Uncoupling protein 3 (UCP3) modulates the activity of Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) by decreasing mitochondrial ATP production

Abstract

The uncoupling proteins UCP2 and UCP3 have been postulated to catalyze Ca(2+) entry across the inner membrane of mitochondria, but this proposal is disputed, and other, unrelated proteins have since been identified as the mitochondrial Ca(2+) uniporter. To clarify the role of UCPs in mitochondrial Ca(2+) handling, we down-regulated the expression of the only uncoupling protein of HeLa cells, UCP3, and measured Ca(2+) and ATP levels in the cytosol and in organelles with genetically encoded probes. UCP3 silencing did not alter mitochondrial Ca(2+) uptake in permeabilized cells. In intact cells, however, UCP3 depletion increased mitochondrial ATP production and strongly reduced the cytosolic and mitochondrial Ca(2+) elevations evoked by histamine. The reduced Ca(2+) elevations were due to inhibition of store-operated Ca(2+) entry and reduced depletion of endoplasmic reticulum (ER) Ca(2+) stores. UCP3 depletion accelerated the ER Ca(2+) refilling kinetics, indicating that the activity of sarco/endoplasmic reticulum Ca(2+) (SERCA) pumps was increased. Accordingly, SERCA inhibitors reversed the effects of UCP3 depletion on cytosolic, ER, and mitochondrial Ca(2+) responses. Our results indicate that UCP3 is not a mitochondrial Ca(2+) uniporter and that it instead negatively modulates the activity of SERCA by limiting mitochondrial ATP production. The effects of UCP3 on mitochondrial Ca(2+) thus reflect metabolic alterations that impact on cellular Ca(2+) homeostasis. The sensitivity of SERCA to mitochondrial ATP production suggests that mitochondria control the local ATP availability at ER Ca(2+) uptake and release sites.

FIGURE 1.

Effect of UCP3 knockdown on mitochondrial and cytosolic Ca²⁺ elevations. A and B, HeLa cells were transiently co-transfected with the mitochondrial Ca²⁺ probe 4mtD3cpv (A) or the cytosolic Ca²⁺ probe YC3.6_cyto (B) and with either scrambled siRNA (control siRNA) or UCP3-specific siRNA (UCP3 siRNA) for 48 h. A, left, averaged [Ca²⁺]_mit recordings in HeLa cells stimulated with 100 μm histamine. Right, statistical evaluation of the UCP3 siRNA effects on the amplitude of the [Ca²⁺]_mit signal evoked by histamine (upper panel) and on the integrated [Ca²⁺]_mit response (lower panel). Bars are mean ± S.E. of 80 (n = 7) and 101 cells (n = 7) for Ctrl and UCP3 siRNA, respectively. AUC, area under the curve. B, left, averaged [Ca²⁺]_cyto recordings in HeLa cells stimulated with 100 μm histamine. Right, statistical evaluation of the UCP3 siRNA effects on the amplitude of the [Ca²⁺]_cyto signal evoked by histamine (upper panel) and on the integrated [Ca²⁺]_cyto response (lower panel). Bars are mean ± S.E. of 40 (n = 3) and 37 cells (n = 3) for Ctrl and UCP3 siRNA, respectively. ***, p < 0.001.

FIGURE 2.

Effect of UCP3 knockdown on Ca²⁺ release and influx. A and B, HeLa cells were transiently co-transfected with the cytosolic Ca²⁺ probe YC3.6_cyto (A) or the mitochondrial Ca²⁺ probe 4mtD3cpv (B) and with Ctrl (scramble) siRNA or the UCP3 siRNA for 48 h, washed, and stimulated with 100 μm histamine in Ca²⁺-free medium to deplete intracellular Ca²⁺ stores. Then Ca²⁺ was readmitted to monitor Ca²⁺ influx from plasma membrane. A, average of cytosolic Ca²⁺ responses (left) elicited by histamine and by Ca²⁺ readmission. Right, statistical evaluation of the UCP3 siRNA on the integrated [Ca²⁺]_cyto response during release from ER and during Ca²⁺ readmission. Bars are mean ± S.E. of 84 (n = 9) and 86 cells (n = 8) for Ctrl and UCP3 siRNA, respectively. AUC, area under the curve. B, average of mitochondrial Ca²⁺responses elicited by histamine and Ca²⁺ readmission (left) and related statistical evaluation (right) on integrated [Ca²⁺]_cyto response during Ca²⁺ release and influx. Bars are mean ± S.E. of 77 (n = 8) and 59 cells(n = 7) for Ctrl and UCP3 siRNA, respectively. *, p < 0.05; ***, p < 0.001. NS, not significant.

FIGURE 3.

