1. Allosteric Regulation of NCLX by Mitochondrial Membrane Potential Links the Metabolic State and Ca2+ Signaling in Mitochondria 
Marko Kostic, Tomer Katoshevski, Israel Sekler 
Cell Reports 
Volume 25, Issue 12, Pages e4 (December 2018)
DOI: /j.celrep
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2. Cell Reports 2018 25, 3465-3475.e4DOI: (10.1016/j.celrep.2018.11.084)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
Mild Mitochondrial Depolarization Triggered by Chemical Uncoupling Allosterically Inhibits Mitochondrial Ca2+ Efflux by NCLX
(A) Representative fluorescent traces of mitochondrial membrane potential (ΔΨm) in BAM15 (5 μM)-pretreated and untreated (control) SH-SY5Y cells, preloaded with TMRM. FCCP (5 μM) was added (marked by black arrows) to calibrate the signal by inducing full depolarization, observed as a drop in TMRM fluorescence.
(B) Quantification of basal ΔΨm of (A), compared with control. Application of BAM15 caused mild mitochondrial depolarization.
(C) Representative fluorescent traces of mitochondrial Ca2+ transients evoked by 100 μM ATP and monitored by the mitochondrial targeted Ca2+ reporter Cepia2mt in untreated SH-SY5Y cells (control) and BAM15-pretreated SH-SY5Y cells.
(D and E) Averaged rates of mitochondrial Ca2+ influx (D) and mitochondrial Ca2+ efflux (E) of (C) compared with control.
(F) Representative fluorescent traces of ΔΨm changes in rhodamine 123-loaded SH-SY5Y cells treated with the indicated concentrations of FCCP. Note that depolarization was observed as an increase in the fluorescence of rhodamine 123 (opposite to TMRM-based measurement of ΔΨm).
(G) Quantification of depolarization induced by 1 μM FCCP, relative to 5 μM FCCP (set as 100% depolarization) from (F). Partial mitochondrial depolarization was induced by 1 μM FCCP.
(H) Representative fluorescent traces of mitochondrial Ca2+ transients evoked by 100 μM ATP and monitored by the mitochondrial targeted Ca2+ reporter Cepia2mt, in untreated SH-SY5Y cells (control) and SHSY-5Y cells pretreated with 1 μM FCCP.
(I and J) Averaged rates of mitochondrial Ca2+ influx (I) and mitochondrial Ca2+ efflux (J) of (H), compared with control.
The n number of each experiment is indicated at the bar graphs. Error bars denote SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, not significant. Note that mild mitochondrial depolarization by BAM15 or FCCP does not change mitochondrial Ca2+ influx but strongly reduces mitochondrial Ca2+ efflux rates.
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4. Figure 2
Phosphorylation of NCLX by PKA Rescues Mitochondrial Ca2+ Efflux during Mild Mitochondrial Depolarization
(A) Representative fluorescent traces of mitochondrial Ca2+ transients recorded by Cepia2-mt in untreated (control) (left), BAM15-pretreated (center), and BAM15 and forskolin co-treated SH-SY5Y cells (right).
(B) Averaged rates of mitochondrial Ca2+ efflux of (A) compared with control.
(C) Western blot of SH-SY5Y cells treated with forskolin for the indicated time and then probed with pSer258-NCLX antibody (see STAR Methods).
(D) Immunoprecipitation by c-myc antibody, followed by western blot with pSer258-NCLX antibody, of lysates form cells expressing c-myc-tagged WT NCLX or S258A NCLX mutant, pretreated with forskolin in the presence or absence of the PKA inhibitor H-89.
(E and F) Representative fluorescent traces of ATP-induced mitochondrial Ca2+ transients in Cepia2mt expressing SH-SY5Y cells co-expressing either WT NCLX (E) or S258D phosphomimetic mutant of NCLX (F) in the presence (+) or absence (-) of BAM15 compared with WT.
(G) Averaged rates of mitochondrial Ca2+ efflux of traces derived from (E) and (F) compared with WT.
The n number of each experiment is indicated at the bar graphs. Error bars denote SEM. ∗p < 0.05 and ∗∗p < 0.01; ns, not significant. Note that NCLX phosphorylation overrides the inhibition of mitochondrial Ca2+ efflux by mild mitochondrial depolarization.
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5. Figure 3
Allosteric Regulation of NCLX by ΔΨm Is Mediated by Interaction of S258 with a Cluster of Positive Residues Located at the Regulatory Domain-Membrane Interface
(A) Predicted membrane topology of NCLX. PKA regulatory site (see STAR Methods and Elazar et al., 2016) Ser258 (in green) lies on the regulatory loop of NCLX. Two clusters of positively charged Arg-R residues (in blue) and Lys-K residues (in purple) lie in the interface of the regulatory domain and transmembrane domains. Conserved catalytic helical loops (α1 and α2) are marked in yellow. Positively charged R and K residues were replaced by charge-neutral Gln-Q in S258D NCLX construct.
