Mitochondria control store-operated Ca2+ entry through Na+ and redox signals

Abstract

Mitochondria exert important control over plasma membrane (PM) Orai1 channels mediating store-operated Ca²⁺ entry (SOCE). Although the sensing of endoplasmic reticulum (ER) Ca²⁺ stores by STIM proteins and coupling to Orai1 channels is well understood, how mitochondria communicate with Orai1 channels to regulate SOCE activation remains elusive. Here, we reveal that SOCE is accompanied by a rise in cytosolic Na⁺ that is critical in activating the mitochondrial Na⁺/Ca²⁺ exchanger (NCLX) causing enhanced mitochondrial Na⁺ uptake and Ca²⁺ efflux. Omission of extracellular Na⁺ prevents the cytosolic Na⁺ rise, inhibits NCLX activity, and impairs SOCE and Orai1 channel current. We show further that SOCE activates a mitochondrial redox transient which is dependent on NCLX and is required for preventing Orai1 inactivation through oxidation of a critical cysteine (Cys195) in the third transmembrane helix of Orai1. We show that mitochondrial targeting of catalase is sufficient to rescue redox transients, SOCE, and Orai1 currents in NCLX-deficient cells. Our findings identify a hitherto unknown NCLX-mediated pathway that coordinates Na⁺ and Ca²⁺ signals to effect mitochondrial redox control over SOCE.

Keywords: NCLX; SOCE; CRAC channel; mitochondrial redox; sodium signaling.

Figure 1. NCLX controls SOCE activity

1. Upper panel: Western blot was performed with 100 μg of protein samples obtained from HEK293T cells transfected with either siControl or siNCLX. Lower panel: Densitometry analysis of immunoblots of siControl‐transfected cells (n = 3) versus siNCLX‐transfected cells (n = 3) representing normalized expression levels (% of control) of NCLX, STIM1 and Orai1.

2. Upper panel: HEK293T cells were transfected with either scrambled siRNA construct (siControl, black) or siNCLX (red) and loaded with Fura‐2. SOCE was triggered by ATP (100 μM) and thapsigargin (TG, 1 μM), and Fura‐2 fluorescence was monitored as described in Materials and Methods. Lowe panel: Averaged rates of Ca²⁺ rise in siNCLX‐silenced cells from several independent recordings (n = 6) versus siControl‐transfected cells (n = 6).

3. Upper panel: Fluorescence of the membrane calcium sensor, GCamp5, targeted to the plasma membrane following treatment as described above using confocal microscopy. The scale bar represents 10 μm. Middle panel: Traces of membrane‐localized Ca²⁺ responses were measured in HEK293T cells co‐transfected with either the siNCLX (red) or siControl (black) and GCamp5‐expressing plasmid. The lower panel shows the averaged rates of membrane‐localized Ca²⁺ rise either in siControl cells (n = 6) or siNCLX cells (n = 10).

4. Electrophysiological recordings were performed on the same batch of transfected cells used for Western blot and shown in panel (A). Representative time courses of whole‐cell CRAC currents activated by dialysis of 20 mM BAPTA through the patch pipette and taken at −100 mV from cells transfected either with siControl (black trace) or siNCLX (red trace).

5. Representative I–V relationships are taken from traces in (D) where indicated by color‐coded asterisks.

6. Statistical analysis on Na⁺ CRAC currents measured at −100 mV is shown.

Data information: The results are presented as the means ± SEM. *P < 0.05; **P < 0.01; ***P < 1E‐03. P‐values indicate the results of an unpaired Student's t‐test.Source data are available online for this figure.

Figure 2. NCLX knockdown has no effect on Orai1 and STIM1 interaction following store depletion

1. A, B

   HEK293T cells stably expressing Orai1‐CFP and STIM1‐YFP were transfected with either control siRNA (A) or NCLX siRNA (B) and incubated for 72 h. E‐FRET experiments were then performed. The scale bar represents 20 μm.

2. C

   Silencing of NCLX by siNCLX (red) has no effect on Orai1 and STIM1 colocalization after store depletion induced by applying 2.5 μM ionomycin to the bath solution, compared to siControl (black; means ± SEM).

