Detecting ATP release by a biosensor method.
Detecting ATP release by a biosensor method.
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DOI：
10.1126/stke.2582004pl14
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发表时间：
2004-11-09
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影响因子：
--
通讯作者：
Okada, Yasunobu
中科院分区：
文献类型：
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作者：
Hayashi, Seiji;Hazama, Akihiro;Okada, Yasunobu
细胞对各种刺激作出反应，将三磷酸腺苷(ATP)释放到细胞外间隙。这种释放的三磷酸腺苷在细胞间信号转导中起着重要的生理作用。可以使用常规的荧光素-荧光素酶分析来检测总体的ATP浓度。然而，细胞表面附近的ATP浓度往往不同于整体浓度，这是因为它被胞外ATPase迅速降解，以及由于非搅拌层效应而延迟扩散。在这里，我们描述了一种简单的生物传感器方法来实时测量细胞表面的局部ATP浓度。该方法是基于在未分化的嗜铬细胞瘤(PC12)细胞或稳定表达重组P2X2受体的人胚胎肾293(HEK293)细胞中表达的嘌呤能P2X受体的配体门控阳离子通道的开放。在膜片钳的全细胞结构下，将传感器PC12细胞或HEK293放置在靶细胞附近，测量靶细胞表面给定位置释放的ATP诱导的P2X介导的电流。通过使用在预设浓度下局部喷洒的ATP的校准程序来量化ATP的释放。
Cells release adenosine 5'-triphosphate (ATP) into the extracellular space in response to various stimuli. This released ATP plays an important physiological role in cell-to-cell signal transduction. The bulk ATP concentration can be detected using a conventional luciferin-luciferase assay. However, the ATP concentration in the vicinity of the cell surface is often different from the bulk concentration because of its rapid degradation by ecto-ATPases and because of delayed diffusion due to unstirred layer effects. Here, we describe a simple biosensor method to measure the local ATP concentration on the cell surface in real time. The method is based on the ATP-dependent opening of ligand-gated cation channels of purinergic P2X receptors expressed in undifferentiated pheochromocytoma (PC12) cells or in human embryonic kidney 293 (HEK293) cells stably transfected with recombinant P2X2 purinergic receptors. Under the whole-cell configuration of patch-clamp, a sensor PC12 cell or HEK293 is positioned within the proximity of a target cell, and the P2X-mediated currents induced by ATP released from a given site on the target cell surface is measured. The ATP release is quantified by a calibration procedure utilizing local puff applications of ATP at preset concentrations.