High‐affinity cooperative Ca2+ binding by MICU1–MICU2 serves as an on–off switch for the uniporter

The mitochondrial calcium uniporter is a Ca²⁺‐activated Ca²⁺ channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca²⁺ signals. It is regulated by two paralogous EF‐hand proteins—MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca²⁺. We reconstitute the MICU1–MICU2 heterodimer and demonstrate that it binds Ca²⁺ cooperatively with high affinity. We discover that both MICU1 and MICU2 exhibit affinity for the mitochondria‐specific lipid cardiolipin. We determine the minimum Ca²⁺ concentration required for disinhibition of the uniporter in permeabilized cells and report a close match with the Ca²⁺‐binding affinity of MICU1–MICU2. We conclude that cooperative, high‐affinity interaction of the MICU1–MICU2 complex with Ca²⁺ serves as an on–off switch, leading to a tightly controlled channel, capable of responding directly to cytosolic Ca²⁺ signals.     

Dynamic buffering of mitochondrial Ca2+ during Ca2+ uptake and Na+-induced Ca2+ release

In cardiac mitochondria, matrix free Ca²⁺ ([Ca²⁺]_m) is primarily regulated by Ca²⁺ uptake and release via the Ca²⁺ uniporter (CU) and Na⁺/Ca²⁺ exchanger (NCE) as well as by Ca²⁺ buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca²⁺ buffering affects these dynamics under various Ca²⁺ uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca²⁺ buffering on the uptake and release of Ca²⁺, we monitored Ca²⁺ dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca²⁺] ([Ca²⁺]_e) and [Ca²⁺]_m. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl₂] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca²⁺]_e and [Ca²⁺]_m were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca²⁺-induced changes in mitochondrial bioenergetics. Our [Ca²⁺]_e and [Ca²⁺]_m measurements demonstrate that Ca²⁺ uptake and release do not show reciprocal Ca²⁺ dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca²⁺ buffering system in the matrix compartment. The Na⁺- induced Ca²⁺ release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca²⁺ uptake in the presence of large amounts of CaCl₂, but not by Na⁺- induced Ca²⁺ release.     

The mitochondrial Ca2+ uptake regulator,MICU1, is involved in cold stress‐induced ferroptosis

Ferroptosis has recently attracted much interest because of its relevance to human diseases such as cancer and ischemia‐reperfusion injury. We have reported that prolonged severe cold stress induces lipid peroxidation‐dependent ferroptosis, but the upstream mechanism remains unknown. Here, using genome‐wide CRISPR screening, we found that a mitochondrial Ca²⁺ uptake regulator, mitochondrial calcium uptake 1 (MICU1), is required for generating lipid peroxide and subsequent ferroptosis under cold stress. Furthermore, the gatekeeping activity of MICU1 through mitochondrial calcium uniporter (MCU) is suggested to be indispensable for cold stress‐induced ferroptosis. MICU1 is required for mitochondrial Ca²⁺ increase, hyperpolarization of the mitochondrial membrane potential (MMP), and subsequent lipid peroxidation under cold stress. Collectively, these findings suggest that the MICU1‐dependent mitochondrial Ca²⁺ homeostasis‐MMP hyperpolarization axis is involved in cold stress‐induced lipid peroxidation and ferroptosis.     

Imaging of mitochondrial Ca2+ dynamics in astrocytes using cell-specific mitochondria-targeted GCaMP5G/6s: Mitochondrial Ca2+ uptake and cytosolic Ca2+ availability via the endoplasmic reticulum store

