Calcium-regulation of mitochondrial respiration maintains ATP homeostasis and requires ARALAR/AGC1-malate aspartate shuttle in intact cortical neurons

Abstract

Neuronal respiration is controlled by ATP demand and Ca2+ but the roles played by each are unknown, as any Ca2+ signal also impacts on ATP demand. Ca2+ can control mitochondrial function through Ca2+-regulated mitochondrial carriers, the aspartate-glutamate and ATP-Mg/Pi carriers, ARALAR/AGC1 and SCaMC-3, respectively, or in the matrix after Ca2+ transport through the Ca2+ uniporter. We have studied the role of Ca2+ signaling in the regulation of mitochondrial respiration in intact mouse cortical neurons in basal conditions and in response to increased workload caused by increases in [Na+]cyt (veratridine, high-K+ depolarization) and/or [Ca2+]cyt (carbachol). Respiration in nonstimulated neurons on 2.5-5 mm glucose depends on ARALAR-malate aspartate shuttle (MAS), with a 46% drop in aralar KO neurons. All stimulation conditions induced increased OCR (oxygen consumption rate) in the presence of Ca2+, which was prevented by BAPTA-AM loading (to preserve the workload), or in Ca2+-free medium (which also lowers cell workload). SCaMC-3 limits respiration only in response to high workloads and robust Ca2+ signals. In every condition tested Ca2+ activation of ARALAR-MAS was required to fully stimulate coupled respiration by promoting pyruvate entry into mitochondria. In aralar KO neurons, respiration was stimulated by veratridine, but not by KCl or carbachol, indicating that the Ca2+ uniporter pathway played a role in the first, but not in the second condition, even though KCl caused an increase in [Ca2+]mit. The results suggest a requirement for ARALAR-MAS in priming pyruvate entry in mitochondria as a step needed to activate respiration by Ca2+ in response to moderate workloads.

Figure 1.

Bioenergetic characterization of aralar WT, aralar KO, SCaMC-3-WT, and SCaMC-3-KO cultured neurons. A, Representative pharmacological profile of oxygen consumption rate in aralar WT neurons showing the sequential injection of agonist/vehicle (Veh) and metabolic inhibitors: oligomycin (Olig, 6 μm), 2,4-dinitrophenol (DNP, 0.5 mm) and antimycin A/rotenone (Ant/Rot, 1.0 μm/1.0 μm) at different time point indicated by dashed lines which allows the determination of basal oxygen consumption (BR), oxygen consumption linked to ATP synthesis (ATP), non-ATP linked oxygen consumption (Leak), maximal uncoupled respiration (MUR) and nonmitochondrial oxygen consumption (NM) as represented graphically. B, C, Effect of glucose and Ca²⁺ on maximal uncoupled respiration (MUR) in aralar WT vs aralar KO neurons and SCaMC-3-WT versus SCaMC-3-KO neurons. D, Oxygen consumption rate in SCaMC-3-WT versus KO neurons in 2.5 mm glucose, 2 mm Ca²⁺. E, F, Effect of glucose and Ca²⁺ on basal respiration capacity in aralar WT versus aralar KO and SCaMC-3-WT versus SCaMC-3-KO neurons. Results are means ± SEM of 3–11 experiments. The effect of Ca²⁺ on MUR was significant for aralar WT (p < 0.05, two-way ANOVA) but not aralar KO neurons, and the lack of ARALAR caused a significant decrease in MUR at all glucose concentrations in the absence (p < 0.05, two-way ANOVA) or presence of Ca²⁺ (p < 0.001, two-way ANOVA; ##p < 0.01, post hoc Bonferroni test). Glucose, but not Ca²⁺, had a significant effect on basal respiration both in aralar WT (p < 0.01, two-way ANOVA) and aralar KO neurons (p < 0.001, two-way ANOVA) and the lack of ARALAR caused a significant decrease in basal respiration both in the presence (p < 0.001, two-way ANOVA) or absence (p < 0.01, two-way ANOVA) of Ca²⁺. G, H, Oxygen consumption profile in aralar WT and aralar KO cultures in 2.5 mm glucose medium supplemented with 2 mm pyruvate (Pyr) in the presence and absence of 2 mm Ca²⁺. I, MUR in the presence or absence of 2 mm pyruvate, expressed as percentage of basal OCR in aralar WT and aralar KO neurons (n = 3–11 experiments, Student's t test; *p ≤ 0.05, ***p ≤ 0.001). Data are expressed as mean ± SEM. Statistical analysis was performed using STATISTICA, version7, StatSoft.

