. Most cells can actively secrete ATP in response to a variety of external stimuli such as gating of the P2X7 receptor. We used Yac-1 murine lymphoma cells to study P2X7-mediated ATP release. These cells co-express P2X7 and ADP-ribosyltransferase ARTC2, permitting gating of P2X7 by NAD(+)-dependent ADP-ribosylation without the need to add exogenous ATP. Yac-1 cells released ATP into the extracellular space within minutes after stimulation with NAD(+). This was blocked by pre-incubation with the inhibitory P2X7-specific nanobody 13A7. Gating of P2X7 for 3h significantly decreased intracellular ATP levels in living cells, but these had returned to normal by 20h. P2X7-mediated ATP release was dependent on a rise in cytosolic calcium and the depletion of intracellular potassium, but was not blocked by inhibitors of pannexins or connexins. We used genetically encoded FRET-based ATP sensors targeted to the cytosol to image P2X7-mediated changes in the distribution ofATP in 3T3 fibroblasts co-expressing P2X7 and ARTC2 and in Yac-1 cells. In response to NAD(+), we observed amarked depletion of ATP in the cytosol. This study demonstrates the potential of ATP sensors as tools to study regulated ATP release by other cell types under other conditions.