Extramitochondrial Ca2+ in the nanomolar range regulates glutamate-dependent oxidative phosphorylation on demand

Abstract

We present unexpected and novel results revealing that glutamate-dependent oxidative phosphorylation (OXPHOS) of brain mitochondria is exclusively and efficiently activated by extramitochondrial Ca(2+) in physiological concentration ranges (S(0.5) = 360 nM Ca(2+)). This regulation was not affected by RR, an inhibitor of the mitochondrial Ca(2+) uniporter. Active respiration is regulated by glutamate supply to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier with regulatory Ca(2+)-binding sites in the mitochondrial intermembrane space providing full access to cytosolic Ca(2+). At micromolar concentrations, Ca(2+) can also enter the intramitochondrial matrix and activate specific dehydrogenases. However, the latter mechanism is less efficient than extramitochondrial Ca(2+) regulation of respiration/OXPHOS via aralar. These results imply a new mode of glutamate-dependent OXPHOS regulation as a demand-driven regulation of mitochondrial function. This regulation involves the mitochondrial glutamate/aspartate carrier aralar which controls mitochondrial substrate supply according to the level of extramitochondrial Ca(2+).

Figure 1. Exclusive activation of glutamate-dependent state 3 respiration of brain mitochondria by extramitochondrial Ca²⁺ in the nanomolar range.

(A,E) Respirograms of rat brain mitochondria were obtained by high-resolution respirometry. (A) Isolated rat brain mitochondria were incubated in EGTA medium (Ca²⁺ _free = 0.15 µM) in the presence of 10 mM glutamate and 2 mM malate as substrates. Additions: M, 0.06 mg/ml brain mitochondria, A, 2.5 mM ADP to activate the phosphorylation-related respiration (state 3); Ca²⁺ _4,9, 4.9 µM Ca²⁺ _free; S, 10 mM succinate as substrate of respiratory chain complex II; C, 5 µM carboxyatractyloside to block the adenine nucleotide translocase. Blue lines indicate the oxygen concentration and red lines represent respiration rates (nmol O₂/mg mitochondrial protein/min). (B) Means of state 3 respiration±S.E. as measured in experiments shown in A without (black columns, n = 6) or with 250 nM RR, an inhibitor of mitochondrial Ca²⁺ uptake (red columns, n = 6). First group of columns, state 3 at Ca²⁺ _free = 0.15 µM. Second group, state 3 with Ca²⁺ _free = 4.9 µM. Third group, state 3 with Ca²⁺ _free = 4.9 µM in the additional presence of 10 µM succinate. *, p<0.05. (C) As B, but derived from experiments with 10 mM pyruvate + 2 mM malate as substrates. *, p<0.05. (D) As B, but derived from experiments with 10 mM succinate + 2 µM rotenone as substrate. (E) Ca²⁺ titration of state 3_glu/mal by stepwise increase of Ca²⁺ as indicated either without (E,F) or with (F) 250 nM RR. (F) Incremental accretions of Ca²⁺-induced state 3_glu/mal were plotted against the fluorimetrically measured Ca²⁺ activity (Fig. 1F), allowing the calculation of the half-activation constant (S_0.5) and the maximum velocity (V_max) using the SigmaPlot kinetic module as given in the text. (G) Rates of state 3_glu/mal respiration obtained by Ca²⁺ titrations under various conditions. (○) Control mitochondria were investigated as in Fig. 1E. (□) As (○), but in the additional presence of 10% dextran 20. (▿) As (○), but in the additional presence of 1 mM CsA. (▵) as (○), but mitochondria isolated without digitonin were used. (◊) as (○), but mitoplasts were used. () as (○), but mitochondria were uncoupled by 50 nM FCCP from the beginning of experiments, and then Ca²⁺ titration was performed. (▴) as (○), but Ca²⁺ was adjusted at the beginning of experiments as indicated. Thereafter, 100 µM ADP was added, causing short transitions between the active and resting states of respiration. After reaching state 4 respiration, FCCP titrations were performed to uncouple respiration and ATP generation. Maximum respiration rates were obtained at 60 or 80 nM FCCP and were plotted against the Ca²⁺ _free value for the respective incubation. Data are means±S.E. of 4 independent experiments.

Figure 2. Exclusive and reversible activation of glutamate-dependent respiration by extramitochondrial Ca²⁺ at low levels of ADP.

(A) Isolated rat brain mitochondria (0.06 mg/ml) were incubated in EGTA-free medium (0.6 µM Ca²⁺ _free) with 10 mM glutamate and 2 mM malate as substrates, but in the presence of 250 nM RR. Additions: M, 0.06 mg/ml rat brain mitochondria; A, 150 µM ADP; EGTA, 100 µM EGTA (0.15 µM Ca²⁺ _free); Ca²⁺ _4.9, 4.9 µM Ca²⁺ _free. Horizontal arrows indicate the actual Ca²⁺ _free concentration. (B–D). Means of phosphorylating respiration±S.E. were calculated as stationary state 3 respiration rate minus state 4 respiration rate from measurements as shown for glutamate and malate in A at defined extramitochondrial Ca²⁺. Different substrates were used as indicated. *P<0.01.

Figure 3. Brain mitochondria do not accumulate, but rather lose, Ca²⁺ in the presence of ruthenium red.

Fluorimetric measurement of extramitochondrial Ca²⁺ with Ca²⁺green. Brain mitochondria were incubated in EGTA-free medium with 10 mM glutamate and 2 mM malate. Additions: BM, 0.25 mg/ml brain mitochondria; RR, 250 nM ruthenium red (RR); Ca²⁺ ₁₀, 10 µM Ca²⁺ _free, Insertion: Control experiment without RR demonstrating normal Ca²⁺ accumulation of brain mitochondria after repeated Ca²⁺ additions.