Mitochondrial Ca²⁺ plays a critical physiological role in cellular energy metabolism and signaling, and its overload contributes to various pathological conditions including neuronal apoptotic death in neurological diseases. Live cell mitochondrial Ca²⁺ imaging is an important approach to understand mitochondrial Ca²⁺ dynamics. Recently developed GCaMP genetically-encoded Ca²⁺ indicators provide unique opportunity for high sensitivity/resolution and cell type-specific mitochondrial Ca²⁺ imaging. In the current study, we implemented cell-specific mitochondrial targeting of GCaMP5G/6s (mito-GCaMP5G/6s) and used two-photon microscopy to image astrocytic and neuronal mitochondrial Ca²⁺ dynamics in culture, revealing Ca²⁺ uptake mechanism by these organelles in response to cell stimulation. Using these mitochondrial Ca²⁺ indicators, our results show that mitochondrial Ca²⁺ uptake in individual mitochondria in cultured astrocytes and neurons can be seen after stimulations by ATP and glutamate, respectively. We further studied the dependence of mitochondrial Ca²⁺ dynamics on cytosolic Ca²⁺ changes following ATP stimulation in cultured astrocytes by simultaneously imaging mitochondrial and cytosolic Ca²⁺ increase using mito-GCaMP5G and a synthetic organic Ca²⁺ indicator, x-Rhod-1, respectively. Combined with molecular intervention in Ca²⁺ signaling pathway, our results demonstrated that the mitochondrial Ca²⁺ uptake is tightly coupled with inositol 1,4,5-trisphosphate receptor-mediated Ca²⁺ release from the endoplasmic reticulum and the activation of G protein-coupled receptors. The current study provides a novel approach to image mitochondrial Ca²⁺ dynamics as well as Ca²⁺ interplay between the endoplasmic reticulum and mitochondria, which is relevant for neuronal and astrocytic functions in health and disease.     

Mitochondrial Calcium Uniporter MCU Supports Cytoplasmic Ca2+ Oscillations,Store-Operated Ca2+ Entry and Ca2+-Dependent Gene Expression in Response to Receptor Stimulation

Ca²⁺ flux into mitochondria is an important regulator of cytoplasmic Ca²⁺ signals, energy production and cell death pathways. Ca²⁺ uptake can occur through the recently discovered mitochondrial uniporter channel (MCU) but whether the MCU is involved in shaping Ca²⁺ signals and downstream responses to physiological levels of receptor stimulation is unknown. Here, we show that modest stimulation of leukotriene receptors with the pro-inflammatory signal LTC₄ evokes a series of cytoplasmic Ca²⁺ oscillations that are rapidly and faithfully propagated into mitochondrial matrix. Knockdown of MCU or mitochondrial depolarisation, to reduce the driving force for Ca²⁺ entry into the matrix, prevents the mitochondrial Ca²⁺ rise and accelerates run down of the oscillations. The loss of cytoplasmic Ca²⁺ oscillations appeared to be a consequence of enhanced Ca²⁺-dependent inactivation of InsP₃ receptors, which arose from the loss of mitochondrial Ca²⁺ buffering. Ca²⁺ dependent gene expression in response to leukotriene receptor activation was suppressed following knockdown of the MCU. In addition to buffering Ca²⁺ release, mitochondria also sequestrated Ca²⁺ entry through store-operated Ca²⁺ channels and this too was prevented following loss of MCU. MCU is therefore an important regulator of physiological pulses of cytoplasmic Ca²⁺.     

Mitochondrial Ca2+ Uptake 1 (MICU1) and Mitochondrial Ca2+ Uniporter (MCU) Contribute to Metabolism-Secretion Coupling in Clonal Pancreatic ��-Cells

In pancreatic β-cells, uptake of Ca²⁺ into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca²⁺ sequestration in clonal INS-1 832/13 pancreatic β-cells: the mitochondrial Ca²⁺ uptake 1 (MICU1), mitochondrial Ca²⁺ uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca²⁺ sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca²⁺ uptake upon both intracellular Ca²⁺ release and Ca²⁺ entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca²⁺ uptake in response to either intracellular Ca²⁺ release or Ca²⁺ entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca²⁺ uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca²⁺ oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca²⁺ uptake in pancreatic β-cells and their involvement in the positive feedback required for sustained insulin secretion.     