(B) Western blot of mutants described in (A). Indicated mutants were expressed in SH-SY5Y cells and detected from purified mitochondrial fraction by anti-NCLX antibody. Mitochondria-specific protein VDAC was used as a loading control.
(C–H) Representative fluorescent traces of ATP-induced mitochondrial Ca2+ transients in Cepia2mt expressing SH-SY5Y cells co-expressing the indicated mutants: S258D (C), R253Q/S258D (D), R255Q/R256Q/S258D (E), K325Q/S258D (F), K328Q/S258D (G), and K311Q/S258D (H) in the presence (+) or absence (-) of BAM15.
(I) Averaged rates of mitochondrial Ca2+ efflux of indicated NCLX mutants from (C)–(H) in the absence (-) and presence (+) of BAM15 compared with S258D.
The n number of each experiment is indicated at the bar graphs. Error bars denote SEM. ∗p < 0.05 and ∗∗p < 0.01; ns, not significant
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6. Figure 4
UCP1 Triggers a Physiological Allosteric Regulation of NCLX by Modulating ΔΨm
(A) Changes in basal ΔΨm were determined in untransfected HEK293-T cells (control) and UCP1-expressing HEK293-T cells after application of palmitate (Palm.). FCCP (5 μM) was added (when indicated by the arrow) to calibrate the signal by inducing full depolarization.
(B) Quantification of the basal ΔΨm of (A) compared with control + Palm. Palmitate treatment caused mild mitochondrial depolarization in UCP1-expressing cells.
(C) Representative fluorescent traces of ATP-induced mitochondrial Ca2+ transients in control and UCP1-expressing HEK293-T cells co-expressing mitoPericam pretreated with palmitate.
(D and E) Averaged rates of mitochondrial Ca2+ influx (D) and mitochondrial Ca2+ efflux (E) of (C) compared with control + Palm.
(F and G) Representative fluorescent traces of mitochondrial Ca2+ recorded by Cepia2mt and induced by ATP and thapsigargin in HEK293-T cells, co-expressing UCP1 and WT NCLX (F) or S258D NCLX (G). Cells were treated either with BSA (control) or palmitate (to induce depolarization).
(H) Averaged rates of mitochondrial Ca2+ efflux derived from traces in (F) and (G) compared with WT NCLX+BSA.
The n number of each experiment is indicated at the bar graphs. Error bars denote SEM. ∗p < 0.05 and ∗∗∗p < 0.001; ns, not significant. Note that mild depolarization induced by UCP1 inhibits mitochondrial efflux but not mitochondrial Ca2+ influx.
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7. Figure 5
Glucose-Induced Change in ΔΨm Physiologically Regulates Mitochondrial Ca2+ Efflux by NCLX in β Cells
(A) A schematic depiction on the link between glucose-induced change in ΔΨm, mitochondrial metabolic rate, and insulin secretion.
(B) Glucose-dependent changes in ΔΨm were recorded in TMRM-loaded INS-1 cells. Representative fluorescent traces of ΔΨm in INS-1 cells under low (3mM) glucose (left), after addition of high glucose (20 mM) (center) or after addition of high glucose (20 mM) in presence of BAM15 (right). High glucose induced mitochondrial repolarization, which was prevented by addition of uncoupler.
(C) Representative traces of ATP-induced mitochondrial Ca2+ transients in INS-1 cells loaded with mitochondrial Ca2+ dye Rhod-2AM and treated as in (B). The black lines are the linear fit of mitochondrial Ca2+ efflux rate.
(D and E) Averaged rates of mitochondrial Ca2+ influx (D) and mitochondrial Ca2+ efflux (E) of (C) compared with control (3 mM glucose).
The n number of each experiment is indicated at the bar graphs. Error bars denote SEM. ∗p < 0.01; ns, not significant.
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8. Figure 6
NCLX Is Dually and Allosterically Regulated by ΔΨm and Phosphorylation at S258 by PKA
(A) In polarized mitochondria, NCLX mediates mitochondrial Ca2+ efflux in exchange for Na+ influx. Serine residue at position 258 is crucial for the allosteric regulation of the exchanger, and replacing it with Ala inactivates NCLX.
(B) Mild mitochondrial depolarization allosterically inhibits mitochondrial Ca2+ efflux by NCLX.
(C) PKA-mediated phosphorylation reactivates NCLX by triggering a functional interaction between S258 and membrane positively charged docking sites that control NCLX activity during mitochondrial depolarization.
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