Fig. 5: NCLX functional determination.

a, Mito-NCX activity in indicated cell lines. Cells were digitonin-permeabilized with calcium green 5N (CG5N) for reporting extra-mitochondrial Ca²⁺. Ca²⁺ (10 µM) increases CG5N fluorescence, followed by a signal reduction reflecting mitochondrial Ca²⁺ uptake. After Ru360 inhibits Ca²⁺ uptake, 10 mM Na⁺ induces mito-NCX, abolished by 5 µM CGP-37157. Mito-NCX rates are summarized in the bar chart. b, Effect of expressing NCLX constructs on mito-NCX in HEK cells. Ca²⁺ efflux traces (top left), efflux rates (bottom left) and Western blots (right) compare activity and expression levels. Control: no NCLX overexpression. c, Mitochondrial Ca²⁺ transport in Sf9 cells. Top: 10 mM Na⁺ fails to elicit mito-NCX with or without human NCLX overexpression in permeabilized Sf9 cells. Bottom: an MCU–EMRE fusion protein (hME) with a D261A substitution induces Ru360-insensitive mitochondrial Ca²⁺ uptake. Ru360 was also added before Ca²⁺ addition to inhibit native uniporter activity. d, ⁴⁵Ca²⁺ influx into Xenopus oocytes expressing indicated human NCLX constructs. Solid lines indicate linear fits used to obtain Ca²⁺ uptake rates. 2DA, D153A–D471A. e, Impact of CGP-37157 or NCLX substitutions on Ca²⁺ uptake into oocytes. Expression levels of mutants are 80–120% of WT. Uninjected, no RNA injection; 2DE, D153E–D471E. P values were obtained by comparing with the WT. f,g, Sensitivity of WT NCLX activity to external pH, Na⁺ (100 mM) or K⁺ (100 mM). The Ca²⁺-uptake rate summary and pH-dependence raw data are shown in f and g, respectively. Solid lines represent linear fits. h, H⁺-coupled Ca²⁺ flux. Oocytes expressing WT NCLX and preloaded with ⁴⁵Ca²⁺ were exposed to the indicated external pH. Intracellular ⁴⁵Ca²⁺ measured at various time points were normalized to the average count at t = 0. CPM, count per minute. i, NICE in HeLa cells. Following CG5N addition (initial signal jump) and digitonin-induced slow signal decline, Ru360 addition inhibits the uniporter and reveals NICE, as also shown in the zoomed-in traces (bottom left) and quantified in the bar chart. RES, NICE rescue by expressing WT NCLX in NCLX KO cells. Numbers in parentheses indicate independent biological repeats. The molecular mass marker unit is kilodaltons. Data show the mean ± s.e.m. Statistics was performed using an unpaired, two-tailed t-test (significant at P < 0.05). Refer to Supplementary Fig. 1 for gel source data. a.u., arbitrary unit.

Source Data