Mitochondrial Ca2+ uptake in skeletal muscle health and disease

Muscle uses Ca²⁺ as a messenger to control contraction and relies on ATP to maintain the intracellular Ca²⁺ homeostasis. Mitochondria are the major sub-cellular organelle of ATP production. With a negative inner membrane potential, mitochondria take up Ca²⁺ from their surroundings, a process called mitochondrial Ca²⁺ uptake. Under physiological conditions, Ca²⁺ uptake into mitochondria promotes ATP production. Excessive uptake causes mitochondrial Ca²⁺ overload, which activates downstream adverse responses leading to cell dysfunction. Moreover, mitochondrial Ca²⁺ uptake could shape spatio-temporal patterns of intracellular Ca²⁺ signaling. Malfunction of mitochondrial Ca²⁺ uptake is implicated in muscle degeneration. Unlike non-excitable cells, mitochondria in muscle cells experience dramatic changes of intracellular Ca²⁺ levels. Besides the sudden elevation of Ca²⁺ level induced by action potentials, Ca²⁺ transients in muscle cells can be as short as a few milliseconds during a single twitch or as long as minutes during tetanic contraction, which raises the question whether mitochondrial Ca²⁺ uptake is fast and big enough to shape intracellular Ca²⁺ signaling during excitation-contraction coupling and creates technical challenges for quantification of the dynamic changes of Ca²⁺ inside mitochondria. This review focuses on characterization of mitochondrial Ca²⁺ uptake in skeletal muscle and its role in muscle physiology and diseases.     

MICU1 and MICU2 play nonredundant roles in the regulation of the mitochondrial calcium uniporter

The mitochondrial uniporter is a selective Ca²⁺ channel regulated by MICU1, an EF hand‐containing protein in the organelle's intermembrane space. MICU1 physically associates with and is co‐expressed with a paralog, MICU2. To clarify the function of MICU1 and its relationship to MICU2, we used gene knockout (KO) technology. We report that HEK‐293T cells lacking MICU1 or MICU2 lose a normal threshold for Ca²⁺ intake, extending the known gating function of MICU1 to MICU2. Expression of MICU1 or MICU2 mutants lacking functional Ca²⁺‐binding sites leads to a striking loss of Ca²⁺ uptake, suggesting that MICU1/2 disinhibit the channel in response to a threshold rise in [Ca²⁺]. MICU2's activity and physical association with the pore require the presence of MICU1, though the converse is not true. We conclude that MICU1 and MICU2 are nonredundant and together set the [Ca²⁺] threshold for uniporter activity.     

Characterization of Mg Inhibition of Mitochondrial Ca Uptake by a Mechanistic Model of Mitochondrial Ca Uniporter

Ca²⁺ is an important regulatory ion and alteration of mitochondrial Ca²⁺ homeostasis can lead to cellular dysfunction and apoptosis. Ca²⁺ is transported into respiring mitochondria via the Ca²⁺ uniporter, which is known to be inhibited by Mg²⁺. This uniporter-mediated mitochondrial Ca²⁺ transport is also shown to be influenced by inorganic phosphate (Pi). Despite a large number of experimental studies, the kinetic mechanisms associated with the Mg²⁺ inhibition and Pi regulation of the uniporter function are not well established. To gain a quantitative understanding of the effects of Mg²⁺ and Pi on the uniporter function, we developed here a mathematical model based on known kinetic properties of the uniporter and presumed Mg²⁺ inhibition and Pi regulation mechanisms. The model is extended from our previous model of the uniporter that is based on a multistate catalytic binding and interconversion mechanism and Eyring's free energy barrier theory for interconversion. The model satisfactorily describes a wide variety of experimental data sets on the kinetics of mitochondrial Ca²⁺ uptake. The model also appropriately depicts the inhibitory effect of Mg²⁺ on the uniporter function, in which Ca²⁺ uptake is hyperbolic in the absence of Mg²⁺ and sigmoid in the presence of Mg²⁺. The model suggests a mixed-type inhibition mechanism for Mg²⁺ inhibition of the uniporter function. This model is critical for building mechanistic models of mitochondrial bioenergetics and Ca²⁺ handling to understand the mechanisms by which Ca²⁺ mediates signaling pathways and modulates energy metabolism.     