Figure EV1. NCLX knockdown has no effect on Orai1/STIM1 interactions measured with FRET

HEK293T cells expressing Orai1‐CFP and STIM1‐YFP were transfected with either control siRNA or NCLX siRNA and effect on Orai1/STIM1 corrected FRET after store depletion with 2.5 μM ionomycin (Iono) were compared. Bar scale: warmer colors indicate high FRET. The scale bar represents 10 μm.

Figure 3. Effect of NCLX knockdown on SOCE in primary vascular smooth muscle cells (VSMCs)

1. A

   Upper panel: Fluorescence traces of cytosolic Ca²⁺ responses in VSMC cells. Lower panel: Averaged rates of cytosolic Ca²⁺ influx in siControl cells (n = 5) and siNCLX cells (n = 6).

2. B, C

   Electrophysiological CRAC recordings in cells as in (A) using the same protocol used in Fig 2. Statistical analysis on Na⁺ CRAC currents measured at −100 mV is shown in (C).

Data information: The results are presented as the means ± SEM. *P < 0.05; ***P < 1E‐03. P‐values indicate the results of an unpaired Student's t‐test.

Figure 4. NCLX does not control CRAC channels through buffering of cytosolic Ca²⁺ or mitochondrial energization

1. A, B

   Analysis of Ca²⁺ (A) and Na⁺ (B) CRAC currents measured at −100 mV in HEK293T cells transfected with either siControl or siNCLX using the indicated concentrations of BAPTA (20 mM versus 50 mM) in the pipette solution to activate CRAC and buffer cytosolic Ca²⁺.

2. C–E

   Electrophysiological CRAC recordings were performed on cells where store depletion was induced by the use of a pipette solution containing low buffering capacity (0.1 mM EGTA) with IP₃ (30 μM) and a cocktail to energize the mitochondria whose composition is listed in the Results section. Representative time courses of whole‐cell current development at −100 mV (D, E) and measured in Ca²⁺‐containing and DVF solutions, respectively, by comparison with siControl. At the end of recordings, 5 μM Gd³⁺ was used to inhibit CRAC currents. Representative I–V relationships of Ca²⁺ (D) and Na⁺ (E) CRAC in cells transfected with siControl or siNCLX are taken from traces in (C) where indicated by color‐coded asterisks. Statistical analysis on Ca²⁺ and Na⁺ CRAC currents measured at −100 mV (D, E).

Data information: The results are presented as the means ± SEM. *P < 0.05; **P < 0.01 ***P < 1E‐03. P‐values indicate the results of a one‐way ANOVA test followed by Tukey post hoc analysis (A, B) or unpaired Student's t‐test (C–E).

Figure EV2. CRAC channels are enhanced under physiological conditions of energized mitochondria

1. A, B

   Electrophysiological CRAC recordings were performed on HEK293T cells where store depletion was induced by the use of a pipette solution containing 0.1 mM EGTA with IP₃ with (B) and without (A) inclusion of a cocktail to energize the mitochondria. At the end of recordings, 5 μM Gd³⁺ was used to inhibit CRAC currents.

2. C, D

   Representative I–V relationships of Ca²⁺ (C) and Na⁺ (D) CRAC in are taken from traces in (A) and (B) where indicated by color‐coded asterisks.

3. E, F

   Statistical analysis on Ca²⁺ and Na⁺ CRAC currents measured at −100 mV.

Data information: The results are presented as the means ± SEM. ***P < 1E‐03. P‐values indicate the results of an unpaired Student's t‐test.

Figure 5. Extracellular Na⁺ signaling controls Ca²⁺ influx through SOCE and CRAC channels

1. A, B

   Upper panel: Cytosolic Ca²⁺, Ca²⁺ [Cyto], responses monitored in HEK293T cells after store depletion by ATP and TG as in Fig 1B and upon extracellular Na⁺ ions replacement by either NMDG⁺ or Li⁺ ions. Lower panel: Averaged rates of cytosolic Ca²⁺ influx. The combined effects of NMDG⁺ together with silencing of NCLX on cytosolic Ca²⁺ influxes via SOCE are presented in (B).

