Fig. 6. Lysosome exocytosis increases P2X₄-mediated currents. (A) Whole-cell currents measured using the perforated patch-clamp technique following application of 30 µM ATP or BzATP at a holding potential of –60 mV in LPS-primed mouse peritoneal macrophages. 3 µM ivermectin was applied 3 minutes before and during ATP application. The histogram shows the normalized mean peak current amplitude +s.e.m. for 30 µM ATP and BzATP (n=4–10). (B) ATP-evoked inward currents before and after a 30 minute pre-incubation with 50 mM methylamine (MA) at 37°C. Pre-incubation with MA caused an approximately fivefold increase in the peak amplitude of ATP-evoked inward currents, whereas MA had no effect when co-applied with ATP. Following MA preincubation, currents remained ivermectin sensitive and were potentiated a further threefold. Pre-incubation with 10 µg/ml brefeldin A for 1 hour before and during incubation with MA did not inhibit MA-induced potentiation of ATP-evoked currents. The histogram shows the normalized mean peak current amplitude plus s.e.m.; n=8–14. (C) Currents evoked by 30 µM ATP or BzATP following treatment with MA. (D) MA pre-treatment did not increase the amplitude of currents evoked by 30 µM ATP in HEK 293 cells transfected with P2X₄. The data shown are the mean+s.e.m.; n=5. (A-D: ^*P<0.05; ^**P<0.01; ^***P<0.001; n.s., not significant.) Patch-clamp measurements were performed at RT.