Mitochondrial Ca2+ Uptake Increases Ca2+ Release from Inositol 1,4,5-Trisphosphate Receptor Clusters in Smooth Muscle Cells

Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP₃)-mediated increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_c). Local Ca²⁺ release from an InsP₃ receptor (InsP₃R) cluster present on the sarcoplasmic reticulum is termed a Ca²⁺ puff. Ca²⁺ released via InsP₃R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca²⁺ rise. In smooth muscle, mitochondrial Ca²⁺ uptake maintains global InsP₃-mediated Ca²⁺ release by preventing a negative feedback effect of high [Ca²⁺] on InsP₃R. Mitochondria may regulate InsP₃-mediated Ca²⁺ signals by operating between or within InsP₃R clusters. In the former mitochondria could regulate only global Ca²⁺ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP₃-mediated Ca²⁺ release by operating within (local) or between (global) InsP₃R clusters has been addressed. Ca²⁺ puffs evoked by localized photolysis of InsP₃ in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5–4.0 F/F₀, durations of ∼112 ms at half-maximum amplitude, and were abolished by the InsP₃R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized ΔΨ_M to prevent mitochondrial Ca²⁺ uptake and attenuated Ca²⁺ puffs by ∼66 or ∼60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca²⁺ puffs by ∼62%. The “fast” Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acted like mitochondria to prolong InsP₃-mediated Ca²⁺ release suggesting that mitochondrial influence is via their Ca²⁺ uptake facility. These results indicate Ca²⁺ uptake occurs quickly enough to influence InsP₃R communication at the intra-cluster level and that mitochondria regulate both local and global InsP₃-mediated Ca²⁺ signals.     

Molecularly Distinct Routes of Mitochondrial Ca2+ Uptake Are Activated Depending on the Activity of the Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA)

The transfer of Ca²⁺ across the inner mitochondrial membrane is an important physiological process linked to the regulation of metabolism, signal transduction, and cell death. While the definite molecular composition of mitochondrial Ca²⁺ uptake sites remains unknown, several proteins of the inner mitochondrial membrane, that are likely to accomplish mitochondrial Ca²⁺ fluxes, have been described: the novel uncoupling proteins 2 and 3, the leucine zipper-EF-hand containing transmembrane protein 1 and the mitochondrial calcium uniporter. It is unclear whether these proteins contribute to one unique mitochondrial Ca²⁺ uptake pathway or establish distinct routes for mitochondrial Ca²⁺ sequestration. In this study, we show that a modulation of Ca²⁺ release from the endoplasmic reticulum by inhibition of the sarco/endoplasmatic reticulum ATPase modifies cytosolic Ca²⁺ signals and consequently switches mitochondrial Ca²⁺ uptake from an uncoupling protein 3- and mitochondrial calcium uniporter-dependent, but leucine zipper-EF-hand containing transmembrane protein 1-independent to a leucine zipper-EF-hand containing transmembrane protein 1- and mitochondrial calcium uniporter-mediated, but uncoupling protein 3-independent pathway. Thus, the activity of sarco/endoplasmatic reticulum ATPase is significant for the mode of mitochondrial Ca²⁺ sequestration and determines which mitochondrial proteins might actually accomplish the transfer of Ca²⁺ across the inner mitochondrial membrane. Moreover, our findings herein support the existence of distinct mitochondrial Ca²⁺ uptake routes that might be essential to ensure an efficient ion transfer into mitochondria despite heterogeneous cytosolic Ca²⁺ rises.     