Figure 2.

Changes in cytosolic and mitochondrial Ca²⁺, cytosolic ATP, oxygen consumption and cytosolic Na⁺ in primary neuronal cultures in response to veratridine. A–C, Changes in [Ca²⁺]_cyt, in Fura-2 loaded neurons obtained by stimulation with 50 μm Veratridine (Ver or Verat) in 2 mm Ca²⁺, Ca²⁺-free medium, or 50 μm BAPTA preloaded neurons in 2 mm Ca²⁺ medium. D–F, Corresponding data in neurons transfected with Mit-GEM-GECO1 probe to determine changes in [Ca²⁺]_mit. Recordings from at least 60 cells per condition and two independent experiments were used for [Ca²⁺]_i and a minimum of 15 cells and eight independent experiments for [Ca²⁺]_mit imaging. Individual cell recordings (gray) and average (thick black trace) were shown. G–I, Cytosolic ATP in neurons transfected with cyt-GO-ATeam1 stimulated with veratridine in 2 mm Ca²⁺ medium, Ca²⁺-free medium plus 100 μm EGTA and comparison of the two conditions. Recordings from individual cells (gray) and average (black) are shown. The drop of ATP values with respect to basal levels 200 s after veratridine addition were 23.9 ± 1.70% in the presence and 30 ± 1.69% in the absence of Ca²⁺.(*p = 0.017 two-tailed unpaired Student's t test). J, Veratridine-induced stimulation of OCR in aralar WT neurons under the mentioned Ca²⁺ and BAPTA conditions. K, L, Stimulation of respiration (as percentage of basal values) and RCR. RCR in nonstimulated state are represented with horizontal lines for each experimental condition (n = 9–11 experiments one-way ANOVA, *p ≤ 0.05, ***p ≤ 0.001, post hoc Bonferroni test). M, N, Changes in [Na⁺]_i, in SBFI-loaded neurons by stimulation with 50 μm veratridine in 2 mm Ca²⁺ medium or Ca²⁺-free medium (∼90 neurons per condition). O, Comparison between response in Ca²⁺ medium (black trace) and Ca²⁺-free (gray trace) is shown. Increases in normalized SBFI ratio 200 s after veratridine were 21.1 ± 1.02% and 20.3 ± 0.81in the presence or absence of Ca²⁺, respectively.

Figure 3.

OCR responses to veratridine in ARALAR- and SCaMC-3-deficient neurons. A–D, Respiratory profiles in the presence or absence of 2 mm Ca²⁺, stimulation of mitochondrial respiration and RCRs of SCaMC-3-WT and SCaMC-3-KO neurons stimulated with 50 μm Veratridne (Ver) in 2.5 mm glucose. E–H, Corresponding data from aralar WT and aralar KO neurons. I–L, Corresponding data for the effect of 2 mm pyruvate (Pyr) on veratridine-induced mitochondrial respiration in aralar WT and aralar KO neurons. RCR in nonstimulated state are represented with horizontal lines for each experimental condition. Data are expressed as mean ± SEM from n = 5–6 experiments in SCaMC-3-WT and SCaMC-3-KO cultures, and from n = 4–12 experiments in aralar WT and aralar KO cultures. Two-way ANOVA, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, post hoc Bonferroni test.

