Fig. 3

Forward and reverse mode Na⁺–Ca²⁺ exchanger. a After knockdown of MCU and MFN2, matrix Ca²⁺ oscillations are induced by LTC₄ in FCCP-treated cells following a small rise in matrix Ca²⁺ by 2 nM ionomycin. Oscillations were seen in 15/24 cells. CGP-37157 suppressed this response (15/15 cells). b As in panel a but LTC₄ is now applied first. Oscillations were seen in 10/15 cells. CGP-37157 blocked the response in 18/18 cells. c Aggregate data are compared. All groups were pre-treated with FCCP and oligomycin and both MCU and MFN2 were knocked down. Ionom-LTC₄ denotes ionomycin followed by LTC₄ (as in panel a). LTC₄-Ionom denotes LTC₄ then ionomycin, as in panel b. d Matrix Ca²⁺ is measured in permeabilised cells following stimulation with InsP₃. Cytosolic Na⁺ was 10 mM and cytosolic Ca²⁺ was weakly buffered at 200 nM. Ionomycin was applied to raise matrix Ca²⁺, then InsP₃ was added. F/O denotes FCCP/oligomycin. BAPTA was perfused to rapidly reduce cytosolic Ca²⁺. e As in panel d, but cytosolic Na⁺ was 1 mM. In the experiments with Li⁺, 9 mM Li⁺ was added to the 1 mM Na⁺ solution. f Aggregate data from experiments as in panels d and e are compared. Each bar denotes between 15 and 23 cells. g Ca²⁺ signals in permeabilised cells are compared in the absence and presence of cyclosporine A (1 μM). Here, mitofusin 2 had been knocked down and FCCP/oligomycin added 5–10 min before simulation with InsP₃. Control trace is in the absence of cyclosporine A. For both traces, 1 μM ruthenium red was present throughout. h Simulations of Na⁺–Ca²⁺ exchanger flux (middle panel, with pink highlighted background showing where NCX is operating in reverse mode) and matrix free Ca²⁺ (bottom panel) following a cytosolic Ca²⁺ rise (upper panel) obtained from fura 2-loaded intact cells. Cells were exposed to 2 nM ionomycin (asterisk) before LTC₄ challenge (marked by arrow; same protocol as in panel a). The cytosolic Ca²⁺ concentration indicated is the measured bulk concentration. i As in panel h but cytosolic Ca²⁺ has been estimated to reach 15 μM (see text). Error bars denote SEM