P2X7-mediated ATP secretion is accompanied by depletion of cytosolic ATP

Purinergic signalling

article

Bjarne Johnsen¹  Klaus Eric Kaschubowski¹  Sorush Nader¹  Enja Schneider¹  Jan-Andrei Nicola¹  Ralf Fliegert²  Insa M. A. Wolf²  Andreas H. Guse²  Viacheslav O. Nikolaev³  Friedrich Koch-Nolte¹  Friedrich Haag¹ 

[1] Institute of Immunology, University Medical Center Hamburg-Eppendorf;The Calcium Signalling Group, Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf;Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf

关键词: P2X7;    Lymphoma;    ATP release;    FRET;    ARTC2;    Nanobodies;   

DOI  :  10.1007/s11302-019-09654-5

学科分类：分子生物学，细胞生物学和基因

来源: Springer

【 摘 要 】

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 3 h significantly decreased intracellular ATP levels in living cells, but these had returned to normal by 20 h. 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 of ATP in 3T3 fibroblasts co-expressing P2X7 and ARTC2 and in Yac-1 cells. In response to NAD+, we observed a marked 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.

【 授权许可】

CC BY