2. C

   Upper panel: After being loaded with Fura‐2, intracellular Na⁺, [Na⁺]_i was preloaded by preincubation for 20 min with ouabain (100 μM) in the presence of Na⁺, following loading of intracellular Na⁺ SOCE protocol was applied as in Fig 1 using NMDG⁺‐containing Ringer. Lower panel: Averaged Ca²⁺ influx rates in NMDG⁺ [Na⁺]_i preloaded cells (n = 6), Na⁺ control cells (n = 14), and cells perfused with NMDG⁺ alone (n = 13).

3. D–H

   Representative Ca²⁺ CRAC current recordings and corresponding color‐matched I–V relationships measured in HEK293T cells with different monovalent cation‐based bath solution is shown in (D) for Na⁺, in (E) for NMDG⁺, and in (F) for Li⁺; 20 mM Ca²⁺ was included in all bath solutions. Statistical analysis on Ca²⁺ CRAC currents measured at −100 mV in all three conditions is shown in (G). The effects of cytosolic Na⁺ concentration on Ca²⁺ CRAC currents. (H) The whole‐cell patch clamp recordings were performed in HEK293T cells transiently expressing eYFP‐STIM1 and CFP‐Orai1. Scatter plots represent Ca²⁺ CRAC currents activated by dialysis through the patch pipette of a solution containing 20 mM BAPTA and the different concentration of Na⁺. Currents were measured at −100 mV and shown as current density pA/pF.

Data information: The results are presented as the means ± SEM. *P < 0.05; **P < 0.01 ***P < 1E‐03. P‐values indicate the results of a one‐way ANOVA test followed by Tukey post hoc analysis.

Figure EV3. Substitution of Na⁺ by NMDG⁺ inhibits CRAC currents in HEK293T and RBL cells

1. A–C

   Scatter plots representing Ca²⁺ CRAC currents activated either by dialysis through the patch pipette of a solution containing either 0.1 mM EGTA+IP₃ (A) or 20 mM BAPTA (B) in HEK293T cells, or in RBL cells activated by dialysis of a solution containing 20 mM BAPTA (C). P‐values (A‐C) indicate the results of an unpaired student's t‐test.

Data information: The results are presented as the means ± SEM. **P < 0.01 ***P < 1E‐03. P‐values indicate the results of an unpaired Student's t‐test.

Figure 6. Depletion of intracellular Ca²⁺ stores triggers cytosolic Na⁺ influx which drives NCLX‐associated mitochondrial Na⁺ influx and Ca²⁺ efflux

1. Upper panel: Fluorescence traces of cytosolic Na⁺ responses, Na⁺ [Cyto], in ATP‐depleted cells (n = 7) versus control non‐depleted HEK293T cells (n = 5) loaded with Asanta Natrium. Na⁺ influx averaged rates are shown in the lower panel.

2. Upper panel: Fluorescence traces of cytosolic Na⁺. Lower panel: Averaged rates of cytosolic Na⁺ influx in cells superfused with either Na⁺‐ (n = 11) or NMDG⁺ (n = 7)‐containing Ringer's solution.

3. Upper panel: Traces of mitochondrial Na⁺ transport in cells preloaded with the mitochondrial Na⁺ dye, CoroNa red in HEK293T cells. Lower panel: Mitochondrial Na⁺ influx rates in the presence (n = 7) or absence (n = 5) of Na⁺.

4. Upper panel: Traces of mitochondrial Ca²⁺, Ca²⁺ [Mito], recorded by monitoring RP‐mt fluorescence following Ca²⁺‐store depletion in HEK293T cells by ATP and TG as in Fig 1B in the presence of Na⁺ or NMDG⁺. Lower panel: Mitochondrial Ca²⁺ efflux in cells in the presence of NMDG⁺ (n = 8) compared to Na⁺ (n = 11).