Figure 4.

Changes of cytosolic and mitochondrial Ca²⁺, cytosolic ATP, oxygen consumption, and cytosolic Na⁺ in primary neuronal in response to KCl. A–C, Changes in [Ca²⁺]_cyt, in Fura-2-loaded neurons obtained by stimulation with 30 mm KCl in 2 mm Ca²⁺, Ca²⁺-free medium, or 50 μm BAPTA preloaded neurons in 2 mm Ca²⁺ medium. D–F, Corresponding data in neurons transfected with Mit-GEM-GECO1 probe to determine changes in [Ca²⁺]_mit. Recordings from at least 60 cells per condition and two independent experiments were used for cytosolic Ca²⁺ imaging and a minimum of 15 cells and eight independent experiments for mitochondrial Ca²⁺ imaging. Individual cell recordings (gray) and average (black) were shown. G, H, Cytosolic ATP levels after a switch from HCSS medium to isosmotic KCl medium in which 30 mm NaCl was replaced by 30 mm KCl either in 2 mm Ca²⁺ medium or 100 μm EGTA medium. I, Comparison between the two conditions. The drop in ATP values 200 s after isosmotic KCl stimulation was 18.6 ± 0.3% and 9 ± 0.1% with respect to basal levels in the presence and absence of Ca²⁺, respectively (**p = 0.009 two-tailed unpaired Student's t test). J–L, Respiratory profiles, stimulation of mitochondrial respiration, and RCRs of neurons stimulated with hyperosmotic KCl in 2 mm Ca²⁺, Ca²⁺-free medium or 50 μm BAPTA preloaded neurons in 2 mm Ca²⁺. M–O, Corresponding data from neurons stimulated with isosmotic KCl using 10 μm BAPTA. RCR in nonstimulated state are represented with horizontal lines for each experimental condition (n = 8–30 experiments in aralar WT neuronal cultures; one-way ANOVA, *p ≤ 0.05, ***p ≤ 0.001, post hoc Bonferroni test). P, Changes in [Na⁺]_i, in SBFI loaded neurons exposed to 5.4 or 30 mm isosmotic KCl in 2 mm Ca²⁺ or 100 μm EGTA medium as indicated. Monensin (Monen; 10 μm) and Ouabain (Oua; 0.1 mm) were added for equilibration of extra- and intracellular [Na⁺] at the end of the experiments. Individual cell recordings (gray) and average (black) were shown (n = 29).

Figure 5.

OCR-responses to 30 mm KCl in ARALAR- or SCaMC-3-deficient cortical neurons. A–C, Respiratory profiles, stimulation of mitochondrial respiration, and RCRs of SCaMC-3-WT and SCaMC-3-KO neurons stimulated with isosmotic 30 mm KCl in the presence of 2 mm Ca²⁺. D–G, Corresponding data for aralar WT and aralar KO cultures in the presence or absence of 2 mm Ca²⁺. H–J, Corresponding data for the effect of 2 mm pyruvate (Pyr) on isosmotic 30 mm KCl-induced respiratory stimulation in aralar WT and aralar KO neurons in the presence of 2 mm Ca²⁺. RCR in nonstimulated state are represented with horizontal lines for each experimental condition. Data correspond to 5–6 experiments in SCaMC-3-WT and SCaMC-3-KO cultures and 4–24 experiments in aralar WT and aralar KO respectively (two-way ANOVA, *p ≤ 0.05; **p ≤ 0.01; post hoc Bonferroni test).

Figure 6.

