Fig. 1: Mitochondrial Ca²⁺ extrusion in BA is mediated by Na⁺/Ca²⁺ exchange and requires PKA.

a Representative traces of mitochondrial Ca²⁺ kinetics in primary brown adipocytes (BA) after application of NE (1.5 μM). Mitochondrial Ca²⁺ was monitored using the mitochondrial Ca²⁺ dye, Rhode2-AM. The dashed lines represent the linear fit used to calculate the Ca²⁺ influx and efflux rates. b Representative traces of mitochondrial Ca²⁺ transients upon application of NE to BA in the presence or absence of Na⁺ (NMDG⁺ iso-osmotically replacing Na⁺). c Quantification of mitochondrial Ca²⁺ efflux rates in the presence and absence of Na⁺. NMDG⁺ was used as a cationic replacement of Na⁺ (n = 4 independent experiments per condition). d Representative traces of mitochondrial Ca²⁺ transients upon application of NE (1.5 μM), ATP (100 μM), or ATP in cells pretreated with PKA activator, Forskolin (FSK (50 μM, 15 min)). e, f Quantification of mitochondrial Ca²⁺ fluxes in response to NE, ATP, or ATP+FSK stimulation. NE (n = 12), ATP (n = 4), ATP+FSK (n = 6). g Representative traces of mitochondrial Ca²⁺ transients in primary BA upon stimulation by NE in the presence and absence of PKA inhibitor, H-89 (5 μM, 1 h pre-incubation). h, i Quantification of mitochondrial Ca²⁺ traces under treatment of H-89 (n = 10 and 14 for control and H-89 in h, n = 8 and 13 for control and H-89 in i). Student’s t-test (c, h, i); one-way ANOVA with Tukey’s post hoc test (e, f). Data are expressed as means ± SEM. ^nsp > 0.05, *p < 0.05, **p < 0.001. Replicates are indicated by individual dots shown for each group in all graphs. Source data are available as a Source Data file.