Mg2+ differentially regulates two modes of mitochondrial Ca2+ uptake in isolated cardiac mitochondria: implications for mitochondrial Ca2+ sequestration

Abstract

The manner in which mitochondria take up and store Ca2+ remains highly debated. Recent experimental and computational evidence has suggested the presence of at least two modes of Ca2+ uptake and a complex Ca2+ sequestration mechanism in mitochondria. But how Mg2+ regulates these different modes of Ca2+ uptake as well as mitochondrial Ca2+ sequestration is not known. In this study, we investigated two different ways by which mitochondria take up and sequester Ca2+ by using two different protocols. Isolated guinea pig cardiac mitochondria were exposed to varying concentrations of CaCl2 in the presence or absence of MgCl2. In the first protocol, A, CaCl2 was added to the respiration buffer containing isolated mitochondria, whereas in the second protocol, B, mitochondria were added to the respiration buffer with CaCl2 already present. Protocol A resulted first in a fast transitory uptake followed by a slow gradual uptake. In contrast, protocol B only revealed a slow and gradual Ca2+ uptake, which was approximately 40 % of the slow uptake rate observed in protocol A. These two types of Ca2+ uptake modes were differentially modulated by extra-matrix Mg2+. That is, Mg2+ markedly inhibited the slow mode of Ca2+ uptake in both protocols in a concentration-dependent manner, but not the fast mode of uptake exhibited in protocol A. Mg2+ also inhibited Na+-dependent Ca2+ extrusion. The general Ca2+ binding properties of the mitochondrial Ca2+ sequestration system were reaffirmed and shown to be independent of the mode of Ca2+ uptake, i.e. through the fast or slow mode of uptake. In addition, extra-matrix Mg2+ hindered Ca2+ sequestration. Our results indicate that mitochondria exhibit different modes of Ca2+ uptake depending on the nature of exposure to extra-matrix Ca2+, which are differentially sensitive to Mg2+. The implications of these findings in cardiomyocytes are discussed.

Structural Insights into Mitochondrial Calcium Uniporter Regulation by Divalent Cations

“…This regulatory model is consistent with a recent study demonstrating Mg 2+ -induced inhibition of MCU (Blomeyer et al, 2016) and work showing Ca 2+ -dependent inactivation of mitochondrial Ca 2+ uptake (Moreau et al, 2006). Further studies are required to determine precisely how MRAP and MCU 72-189 is involved in heteromeric protein-protein interactions known to mediate MCU complex assembly and activity.…”

Identity and function of a cardiac mitochondrial small conductance Ca 2+ -activated K + channel splice variant

“…This is apparent from a previous report [33] in which we measured external and matrix [Ca 2+ ] using a similar respiration buffer but containing 0.6 mM CaCl 2 and 1 mM EGTA; this resulted in an external [Ca 2+ ] of 300 nM and a matrix [Ca 2+ ] of 1000 nM. In buffer containing approximately 40 μM EGTA carried over with the mitochondria from the isolation buffer, with no added CaCl 2 , we observed a basal external [Ca 2+ ] of 80–100 nM and a matrix [Ca 2+ ] of 210–250 nM [34,35]. Mitochondrial K + was monitored in a cuvettebased spectrophotometer (QM-8, Photon Technology International, PTI) with excitation and emission light, λ ex 340 and 380 nm; λ em 500 nm, while adding 0–3 mM CaCl 2 to the buffer.…”

Endogenous and Agonist-induced Opening of Mitochondrial Big Versus Small Ca2+-sensitive K+ Channels on Cardiac Cell and Mitochondrial Protection

“…At 20 min of reperfusion some hearts (6 groups of approximately 5 each) that were not used to measure infarct size were removed from the perfusion apparatus and the ventricles were minced in cold mitochondrial isolation buffer on ice. Mitochondria were isolated by differential centrifugation as described previously 5, 6, 41–46 and the mitochondria (0.5 mg protein/mL) were suspended in respiration buffer containing 130 mM KCl, 5 mM K 2 HPO 4 , 20 mM MOPS, 40 μM EGTA (carried over from the isolation buffer), 1 μM Na 4 P 2 O 7 , 0.1% BSA, pH 7.15 adjusted with KOH. To test mitochondrial viability and function, the respiratory control index (RCI, state 3/state 4) was determined under different substrate conditions: Na-pyruvate (P, 10 mM) or Na-succinate (S, 10 mM) or S + rotenone (R, 4 μM).…”