Mitochondrial Ca2+ uptake with and without the formation of high-Ca2+ microdomains

The mitochondrial Ca²⁺ uniporter has low affinity for Ca²⁺, therefore it has been assumed that submicromolar Ca²⁺ signals cannot induce mitochondrial Ca²⁺ uptake. The close apposition of the plasma membrane or the endoplamic reticulum (ER) to the mitochondria and the limited Ca²⁺ diffusion in the cytoplasm result in the formation of perimitochondrial high-Ca²⁺ microdomains (HCMDs) capable of activating mitochondrial Ca²⁺ uptake. The possibility of mitochondrial Ca²⁺ uptake at low submicromolar [Ca²⁺]_c has not yet been generally accepted.Earlier we found in permeabilized glomerulosa, luteal and pancreatic β cells that [Ca²⁺]_m increased when [Ca²⁺]_c was raised from 60 nM to less than 200 nM. Here we report data obtained from H295R (adrenocortical) cells transfected with ER-targeted GFP. Cytoplasmic Ca²⁺ response to angiotensin II was different in mitochondrion-rich and mitochondrion-free domains. The mitochondrial Ca²⁺ response to angiotensin II correlated with GFP fluorescence indicating the vicinity of ER. When the cells were exposed to K⁺ (inducing Ca²⁺ influx), no correlation was found between the mitochondrial Ca²⁺ signal and the vicinity of the plasma membrane or the ER. The results presented here provide evidence that mitochondrial Ca²⁺ uptake may occur both with and without the formation of HCMDs within the same cell.     

Structural and mechanistic insights into MICU1 regulation of mitochondrial calcium uptake

Mitochondrial calcium uptake is a critical event in various cellular activities. Two recently identified proteins, the mitochondrial Ca²⁺ uniporter (MCU), which is the pore‐forming subunit of a Ca²⁺ channel, and mitochondrial calcium uptake 1 (MICU1), which is the regulator of MCU, are essential in this event. However, the molecular mechanism by which MICU1 regulates MCU remains elusive. In this study, we report the crystal structures of Ca²⁺‐free and Ca²⁺‐bound human MICU1. Our studies reveal that Ca²⁺‐free MICU1 forms a hexamer that binds and inhibits MCU. Upon Ca²⁺ binding, MICU1 undergoes large conformational changes, resulting in the formation of multiple oligomers to activate MCU. Furthermore, we demonstrate that the affinity of MICU1 for Ca²⁺ is approximately 15–20 μM. Collectively, our results provide valuable details to decipher the molecular mechanism of MICU1 regulation of mitochondrial calcium uptake.     

MICU3 regulates mitochondrial Ca2+-dependent antioxidant response in skeletal muscle aging

Age-related loss of skeletal muscle mass and function, termed sarcopenia, could impair the quality of life in the elderly. The mechanisms involved in skeletal muscle aging are intricate and largely unknown. However, more and more evidence demonstrated that mitochondrial dysfunction and apoptosis also play an important role in skeletal muscle aging. Recent studies have shown that mitochondrial calcium uniporter (MCU)-mediated mitochondrial calcium affects skeletal muscle mass and function by affecting mitochondrial function. During aging, we observed downregulated expression of mitochondrial calcium uptake family member3 (MICU3) in skeletal muscle, a regulator of MCU, which resulted in a significant reduction in mitochondrial calcium uptake. However, the role of MICU3 in skeletal muscle aging remains poorly understood. Therefore, we investigated the effect of MICU3 on the skeletal muscle of aged mice and senescent C2C12 cells induced by d-gal. Downregulation of MICU3 was associated with decreased myogenesis but increased oxidative stress and apoptosis. Reconstitution of MICU3 enhanced antioxidants, prevented the accumulation of mitochondrial ROS, decreased apoptosis, and increased myogenesis. These findings indicate that MICU3 might promote mitochondrial Ca²⁺ homeostasis and function, attenuate oxidative stress and apoptosis, and restore skeletal muscle mass and function. Therefore, MICU3 may be a potential therapeutic target in skeletal muscle aging.Subject terms: Ageing, Calcium and phosphate metabolic disorders     

