Ca2+ uptake by mitochondria regulates bioenergetics, apoptosis, and Ca2+ signaling. The primary pathway for mitochondrial Ca2+ uptake is the mitochondrial calcium uniporter (MCU), a Ca2+-selective ...ion channel in the inner mitochondrial membrane. MCU-mediated Ca2+ uptake is driven by the sizable inner-membrane potential generated by the electron-transport chain. Despite the large thermodynamic driving force, mitochondrial Ca2+ uptake is tightly regulated to maintain low matrix Ca2+ and prevent opening of the permeability transition pore and cell death, while meeting dynamic cellular energy demands. How this is accomplished is controversial. Here we define a regulatory mechanism of MCU-channel activity in which cytoplasmic Ca2+ regulation of intermembrane space-localized MICU1/2 is controlled by Ca2+-regulatory mechanisms localized across the membrane in the mitochondrial matrix. Ca2+ that permeates through the channel pore regulates Ca2+ affinities of coupled inhibitory and activating sensors in the matrix. Ca2+ binding to the inhibitory sensor within the MCU amino terminus closes the channel despite Ca2+ binding to MICU1/2. Conversely, disruption of the interaction of MICU1/2 with the MCU complex disables matrix Ca2+ regulation of channel activity. Our results demonstrate how Ca2+ influx into mitochondria is tuned by coupled Ca2+-regulatory mechanisms on both sides of the inner mitochondrial membrane.
The mitochondrial uniporter (MCU) is an ion channel that mediates Ca2+ uptake into the matrix to regulate metabolism, cell death, and cytoplasmic Ca2+ signaling. Matrix Ca2+ concentration is similar ...to that in cytoplasm, despite an enormous driving force for entry, but the mechanisms that prevent mitochondrial Ca2+ overload are unclear. Here, we show that MCU channel activity is governed by matrix Ca2+ concentration through EMRE. Deletion or charge neutralization of its matrix-localized acidic C terminus abolishes matrix Ca2+ inhibition of MCU Ca2+ currents, resulting in MCU channel activation, enhanced mitochondrial Ca2+ uptake, and constitutively elevated matrix Ca2+ concentration. EMRE-dependent regulation of MCU channel activity requires intermembrane space-localized MICU1, MICU2, and cytoplasmic Ca2+. Thus, mitochondria are protected from Ca2+ depletion and Ca2+ overload by a unique molecular complex that involves Ca2+ sensors on both sides of the inner mitochondrial membrane, coupled through EMRE.
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•The inner membrane mitochondrial MCU ion channel mediates Ca2+ uptake into the matrix•MCU channel activity is governed by matrix Ca2+ concentration through EMRE•EMRE-dependent regulation requires MICU1, MICU2, and cytoplasmic Ca2+•EMRE couples Ca2+ sensors on both sides of the inner membrane to regulate MCU
Using patch clamp electrophysiology, Vais et al. demonstrate that mitochondrial matrix Ca2+ concentration regulates the activity of MCU, the major mitochondrial Ca2+ influx pathway. Mitochondria are protected from Ca2+ depletion and overload by a unique complex involving Ca2+ sensors on both sides of the inner mitochondrial membrane, coupled through EMRE.
The mitochondrial calcium uniporter complex is essential for calcium (Ca2+) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca2+ signal ...transduction. Despite its involvement in several human diseases, we currently lack pharmacological agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-molecule modulators in primary drug screens. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate- and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-positive hits, we identify mitoxantrone out of more than 600 clinically approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compounds that target mitochondrial functions.
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•Targeting human MCU in yeast mitochondria is an effective drug discovery strategy•A yeast-specific bioenergetic shunt minimizes detection of false-positive hits•An orthogonal, interspecies drug screening can identify specific MCU modulators•Mitoxantrone is a selective and direct inhibitor of MCU
Arduino et al. develop a high-throughput drug discovery strategy to identify chemical modulators of the mitochondrial calcium uniporter. They find that mitoxantrone is a selective and direct inhibitor of the MCU channel.
