Date Published: December 9, 2009
Publisher: Public Library of Science
Author(s): Frank Norbert Gellerich, Zemfira Gizatullina, Odeta Arandarcikaite, Doreen Jerzembek, Stefan Vielhaber, Enn Seppet, Frank Striggow, Mark R. Cookson. http://doi.org/10.1371/journal.pone.0008181
Abstract: We present unexpected and novel results revealing that glutamate-dependent oxidative phosphorylation (OXPHOS) of brain mitochondria is exclusively and efficiently activated by extramitochondrial Ca2+ in physiological concentration ranges (S0.5 = 360 nM Ca2+). This regulation was not affected by RR, an inhibitor of the mitochondrial Ca2+ uniporter. Active respiration is regulated by glutamate supply to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier with regulatory Ca2+-binding sites in the mitochondrial intermembrane space providing full access to cytosolic Ca2+. At micromolar concentrations, Ca2+ can also enter the intramitochondrial matrix and activate specific dehydrogenases. However, the latter mechanism is less efficient than extramitochondrial Ca2+ 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 Ca2+.
Partial Text: It has been assumed that ADP formed by ATP-consuming enzymes activates OXPHOS . However, cytosolic ADP of the heart muscle is only insignificantly increased in vivo during elevated work loads , . Therefore, two hypotheses have been proposed, (i) the dynamic compartmentation of ADP, assuming that necessary ADP augmentations occur exclusively within the mitochondrial intermembrane space ,  and (ii) the stimulation of OXPHOS due to Ca2+ influx into the mitochondrial matrix via Ca2+ uniporter, followed by the activation of distinct intramitochondrial dehydrogenases , . Some authors also assume a Ca2+ stimulation of F0F1-ATP synthase , . However, both scenarios comply only partially with the in vivo findings outlined above .
First, we investigated the influence of Ca2+ on OXPHOS of isolated rat brain mitochondria in a medium containing 150 nM free Ca2+ (Ca2+free), corresponding to basal levels of cytosolic Ca2+ under physiological conditions . ADP was added so as to fully activate phosphorylation-related respiration (state 3). Using glutamate/malate as substrate, a relatively low state 3glu/mal was obtained (Fig. 1A,B). However, state 3glu/mal nearly doubled immediately after a pulse addition of 4.9 µM Ca2+free (Fig. 1A,B). This Ca2+ activation was not limited by the mitochondrial capacity of OXPHOS, but rather was due to its efficacy in metabolizing glutamate, as succinate conspicuously enhanced respiration above the level of state 3glu/mal. With pyruvate/malate (Fig. 1C), state 3pyr/mal significantly exceeded state 3glu/mal (Fig. 1A,B). However, added Ca2+ did not augment state 3pyr/mal, whereas added succinate did (Fig. 1C). Fig. 1D demonstrates that there was also no Ca2+ effect on complex II-dependent state 3suc with succinate/rotenone. Overall, these results show that Ca2+ activation of OXPHOS in isolated brain mitochondria is a glutamate-specific phenomenon. The next series of experiments revealed that RR, an inhibitor of the mitochondrial Ca2+ uniporter , is not able to modulate Ca2+ effects on state 3 with any substrate (Fig. 1B–D). We performed these experiments in the presence of relatively low RR concentrations (250 nM) in order to avoid possible unspecific RR effects. Nevertheless, even in the presence of up to 5 µM RR, extramitochondrial Ca2+-induced stimulation of state 3glu/mal was detectable (Data not shown).
It is widely believed that increased cytosolic Ca2+ exerts a parallel activation of extramitochondrial ATPases and OXPHOS, thereby balancing exactly ATP consumption and production without major changes in ADP concentration , , , , , . Ca2+ transport into the mitochondrial matrix and subsequent activation of distinct intramitochondrial dehydrogenases , , , , ,  and F0F1ATPase ,  are assumed to constitute the regulatory mechanism of mitochondrial respiration and OXPHOS. However, an exclusive activation of OXPHOS by intramitochondrial Ca2+ is questionable in the light of following arguments. (i) Computer modeling of intramitochondrial Ca2+ activation of OXPHOS was unable to simulate the OXPHOS activation in response to physiological changes of work load in vivo. (ii) The low affinity of the mitochondrial Ca2+ uniporter to Ca2+free (KM = 3.7±0.9 µM) should not allow an effective increase in intramitochondrial Ca2+ effectively under conditions of only slightly elevated Ca2+. Therefore, detectable mitochondrial Ca2+ uptake at nanomolar Ca2+ levels was explained by spatial heterogeneity of cytosolic Ca2+ concentration  and/or by a spermine-induced increase in the uniporter’s affinity for extramitochondrial Ca2+, . (iii) Moreover, the relative insensitivities of intramitochondrial dehydrogenases to Ca2+ (S0.5 = 0.4 – 13 µM Ca2+free) ,  require significant higher Ca2+free levels for their activation compared with extramitochondrial Ca2+ activation of state 3glu/mal and OXPHOS. Thus, the function of mitochondrial Ca2+ uptake and accumulation appears rather to serve as reversible Ca2+ buffer, ensuring intracellular Ca2+ homeostasis, than to regulate state 3glu/mal and OXPHOS .