A Biophysically Based Mathematical Model for the Kinetics of Mitochondrial Calcium Uniporter

Ca²⁺ transport through mitochondrial Ca²⁺ uniporter is the primary Ca²⁺ uptake mechanism in respiring mitochondria. Thus, the uniporter plays a key role in regulating mitochondrial Ca²⁺. Despite the importance of mitochondrial Ca²⁺ to metabolic regulation and mitochondrial function, and to cell physiology and pathophysiology, the structure and composition of the uniporter functional unit and kinetic mechanisms associated with Ca²⁺ transport into mitochondria are still not well understood. In this study, based on available experimental data on the kinetics of Ca²⁺ transport via the uniporter, a mechanistic kinetic model of the uniporter is introduced. The model is thermodynamically balanced and satisfactorily describes a large number of independent data sets in the literature on initial or pseudo-steady-state influx rates of Ca²⁺ via the uniporter measured under a wide range of experimental conditions. The model is derived assuming a multi-state catalytic binding and Eyring's free-energy barrier theory-based transformation mechanisms associated with the carrier-mediated facilitated transport and electrodiffusion. The model is a great improvement over the previous theoretical models of mitochondrial Ca²⁺ uniporter in the literature in that it is thermodynamically balanced and matches a large number of independently published data sets on mitochondrial Ca²⁺ uptake. This theoretical model will be critical in developing mechanistic, integrated models of mitochondrial Ca²⁺ handling and bioenergetics which can be helpful in understanding the mechanisms by which Ca²⁺ plays a role in mediating signaling pathways and modulating mitochondrial energy metabolism.     

Mechanisms of Rapid Reactive Oxygen Species Generation in Response to Cytosolic Ca2+ or Zn2+ Loads in Cortical Neurons

Excessive “excitotoxic” accumulation of Ca²⁺ and Zn²⁺ within neurons contributes to neurodegeneration in pathological conditions including ischemia. Putative early targets of these ions, both of which are linked to increased reactive oxygen species (ROS) generation, are mitochondria and the cytosolic enzyme, NADPH oxidase (NOX). The present study uses primary cortical neuronal cultures to examine respective contributions of mitochondria and NOX to ROS generation in response to Ca²⁺ or Zn²⁺ loading. Induction of rapid cytosolic accumulation of either Ca²⁺ (via NMDA exposure) or Zn²⁺ (via Zn²⁺/Pyrithione exposure in 0 Ca²⁺) caused sharp cytosolic rises in these ions, as well as a strong and rapid increase in ROS generation. Inhibition of NOX activation significantly reduced the Ca²⁺-induced ROS production with little effect on the Zn²⁺- triggered ROS generation. Conversely, dissipation of the mitochondrial electrochemical gradient increased the cytosolic Ca²⁺ or Zn²⁺ rises caused by these exposures, consistent with inhibition of mitochondrial uptake of these ions. However, such disruption of mitochondrial function markedly suppressed the Zn²⁺-triggered ROS, while partially attenuating the Ca²⁺-triggered ROS. Furthermore, block of the mitochondrial Ca²⁺ uniporter (MCU), through which Zn²⁺ as well as Ca²⁺ can enter the mitochondrial matrix, substantially diminished Zn²⁺ triggered ROS production, suggesting that the ROS generation occurs specifically in response to Zn²⁺ entry into mitochondria. Finally, in the presence of the sulfhydryl-oxidizing agent 2,2''-dithiodipyridine, which impairs Zn²⁺ binding to cytosolic metalloproteins, far lower Zn²⁺ exposures were able to induce mitochondrial Zn²⁺ uptake and consequent ROS generation. Thus, whereas rapid acute accumulation of Zn²⁺ and Ca²⁺ each can trigger injurious ROS generation, Zn²⁺ entry into mitochondria via the MCU may do so with particular potency. This may be of particular relevance to conditions like ischemia in which cytosolic Zn²⁺ buffering is impaired due to acidosis and oxidative stress.     

