Figure 3.

Mild mitochondrial depolarization, triggered by chemical uncoupling, allosterically inhibits [Ca2+]m efflux by NCLX and is prevented by phosphomimetic mutant of NCLX^S271D. (A) Representative fluorescent traces of ΔΨm on SH-SY5Y cells co-expressing shNCLX and NCLX^WT (black) or mutant NCLX^S271A (purple)\NCLX^S271D (turquoise) preloaded with TMRM (30 μM for loading and 10 μM for washing in all experimental solutions). FCCP (5 μM) was added to calibrate the signal by inducing complete depolarization, observed as a drop in TMRM fluorescence. (B) Quantification of basal ΔΨm in (A) (Welch and Brown-Forsythe ANOVA, F(2,135.3) = 1.414;Dunnett’s T3 multiple comparisons test; NCLX^WT vs. NCLX^S271A: ns; NCLX^WT vs. NCLX^S271D: ns; n > 3). (C–E) Representative fluorescent traces of ATP (100 μM) induced [Ca²⁺]m transients in Cepia2mt expressing SH-SY5Y cells co-expressing shNCLX and NCLX^WT (black or red with BAM15), or mutant NCLX^S271A (purple or blue with BAM15)\NCLX^S271D (turquoise or green with BAM15) treated and untreated with BAM15 (5 μM) for 15 min. (F) Quantification of [Ca²⁺]m efflux rates (C–E) (Welch and Brown-Forsythe ANOVA, F(5,279.1) = 4.762; Dunnett’s T3 multiple comparisons test; NCLX^WT vs. NCLX^WT+BAM15: *p = 0.0380; NCLX^WT vs. NCLX^S271A: *p = 0.0195; NCLX^WT vs. NCLX^S271A+BAM15: ****p > 0.0001; NCLX^WT vs. NCLX^S271D: ns; NCLX^WT vs. NCLX^S271D+BAM15: ns; n > 3). ns—not significant. Error bars denote SEM.