Mitochondrial calcium uptake

Abstract

Calcium (Ca(2+)) uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca(2+) levels, this flux influences energy production and can initiate cell death. If large, this flux could potentially alter intracellular Ca(2+) ([Ca(2+)]i) signals. Despite years of study, fundamental disagreements on the extent and speed of mitochondrial Ca(2+) uptake still exist. Here, we review and quantitatively analyze mitochondrial Ca(2+) uptake fluxes from different tissues and interpret the results with respect to the recently proposed mitochondrial Ca(2+) uniporter (MCU) candidate. This quantitative analysis yields four clear results: (i) under physiological conditions, Ca(2+) influx into the mitochondria via the MCU is small relative to other cytosolic Ca(2+) extrusion pathways; (ii) single MCU conductance is ∼6-7 pS (105 mM [Ca(2+)]), and MCU flux appears to be modulated by [Ca(2+)]i, suggesting Ca(2+) regulation of MCU open probability (P(O)); (iii) in the heart, two features are clear: the number of MCU channels per mitochondrion can be calculated, and MCU probability is low under normal conditions; and (iv) in skeletal muscle and liver cells, uptake per mitochondrion varies in magnitude but total uptake per cell still appears to be modest. Based on our analysis of available quantitative data, we conclude that although Ca(2+) critically regulates mitochondrial function, the mitochondria do not act as a significant dynamic buffer of cytosolic Ca(2+) under physiological conditions. Nevertheless, with prolonged (superphysiological) elevations of [Ca(2+)]i, mitochondrial Ca(2+) uptake can increase 10- to 1,000-fold and begin to shape [Ca(2+)]i dynamics.

Keywords: NCLX; NCX; SERCA; inner mitochondrial membrane; microdomain.

Fig. 1.

Schematic diagram shows the spatial distribution of cardiac mitochondrial Ca²⁺ signaling components. A spatial representation of a Ca²⁺ spark (red gradient) initiated at the CRU, which is located between the TT and JSR membranes, is shown. At the peak of a Ca²⁺ spark, [Ca²⁺]_i briefly (10 ms) bathes the mitochondrion at levels indicated by the red line (note that the y axis is log scale). During a [Ca²⁺]_i transient, multiple CRUs release Ca²⁺, causing the mitochondrion to experience a [Ca²⁺]_i profile similar to the green line. Note that the JSR ends of the mitochondria are in high [Ca²⁺]_i microdomains for brief periods during both Ca²⁺ sparks and Ca²⁺ transients. The green arrow indicates the approximate global average [Ca²⁺]_i at the peak of a systolic [Ca²⁺]_i transient. LCCs, L-type Ca²⁺ channels; RyRs, ryanodine receptor 2’s.

Fig. 2.

Whole-cell MCU fluxes from heart, liver, and skeletal muscle tissue. (A) Peak cardiac whole-cell MCU fluxes from different research groups (colored data points). Filled circles indicate measurements from intact mitochondria within cells, and empty circles indicate results from suspensions of isolated mitochondria. The solid black line is a “best-fit” line to all experimental data. (B) Peak liver whole-cell MCU fluxes from several different research groups (colored data points) plotted alongside the “Cardiac MCU” line from A. (C) Peak skeletal muscle whole-cell MCU fluxes from several different research groups (colored data points) plotted alongside the Cardiac MCU line from A. FT, fast-twitch muscle; ST, slow-twitch muscle. Also see Figs. S1–S3. (D) Cardiac whole-cell MCU flux (black line and black circle) compared with other cardiac Ca²⁺ extrusion pathways: SERCA (red line and red circle) and NCX (blue line and blue circle). Solid lines indicate theoretical fluxes from the studies of Tran et al. (46) and Weber et al. (47), whereas filled circles indicate experimental results from Bassani et al. (25). Also see Fig. S5. in SI Material. (E) Comparison of the relative contribution of each flux when scaled to total cytosolic extrusion. For example, the bar labeled MCU is calculated as the whole-cell MCU flux divided by the sum of the three whole-cell fluxes (i.e., MCU, SERCA, and NCX) shown in Fig. 2D. Conversions and scaling for the heart are 40 mg of mitochondrial protein per milliliter of cell, a cytosolic-to-cellular volume ratio of 0.5, and 10,000 mitochondria per cell (50). Details regarding other unit conversions and scaling are provided in SI Material (see Eqs. S1–S3 and Table S1). Note that the SERCA and NCX fluxes shown in D and E are for small rodents, but the result is qualitatively similar (Fig. S4) for larger animals (e.g., rabbit) that have less SERCA and more NCX activity. Cardiac uptake measurements (–27), liver uptake measurements (–35), and skeletal muscle uptake measurements (–30) are taken from the literature.

Fig. 3.

Apparent Ca²⁺-dependent activation of mitochondrial Ca²⁺ uptake. (A) Measured whole-cell MCU fluxes (gray circles) are replotted from Fig. 2A. The potential influence of MCU P_O on cardiac MCU uptake is shown using a theoretical flux equation with fixed P_O = 0.9 (blue line; Eq. 1) or when P_O is a function of [Ca²⁺]_i (red line; Eq. 2). The filled blue circle represents the whole-cell MCU flux converted from the study by De Stefani et al. (17), and the filled green circles represent whole-cell MCU fluxes converted from the study by Fieni et al. (21) (see Eqs. S4–10 in SI Material). (B, Inset) Relationship between P_O (black line) and [Ca²⁺]_i over a wide range of [Ca²⁺]_i (0 to 1 mM), which produces the red line in Fig. 3A.

Fig. 4.

Mitochondrial Ca²⁺ uptake plasticity. The whole-mitoplast MCU current density density (I_mcu measured as pA/pF) measured by Fieni et al. (21) in different tissues is plotted vs. the fraction of the cell composed of mitochondria in six different tissue types (i.e., skeletal muscle, kidney, liver, neonate heart, adult heart, and flight wing muscle). Additional details of the tissue-specific mitochondrial densities are provided in Table S2. The fit line is a first-order exponential decay with extra weight applied to skeletal and heart tissues.