Pellegatti Patrizia, Falzoni Simonetta, Pinton Paolo, Rizzuto Rosario, Di Virgilio Francesco
Department of Experimental and Diagnostic Medicine, Section of General Pathology and Interdisciplinary Center for the Study of Inflammation, University of Ferrara, Ferrara 44100, Italy.
Mol Biol Cell. 2005 Aug;16(8):3659-65. doi: 10.1091/mbc.e05-03-0222. Epub 2005 Jun 8.
ATP is emerging as an ubiquitous extracellular messenger. However, measurement of ATP concentrations in the pericellular space is problematic. To this aim, we have engineered a firefly luciferase-folate receptor chimeric protein that retains the N-terminal leader sequence and the C-terminal GPI anchor of the folate receptor. This chimeric protein, named plasma membrane luciferase (pmeLUC), is targeted and localized to the outer aspect of the plasma membrane. PmeLUC is sensitive to ATP in the low micromolar to millimolar level and is insensitive to all other nucleotides. To identify pathways for nonlytic ATP release, we transfected pmeLUC into cells expressing the recombinant or native P2X7 receptor (P2X7R). Both cell types release large amounts of ATP (100-200 microM) in response to P2X7R activation. This novel approach unveils a hitherto unsuspected nonlytic pathway for the release of large amounts of ATP that might contribute to spreading activation and recruitment of immune cells at inflammatory sites.
三磷酸腺苷（ATP）正逐渐成为一种普遍存在的细胞外信使。然而，测量细胞周围空间中的ATP浓度存在问题。为此，我们构建了一种萤火虫荧光素酶-叶酸受体嵌合蛋白，该蛋白保留了叶酸受体的N端前导序列和C端糖基磷脂酰肌醇（GPI）锚定。这种嵌合蛋白名为质膜荧光素酶（pmeLUC），靶向并定位于质膜外侧。PmeLUC对低微摩尔至毫摩尔水平的ATP敏感，对所有其他核苷酸不敏感。为了确定非裂解性ATP释放的途径，我们将pmeLUC转染到表达重组或天然P2X7受体（P2X7R）的细胞中。两种细胞类型在P2X7R激活后都会释放大量ATP（100 - 200微摩尔）。这种新方法揭示了一种迄今未被怀疑的非裂解途径，可释放大量ATP，这可能有助于在炎症部位传播激活信号并募集免疫细胞。
