Anales RANF

P.08 POINT MUTATIONS IN P2X7 RECEPTORS H. Dentler 1 , A. Pippel 1 , M. Klapperstück 1 , G. Schmalzing 2 and F. Markwardt 1 1 . Martin-Luther-University, Halle (Saale), Germany; 2 . RWTH University, Aachen, Germany P2X7 receptors are predominately expressed in the plasma membrane of cells of the hematopoietic lineages and of epithelial cells. They are nonselective Ca 2+ -permeable cation channels activated by extracellular ATP. P2X7 receptors are involved in inflammation, pain sensation and bone homeostasis. Several point mutations of P2X7 have been associated with mental and infectious diseases, inflammatory and bone disorders and malignancies. After heterologous expression of different P2X7 point mutation constructs in Xenopus oocytes and HEK-293 cells we measured ATP-induced P2X7-mediated ion currents by two microelectrode voltage clamp in oocytes and by the tight seal whole cell voltage clamp method in HEK-293 cells. ATP-induced intracellular Ca 2+ signals were measured in HEK-293 cells by single cell fluorimetry using the Ca 2+ - indicator Fluo-4. Fluorimetry was also used to quantify the P2X7-dependent uptake of the large cationic fluorescent dye Yo-Pro by HEK-293 cells. The hR307Q, hT357S, rF218K and rF288S mutations significantly decreased ATP-induced ion currents, Ca 2+ signals and Yo-Pro fluorescence compared to the P2X7 wt. The E496A mutation, known as loss of function variant, displayed no significant change in ATP-dependent ion currents and Ca 2+ signals but a reduced Yo-Pro uptake. Cells expressing the construct H155Y, known as gain of function mutation, had unchanged ion currents and Ca 2+ signals but an increased Yo-Pro uptake showing that different signaling pathways are involved in P2X7-mediated Ca 2+ of Yo-Pro uptake, respectively. These results indicate that point mutations of P2X7 receptors may have different effects on P2X7- dependent cellular signaling and that the characterization of the mutation effect may depend on the experimental readout.