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Vol. 16, Issue 8, 3659-3665, August 2005
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Department of Experimental and Diagnostic Medicine, Section of General Pathology and Interdisciplinary Center for the Study of Inflammation, University of Ferrara, Ferrara 44100, Italy
Submitted March 17, 2005;
Revised May 25, 2005;
Accepted June 1, 2005
Monitoring Editor: Guido Guidotti
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 µM) 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.
Abbreviations used: PmeLUC, plasma membrane luciferase; P2X7R, P2X7 receptor; BzATP, benzoyl ATP; oATP, oxidized ATP; DTT, dithiothreitol; HEK293-hP2X7, HEK293 cells transfected with the human P2X7R; HEK293-rP2X7, HEK293 cells transfected with the rat P2X7R; HEK293-P2X7/pmeLUC, HEK293 cells transfected with both P2X7R and pmeLUC; HEK293-mock, mock-transfected HEK293 cells.
Address correspondence to: Francesco Di Virgilio (fdv{at}unife.it).
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