5. Upper panel: Traces of Ca²⁺ [Mito] of cell superfused with ATP alone in Ca²⁺‐free Ringer followed by superfusion with Ca²⁺ Ringer with or without Na⁺ in shControl cells versus shNCLX cells. Lower panel: Rates of traces shown in the upper panel in the presence (n = 4) or absence (n = 3) of Na⁺ ions in cells transfected with shControl (n = 3) or shNCLX (n = 3).

6. Upper panel: Cytosolic Na⁺‐dose dependence of Ca²⁺ [Mito] efflux. Lower panel: Mitochondrial Ca²⁺ efflux rates at the indicated concentrations of Na⁺ [mM] [0 (n = 4); 1, 5 and 20 (n = 6)].

Data information: The results are presented as the means ± SEM. *P < 0.05; **P < 0.01; ***P < 1E‐03; ****P < 1E‐04. P‐values indicate the results of an unpaired Student's t‐test (A, B and D) or one‐way ANOVA test followed by Tukey post hoc analysis (C, E and F).

Figure EV4. Knockdown of NCLX expression inhibits mitochondrial Ca²⁺ efflux and Na⁺ uptake

1. Upper panel: Mitochondrial Ca²⁺ was monitored in cells expressing the mitochondrial Ca²⁺ sensor RP‐mt. Mitochondrial Ca²⁺ transient was evoked by application of ATP and TG, added when indicated. Lower right panel: Rates of mitochondrial Ca²⁺ efflux derived from the upper panel. Lower left panel: The basal Ca²⁺ level (arbitrary units, AU) of shControl (n = 3) versus shNCLX (n = 4).

2. Upper panel: Mitochondrial Na⁺ uptake in HEK293T cells preloaded with the mitochondrial Na⁺ sensor, CoroNa red. Na⁺ signals were evoked by ATP and TG added when indicated. Lower panel: Rates of mitochondrial Na⁺ influx derived from the upper panel.

Data information: The results are presented as the means ± SEM. *P < 0.05. P‐values indicate the results of an unpaired Student's t‐test.

Figure 7. Mitochondrial catalase (m‐catalase) rescues SOCE, CRAC, and mitochondrial redox responses after NCLX knockdown

1. A

   Upper panel: Traces of mitochondrial roGFP1 fluorescence in HEK293T cells co‐transfected with either siControl or siNCLX and Ca²⁺‐ store depleted as described in Fig 1B. Changes in roGFP1 410/480 ratio fluorescence were determined using minimal and maximal values obtained by application of 100 mM DTT and 10 mM H₂O_2, respectively, as described in Materials and Methods. Lower panel: ΔF ratio of redox change after Ca²⁺ restoration to the external milieu in siControl cells (n = 8) compared to siNCLX cells (n = 12).

2. B

   Upper panel: Effect of NCLX on redox state determined by monitoring NAD(P)H intrinsic fluorescence in HEK293T cells, transfected with either siNCLX or siControl, and treated as described in Fig 1B. Oligomycin or FCCP were used for calibration and added where indicated. Lower panel: Averaged autofluorescence rates after adding Ringer's solution containing Ca²⁺.

3. C

   Upper panel: Effect of mitochondrial catalase (pZeoSV2 + mCat) expression. Lower panel: Rates of redox change in siNCLX + pZeoSV2 + (pZeo) empty vector transfected cells (n = 4) compared to siNCLX cells transfected with m‐catalase (n = 6).

4. D

   Upper panel: Fluorescence traces of cytosolic Ca²⁺ responses in HEK293T cells co‐transfected with either siControl or siNCLX together with pZeo or m‐catalase. Lower panel: Averaged rates of Ca²⁺ influx in cells transfected with siNCLX (n = 9) or cells co‐transfected with siNCLX and either m‐catalase (n = 9) or pZeo (n = 5) compared to cells transfected with siControl (n = 6) or co‐transfected with siControl and m‐catalase (n = 5).

5. E–H

   CRAC electrophysiological recordings in HEK293T cells activated by dialysis of 20 mM BAPTA through the patch pipette under the same conditions of transfection used in (B). I–V relationships are shown in (G) and statistical analysis on Na⁺ CRAC currents measured at −100 mV is shown in (H). m‐Catalase is routinely co‐transfected with a plasmid encoding eGFP for identification of transfected cells.