Mitochondria in Ca2+ Signaling and Apoptosis

Cellular Ca²⁺ signals are crucial in the control of most physiological processes, cell injuryand programmed cell death; mitochondria play a pivotal role in the regulation of such cytosolicCa²⁺ ([Ca²⁺]_c) signals. Mitochondria are endowed with multiple Ca²⁺ transport mechanismsby which they take up and release Ca²⁺ across their inner membrane. These transport processesfunction to regulate local and global [Ca²⁺]_c, thereby regulating a number of Ca²⁺-sensitivecellular mechanisms. The permeability transition pore (PTP) forms the major Ca²⁺ effluxpathway from mitochondria. In addition, Ca²⁺ efflux from the mitochondrial matrix occursby the reversal of the uniporter and through the inner membrane Na⁺/Ca²⁺ exchanger. Duringcellular Ca²⁺ overload, mitochondria take up [Ca²⁺]_c, which, in turn, induces opening of PTP,disruption of mitochondrial membrane potential (_m) and cell death. In apoptosis signaling,collapse of ;_m and cytochrome c release from mitochondria occur followed by activationof caspases, DNA fragmentation, and cell death. Translocation of Bax, an apoptotic signalingprotein from the cytosol to the mitochondrial membrane, is another step during thisapoptosis-signaling pathway. The role of permeability transition in the context of cell death in relationto Bcl-2 family of proteins is discussed.     

Mitochondrial Calcium Uniporter Activity Is Dispensable for MDA-MB-231 Breast Carcinoma Cell Survival

Calcium uptake through the mitochondrial Ca²⁺ uniporter (MCU) is thought to be essential in regulating cellular signaling events, energy status, and survival. Functional dissection of the uniporter is now possible through the recent identification of the genes encoding for MCU protein complex subunits. Cancer cells exhibit many aspects of mitochondrial dysfunction associated with altered mitochondrial Ca²⁺ levels including resistance to apoptosis, increased reactive oxygen species production and decreased oxidative metabolism. We used a publically available database to determine that breast cancer patient outcomes negatively correlated with increased MCU Ca²⁺ conducting pore subunit expression and decreased MICU1 regulatory subunit expression. We hypothesized breast cancer cells may therefore be sensitive to MCU channel manipulation. We used the widely studied MDA-MB-231 breast cancer cell line to investigate whether disruption or increased activation of mitochondrial Ca²⁺ uptake with specific siRNAs and adenoviral overexpression constructs would sensitize these cells to therapy-related stress. MDA-MB-231 cells were found to contain functional MCU channels that readily respond to cellular stimulation and elicit robust AMPK phosphorylation responses to nutrient withdrawal. Surprisingly, knockdown of MCU or MICU1 did not affect reactive oxygen species production or cause significant effects on clonogenic cell survival of MDA-MB-231 cells exposed to irradiation, chemotherapeutic agents, or nutrient deprivation. Overexpression of wild type or a dominant negative mutant MCU did not affect basal cloning efficiency or ceramide-induced cell killing. In contrast, non-cancerous breast epithelial HMEC cells showed reduced survival after MCU or MICU1 knockdown. These results support the conclusion that MDA-MB-231 breast cancer cells do not rely on MCU or MICU1 activity for survival in contrast to previous findings in cells derived from cervical, colon, and prostate cancers and suggest that not all carcinomas will be sensitive to therapies targeting mitochondrial Ca²⁺ uptake mechanisms.     

Essential Role of Mitochondrial Ca2+ Uniporter in the Generation of Mitochondrial pH Gradient and Metabolism-Secretion Coupling in Insulin-releasing Cells

In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca²⁺ influx, which triggers insulin exocytosis. The mitochondrial Ca²⁺ uniporter (MCU) mediates Ca²⁺ uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca²⁺ rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca²⁺. Suppression of the putative Ca²⁺/H⁺ antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca²⁺-induced matrix acidification. These results demonstrate that MCU-mediated Ca²⁺ uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells.