Anales RANF

P3. P HARMACOLOGY AND B IOCHEMISTRY OF P2 R ECEPTORS P.14 FLUORESCENT SENSORS FOR THE IMAGING OF ATP AT CELL SURFACES A. Laubach 1 , K. Kaschubowski 2 , F. Koch-Nolte 2 , C. Meier 1 , F. Haag 2 * 1 . University of Hamburg, Hamburg, Germany; 2 . University Medical Center Hamburg- Eppendorf, Hamburg, Germany. Signalling by extracellular adenine nucleotides is an important mechanism regulating activation and quiescence in the immune system. The prime event in this process is the release of ATP by stressed or damaged cells or by regulated secretion. Extracellular ATP either acts directly on P2 receptors (e.g. P2X7) to mediate activating effects or is converted to adenosine (ADO) by the sequential action of the ecto-nucleotidases CD39 and CD73 to mediate suppressive effects by acting on P1 ADO receptors (ARs). Since the relevant concentrations of eATP at the site of receptor signalling differ from bulk ATP levels measured in cell supernatants, it is important to develop methods to monitor ATP concentrations at distinct subcellular locations like the cell surface. We engineered genetically encoded FRET-based sensors for expression in the cytoplasm or at the cell surface and monitored ATP levels in the cytoplasm by flow cytometry or live cell imaging. To avoid the dependence on genetic manipulation we also synthesized several ATP-responsive small-molecule fluorescent probes. Transfection of the cytoplasmic FRET sensors into Yac-1 lymphoma cells revealed that gating of P2X7 resulted in a constant decrease in cytosolic ATP levels for the duration of exposure to eATP that was blocked if the cells were pre-incubated with the P2X7- inhibitory nanobody 13A7. This response could be monitored both by flow cytometry on a population level and by fluorescence microscopy imaging on the level of individual cells. In a complementary approach we synthesized small-molecule fluorescent probes that change their emission intensity or spectra in response to ATP. Work is currently in progress to improve the sensitivity of these probes as well as their selectivity towards ATP and to target these probes to the cell surface. * This work is part of SFB1328 “Adenine Nucleotides in Immunity and Inflammation” funded by the DFG