K-Ras4B is targeted to the plasma membrane by a farnesyl modification that operates in conjunction with a polybasic domain. We characterized a farnesyl-electrostatic switch whereby protein kinase C ...phosphorylates K-Ras4B on serine 181 in the polybasic region and thereby induces translocation from the plasma membrane to internal membranes that include the endoplasmic reticulum (ER) and outer mitochondrial membrane. This translocation is associated with cell death. Here we have explored the mechanism of phospho–K-Ras4B toxicity and found that GTP-bound, phosphorylated K-Ras4B associates with inositol trisphosphate receptors on the ER in a Bcl-xL–dependent fashion and, in so doing, blocks the ability of Bcl-xL to potentiate the InsP ₃ regulated flux of calcium from ER to mitochondria that is required for efficient respiration, inhibition of autophagy, and cell survival. Thus, we have identified inositol trisphosphate receptors as unique effectors of K-Ras4B that antagonize the prosurvival signals of other K-Ras effectors.
Modulating cytoplasmic Ca2+ concentration (Ca2+i) by endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+-release channels is a universal signaling pathway that ...regulates numerous cell-physiological processes. Whereas much is known regarding regulation of InsP3R activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca2+ concentration (Ca2+ER) is poorly understood and controversial. We discovered that the InsP3R is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological Ca2+ER. The widely-expressed Ca2+-binding protein annexin A1 (ANXA1) is present in the nuclear envelope lumen and, through interaction with a luminal region of the channel, can modify high-Ca2+ER inhibition of InsP3R activity. Genetic knockdown of ANXA1 expression enhanced global and local elementary InsP3-mediated Ca2+ signaling events. Thus, Ca2+ER is a major regulator of InsP3R channel activity and InsP3R-mediated Ca2+i signaling in cells by controlling an interaction of the channel with a peripheral membrane-associated Ca2+-binding protein, likely ANXA1.
Members of the Bcl-2 family of proteins regulate apoptosis, with some of their physiological effects mediated by their modulation of endoplasmic reticulum (ER) Ca²⁺ homeostasis. Antiapoptotic Bcl-xL ...binds to the inositol trisphosphate receptor (InsP₃R) Ca²⁺ release channel to enhance Ca²⁺- and InsP₃-dependent regulation of channel gating, resulting in reduced ER Ca²⁺, increased oscillations of cytoplasmic Ca²⁺ concentration (Ca²⁺i), and apoptosis resistance. However, it is controversial which InsP₃R isoforms mediate these effects and whether reduced ER Ca²⁺ or enhanced Ca²⁺i signaling is most relevant for apoptosis protection. DT40 cell lines engineered to express each of the three mammalian InsP₃R isoforms individually displayed enhanced apoptosis sensitivity compared with cells lacking InsP₃R. In contrast, coexpression of each isoform with Bcl-xL conferred enhanced apoptosis resistance. In single-channel recordings of channel gating in native ER membranes, Bcl-xL increased the apparent sensitivity of all three InsP₃R isoforms to subsaturating levels of InsP₃. Expression of Bcl-xL reduced ER Ca²⁺ in type 3 but not type 1 or 2 InsP₃R-expressing cells. In contrast, Bcl-xL enhanced spontaneous Ca²⁺i signaling in all three InsP₃R isoform-expressing cell lines. These results demonstrate a redundancy among InsP₃R isoforms in their ability to sensitize cells to apoptotic insults and to interact with Bcl-xL to modulate their activities that result in enhanced apoptosis resistance. Furthermore, these data suggest that modulation of ER Ca²⁺ is not a specific requirement for ER-dependent antiapoptotic effects of Bcl-xL. Rather, apoptosis protection is conferred by enhanced spontaneous Ca²⁺i signaling by Bcl-xL interaction with all isoforms of the InsP₃R.
Antiapoptotic Bcl-2 family members interact with inositol trisphosphate receptor (InsP₃R) Ca2+ release channels in the endoplasmic reticulum to modulate Ca2+ signals that affect cell viability. ...However, the molecular details and consequences of their interactions are unclear. Here, we found that Bcl-xL activates single InsP₃R channels with a biphasic concentration dependence. The Bcl-xL Bcl-2 homology 3 (BH3) domain-binding pocket mediates both high-affinity channel activation and low-affinity inhibition. Bcl-xL activates channel gating by binding to two BH3 domain-like helices in the channel carboxyl terminus, whereas inhibition requires binding to one of them and to a previously identified Bcl-2 interaction site in the channel-coupling domain. Disruption of these interactions diminishes cell viability and sensitizes cells to apoptotic stimuli. Our results identify BH3-like domains in an ion channel and they provide a unifying model of the effects of antiapoptotic Bcl-2 proteins on the InsP₃R that play critical roles in Ca2+ signaling and cell viability.
