Fig. 5: Activation of LRRC8 mediates ATP release by THP-1 macrophages.

a ATP levels in supernatants of control and LRRC8A-KD THP-1 cells under isotonic or hypotonic solutions ± DCPIB (n = 6–15 from 6 independent experiments). b Illustration of the PNG6 biosensor fluorescence change upon extracellular ATP binding. c Kinetics of ATP release evoked by hypotonic solution in control and LRRC8A-KD THP-1 cells expressing the PNG6 biosensor with/without DCPIB. Maximum fluorescence was evoked by ATP application at the end of the recordings (n = 5 recordings from 3 independent experiments). d–f ATP whole-cell currents in control THP-1 cells in isotonic and hypotonic ATP bath solutions with/without DCPIB (d). I/V curves (e) and mean currents at -100 mV (f) in isotonic (black) and hypotonic solution (green, 5 min) and with DCPIB (light green). n = 6 independent recordings. g Lentiviral construction expressing PNG6-P2A-Scarlet and images showing ATP release (green PNG6) and surface/volume decrease (red mScarlet) in control THP-1 cells under hypotonic solution. Yellow and blue arrows identified cells with different time responses. Scale bar 20 μm. n = 3 independent experiments. h Single-cell video recording of surface change and ATP release in control and LRRC8A-KD THP-1 cells expressing PNG6. Colored lines represent individual cells; black lines show mean surface change and ATP-evoked fluorescence. i Surface change (upper) and ATP release (lower) in 3 cells during a hypotonic challenge. j Time correlation between ATP release event and RVD induction (n = 32 cells from 2 independent experiments). k ATP levels in supernatants of control and LRRC8A-KD THP-1 cells exposed to MSU or CPP crystals with/without DCPIB. n = 8–23 from 8 independent experiments (control THP-1) and n = 9–21 from 7 independent experiments (LRRC8A-KD cells). Values are mean ± SEM. One-way ANOVA (f), 2-way ANOVA, Sidak’s multiple comparisons test (a, k). Spearman correlation test (j).