Effect of UCP3 knockdown on ER Ca²⁺ release. HeLa cells were transiently co-transfected with the ER Ca²⁺ probe D1_ER and with the Ctrl (scramble) siRNA or the UCP3 siRNA for 48 h and washed, and Ca²⁺ responses were measured. A, resting D1_ER ratio values of 73 (n = 8) and 77 (n = 8) cells, transfected with Ctrl and UCP3 siRNA, respectively. NS, not stimulated. B, averaged [Ca²⁺]_ER recordings of HeLa cells stimulated with 100 μm histamine in Ca²⁺-free medium and related statistical evaluation (inset) of the UCP3 siRNA effects on the kinetics of Ca²⁺ release. For the latter statistics, the D1_ER responses were fitted with a one-phase exponential decay function to extract the half-time. Bars are mean ± S.E. of 73 (n = 8) and 77 cells (n = 8) for Ctrl and UCP3 siRNA, respectively. ***, p < 0.001. C, the same as in B, but stimulating the cells with 1 μm TG. Bars are mean ± S.E. of 69 (n = 8) and 74 cells (n = 9) for Ctrl and UCP3 siRNA, respectively. D, the same as in B, but preincubating the cells with 1 μm TG 40 s before histamine stimulation. Bars are mean ± S.E. of 59 (n = 5) and 54 cells (n = 5) for Ctrl and UCP3 siRNA, respectively.

FIGURE 4.

Effect of UCP3 knockdown on ER Ca²⁺ refilling in intact and permeabilized cells. A, SERCA2 immunoblot of HeLa cells transfected with Ctrl or UCP3 siRNA for 48 h. 50 μg/lane of protein from cell extracts was analyzed, using actin as loading control. B, left, averaged [Ca²⁺]_ER recordings of intact HeLa cells during ER Ca²⁺ refilling. After 15 μm BHQ induced Ca²⁺ release in Ca²⁺-free medium, cells were washed, and Ca²⁺ was then added to assess the kinetics of store refilling. Data were fitted with the sigmoidal equation to extract the EC₅₀ (right), and they are mean ± S.E. of 40 (n = 5) and 30 cells (n = 4) for Ctrl and UCP3 siRNA, respectively. **, p < 0.01. C, left, averaged [Ca²⁺]_ER recordings during ER Ca²⁺ release and refilling in permeabilized cells. The K⁺-rich intracellular buffer was supplemented with 1 mm Mg-ATP and 1 mm MgCl₂ to allow ER Ca²⁺ refilling. Where indicated, 100 μm digitonin (dig), 15 μm BHQ, and 100 nm free Ca²⁺ were added. Right, statistical evaluation of the ER Ca²⁺ refilling kinetics. EC₅₀ was determined as described for B. Data are mean ± S.E. of 47 (n = 3) and 33 cells (n = 3) for Ctrl and UCP3 siRNA, respectively. NS, not stimulated.

FIGURE 5.

Effect of SERCA inhibition or cell permeabilization on mitochondrial Ca²⁺ elevation. A, the same protocols and conditions as in Fig. 2 were used to deplete Ca²⁺ stores and to monitor Ca²⁺ release and then the influx component in mitochondria, but in the presence of the SERCA pump inhibitor 1 μm TG. Histamine was 100 μm. Left, average of [Ca²⁺]_mit recordings from Ctrl (scramble) siRNA or the UCP3 siRNA cells. Right, statistical evaluation of the UCP3 knockdown on the integrated [Ca²⁺]_mit responses evoked by histamine or after Ca²⁺ readmission, from data shown in the left panel. Bars are mean ± S.E. of 51 (n = 5) and 45 cells (n = 4) for Ctrl and UCP3 siRNA, respectively. NS, not stimulated. AUC, area under the curve. B, Ru360-sensitive mitochondrial Ca²⁺-uptake in permeabilized cells, in ATP-depleted medium. HeLa cells were transiently co-transfected with the mitochondrial calcium probe 4mtD3cpv and the indicated siRNAs. After permeabilization with digitonin, [Ca²⁺]_mit was measured in intracellular buffer. Left, original [Ca²⁺]_mit recordings of permeabilized HeLa cells during the addition of 3.5 μm free Ca²⁺ in the presence of or after wash-out of the mitochondrial Ca²⁺ uniporter inhibitor Ru360 (10 μm). Right, statistical evaluation of UCP3 siRNA effects on the slope of Ca²⁺ elevation. For the latter statistics, the Ca²⁺ responses for each trace were fitted with a linear function, and the Ru360-dependent slope was subtracted from the following one in the absence of the inhibitor. Bars are mean ± S.E. of 79 (n = 6) and 67 (n = 6) cells for Ctrl and UCP3 siRNA, respectively.

FIGURE 6.

Effect of UCP3 knockdown on mitochondrial and cytosolic [ATP]. HeLa cells were transiently transfected with the mitochondrial or the cytosolic ATP probes ATeam_mito or ATeam_cyto, respectively, together with the indicated siRNA for 48 h. A, averaged [ATP]_mit changes elicited by 100 μm histamine and inhibition of mitochondrial ATP synthesis with oligomycin A (10 μg/ml) in cells perfused with 10 mm glucose as metabolic substrate and after inhibition of glycolysis with 10 mm 2-deoxyglucose. B, upper panels, mitochondrial (left) and cytosolic (right) ATeam signals recorded at 535 nm. Left lower panel, statistical evaluation of the histamine-induced, oligomycin A-sensitive [ATP]_mit changes. Bars are mean ± S.E. of n = 4 (44 cells) and n = 5 (59 cells) for Ctrl and UCP3 siRNA, respectively. Right lower panel, statistical evaluation of the histamine-induced, oligomycin A-sensitive [ATP]_cyt changes. Bars are mean ± S.E. of n = 5 (60 cells) and n = 5 (64 cells) for Ctrl and UCP3 siRNA, respectively. [ATP]_cyt changes were recorded by applying the same protocol as in A. *, p < 0.05. NS, not stimulated.