Modulation of cytoplasmic free Ca 2 + concentration (Ca 2 + i ) by receptor-mediated generation of inositol 1,4,5-trisphosphate (InsP 3 ) and activation of its receptor (InsP 3 R), a Ca 2 + -release ...channel in the endoplasmic reticulum, is a ubiquitous signalling mechanism. A fundamental aspect of InsP 3 -mediated signalling is the graded release of Ca 2 + in response to incremental levels of stimuli. Ca 2 + release has a transient fast phase, whose rate is proportional to InsP 3 , followed by a much slower one even in constant InsP 3 . Many schemes have been proposed to account for quantal Ca 2 + release, including the presence of heterogeneous channels and Ca 2 + stores with various mechanisms of release termination. Here, we demonstrate that mechanisms intrinsic to the single InsP 3 R channel can account for quantal Ca 2 + release. Patch-clamp electrophysiology of isolated insect Sf9 cell nuclei revealed a consistent and high probability of detecting
functional endogenous InsP 3 R channels, enabling InsP 3 -induced channel inactivation to be identified as an inevitable consequence of activation, and allowing the average number
of activated channels in the membrane patch ( N A ) to be accurately quantified. InsP 3 -activated channels invariably inactivated, with average duration of channel activity reduced by high Ca 2 + i and suboptimal InsP 3 . Unexpectedly, N A was found to be a graded function of both Ca 2 + i and InsP 3 . A qualitative model involving Ca 2 + -induced InsP 3 R sequestration and inactivation can account for these observations. These results suggest that apparent heterogeneous ligand
sensitivity can be generated in a homogeneous population of InsP 3 R channels, providing a mechanism for graded Ca 2 + release that is intrinsic to the InsP 3 R Ca 2 + release channel itself.
The type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) is a ubiquitous intracellular Ca2+ release channel that is vital to intracellular Ca2+ signaling. InsP3R1 is a proteolytic target of ...calpain, which cleaves the channel to form a 95-kDa carboxyl-terminal fragment that includes the transmembrane domains, which contain the ion pore. However, the functional consequences of calpain proteolysis on channel behavior and Ca2+ homeostasis are unknown. In the present study we have identified a unique calpain cleavage site in InsP3R1 and utilized a recombinant truncated form of the channel (capn-InsP3R1) corresponding to the stable, carboxyl-terminal fragment to examine the functional consequences of channel proteolysis. Single-channel recordings of capn-InsP3R1 revealed InsP3-independent gating and high open probability (Po) under optimal cytoplasmic Ca2+ concentration (Ca2+i) conditions. However, some Ca2+i regulation of the cleaved channel remained, with a lower Po in suboptimal and inhibitory Ca2+i. Expression of capn-InsP3R1 in N2a cells reduced the Ca2+ content of ionomycin-releasable intracellular stores and decreased endoplasmic reticulum Ca2+ loading compared with control cells expressing full-length InsP3R1. Using a cleavage-specific antibody, we identified calpain-cleaved InsP3R1 in selectively vulnerable cerebellar Purkinje neurons after in vivo cardiac arrest. These findings indicate that calpain proteolysis of InsP3R1 generates a dysregulated channel that disrupts cellular Ca2+ homeostasis. Furthermore, our results demonstrate that calpain cleaves InsP3R1 in a clinically relevant injury model, suggesting that Ca2+ leak through the proteolyzed channel may act as a feed-forward mechanism to enhance cell death.
The mitochondrial uniporter (MCU) is an ion channel that mediates Ca(2+) uptake into the matrix to regulate metabolism, cell death, and cytoplasmic Ca(2+) signaling. Matrix Ca(2+) concentration is ...similar to that in cytoplasm, despite an enormous driving force for entry, but the mechanisms that prevent mitochondrial Ca(2+) overload are unclear. Here, we show that MCU channel activity is governed by matrix Ca(2+) concentration through EMRE. Deletion or charge neutralization of its matrix-localized acidic C terminus abolishes matrix Ca(2+) inhibition of MCU Ca(2+) currents, resulting in MCU channel activation, enhanced mitochondrial Ca(2+) uptake, and constitutively elevated matrix Ca(2+) concentration. EMRE-dependent regulation of MCU channel activity requires intermembrane space-localized MICU1, MICU2, and cytoplasmic Ca(2+). Thus, mitochondria are protected from Ca(2+) depletion and Ca(2+) overload by a unique molecular complex that involves Ca(2+) sensors on both sides of the inner mitochondrial membrane, coupled through EMRE.
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