Changes in cytosolic and mitochondrial Ca²⁺, cytosolic ATP and oxygen consumption in response to carbachol. A–C, Changes in [Ca²⁺]_cyt, in Fura-2-loaded neurons obtained by stimulation with 250 μm Carbachol (Cch) in 2 mm Ca²⁺, Ca²⁺-free medium, or 50 μm BAPTA preloaded neurons in 2 mm Ca²⁺ medium. D, E, Corresponding data in neurons transfected with Mit-GEM-GECO1 to determine changes in [Ca²⁺]_mit. Recordings from at least 60 cells per condition and two independent experiments were used for cytosolic Ca²⁺ and a minimum of 15 cells and eight independent experiments for mitochondrial Ca²⁺. Individual cell recordings (gray) and average (black) were shown. F, Corresponding data for cytosolic ATP in neurons transfected with cyt-GO-ATeam1. Drop in ATP values 200 s after carbachol addition were 4.9 ± 0.8% and 2.1 ± 0.4% with respect to basal levels in the presence or absence of Ca²⁺ (p = 0.92, two-tailed unpaired Student's t test). G–I, Respiratory profiles, stimulation of mitochondrial respiration, and RCRs of neurons stimulated with 250 μm carbachol in 2 mm Ca²⁺, Ca²⁺-free medium, or 50 μm BAPTA preloaded neurons in 2 mm Ca²⁺. RCR in nonstimulated state were represented with horizontal lines for each experimental condition. Data from 18 to 22 experiments in aralar WT cultures (one-way ANOVA, ***p ≤ 0.001, post hoc Bonferroni test).

Figure 7.

OCR response to Carbachol (Cch) in ARALAR- and SCaMC-3-deficient neuronal cultures. A–D, Respiratory profiles in the presence or absence of 2 mm Ca²⁺, stimulation of mitochondrial respiration, and RCRs of SCaMC-3-WT and SCaMC-3-KO neurons stimulated with 250 μm carbachol in 2.5 mm glucose. E–H, Corresponding data from aralar WT and aralar KO neurons. G–I, Corresponding data of the effect of 2 mm pyruvate (Pyr) on carbachol-induced mitochondrial respiration in aralar WT and aralar KO neurons. RCR in nonstimulated state are represented with horizontal lines for each experimental condition. Data from 5 to 6 experiments in SCaMC-3-WT and SCaMC-3-KO cultures, and from 4 to 12 experiments in aralar WT and aralar KO cultures (two-way ANOVA, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, post hoc Bonferroni test).

Figure 8.

Schematic representation of the effects of the different workloads on Ca²⁺ regulation of neuronal oxygen consumption. Pathways indicated in green, purple, and red are activated by Veratridine (Ver), KCl, and Carbachol (Cch), respectively. All three stimuli activate ARALAR-MAS, increasing pyruvate supply in mitochondria, whereas only veratridine activates adenine nucleotide uptake through SCaMC-3. Veratridine also stimulates respiration after mitochondrial Ca²⁺ uptake through the MCU and activation of mitochondrial dehydrogenases. SOCE, Store operated Ca²⁺ entry; ETC, electron transport chain; AAT,aspartate aminotranferase; AcCoA, acetyl coenzyme A; Asp, aspartate; Cch, carbachol; ER, endoplasmic reticulum; ETC, electron transport chain; Glu, glutamate; IP₃, inositol trisphosphate; IP₃R, inositol 3-phosphate receptor; α-KG, α-ketoglutarate; mAChR, muscarinic cholinergic G-protein-coupled receptor; MCU, mitochondrial calcium uniporter; MDH, malate dehydrogenase; NCX, sodium calcium exchanger; OAA, oxaloacetate; OGC, oxoglutarate carrier; PDH, pyruvate dehydrogenase; PDKase, pyruvate dehydrogenase kinase; PDPase, pyruvate dehydrogenase phosphatase; PIP₂, phosphatidylinositol 4,5-bisphosphate; PLC-β, phospholipase C-β; PMCA, plasma membrane calcium ATPase; PyrC, pyruvate carrier; SERCA, sarco/endoplasmic reticulum Ca²⁺-ATPase; SOCE, store-operated calcium entry; TCA, tricarboxylic acid cycle; Ver, veratridine; VOCC, voltage operated calcium channel; VONC, voltage operated sodium channel.