Data information: The results are presented as the means ± SEM. *P < 0.05; **P < 0.01 ***P < 1E‐03. P‐values indicate the results of an unpaired Student's t‐test (A–C) or one‐way ANOVA test followed by Tukey post hoc analysis (D–H).

Figure EV5. Na⁺ has no effect on SOCE in the presence of m‐catalase

1. A

   HEK293T cells were transfected with either siControl or siNCLX with or without m‐catalase. Cytosolic Ca²⁺ responses were monitored in HEK293T cells after store depletion by ATP and TG as in Fig 1B in the presence or absence of Na⁺ ions.

2. B, C

   Averaged rates (means ± SEM) of Ca²⁺ influx in either siControl cells (n = 4), siNCLX (n = 4), siNCLX+m‐catalase with Ringer containing Na⁺ (n = 4), or NMDG⁺ (n = 4) are shown in (B). P‐values indicate the results of a one‐way ANOVA test followed by Tukey post hoc analysis. *P < 0.05. The basal redox levels of siNCLX+pZeo cells (n = 6) versus siNCLX+m‐catalase (n = 4) are shown in (C).

Figure 8. C195S mutation in Orai1 prevents the inhibitory effect of SOCE and CRAC current caused by NCLX knockdown

1. A–C

   HEK293T cells were transfected with either control siRNA (black) or NCLX siRNA (red) and incubated for 72 h (A). Cells were then transfected again with siRNA along with either plasmids, STIM1 and Orai1 (B), or plasmids, STIM1 and C195S‐Orai1 (C), and incubated for another 24 h. The numbers in bar graphs (i.e., (x, y)) represent the total number of independent recording (x) and the total number of cells from all recordings (y). SOCE was triggered as described in Fig 1B, and Fura‐2 fluorescence was monitored as described in Materials and Methods. Averaged rates of Ca²⁺ influx were shown in the lower panels.

2. D–L

   Representative time course traces (D and G) of Orai1‐ and Orai1‐C195S‐mediated CRAC currents activated by dialysis through the patch pipette of a solution containing 20 mM BAPTA and recorded in a bath solution containing 20 mM Ca²⁺. I–V curves are shown in (E and H) which are taken from traces as indicated by color‐coded asterisks. Scatter blots (F and I) depict maximal CRAC currents values taken at −100 mV and represented as current densities (pA/pF). Similar CRAC current recordings from HEK293T cells expressing STIM1 and C126S/C143S/C195S‐Orai1 triple mutant are shown in (J–L).

Data information: The results are presented as the means ± SEM. *P < 0.05; ns, not significant. P‐values indicate the results of an unpaired Student's t‐test.

Figure 9. Na⁺ and Ca²⁺ signals are regulated by NCLX and mediate mitochondrial redox control of SOCE

A schematic illustration of the Na⁺ and Ca²⁺ signaling networks involving NCLX and SOCE cross talk. Ca²⁺ depletion of intracellular stores activates store‐operated Ca²⁺ entry (SOCE) pathway mediated through the highly Ca²⁺ selective, Ca²⁺ release‐activated Ca²⁺ (CRAC) currents. SOCE activation involves interaction upon store depletion of the endoplasmic reticulum (ER) Ca²⁺ STIM1 with the plasma membrane channel protein Orai1. Upon store depletion, rise of cytosolic Na⁺ also occurs, apparently, reflecting the activation of non‐selective transient potential channel canonical (TRPC) channel isoforms. Cytosolic Na⁺ is required for powering NCLX to activate mitochondrial Ca²⁺ efflux, thus ensuring proper mitochondrial Ca²⁺ shuttling. This tight regulation by NCLX of mitochondrial matrix Ca²⁺ homeostasis prevents the excessive Ca²⁺ rise in mitochondrial matrix that would otherwise lead to increases in mitochondrial reactive oxygen species (ROS) and subsequent inhibition of SOCE via oxidation of a reactive cysteine at position C195 on an extracellular loop of Orai1.