SUMMARY Mitochondrial Ca2+ (Ca2+m) uptake is mediated by an inner membrane Ca2+ channel called the uniporter. Ca2+ uptake is driven by the considerable voltage present across the inner membrane (ΔΨm) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca2+ concentration is maintained 5–6 orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca2+]m under basal conditions. In its absence, mitochondria become constitutively loaded with Ca2+, triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca2+ threshold for Ca2+m uptake without affecting the kinetic properties of MCU-mediated Ca2+ uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca2+m uptake that is essential to prevent [Ca2+]m overload and associated stress.

Ca2+ flux across the mitochondrial inner membrane regulates bioenergetics, cytoplasmic Ca2+ signals and activation of cell death pathways1–11. Mitochondrial Ca2+ uptake occurs at regions of close apposition with intracellular Ca2+ release sites 12–14, driven by the inner membrane voltage generated by oxidative phosphorylation and mediated by a Ca2+ selective ion channel (MiCa15) called the uniporter16–18 whose complete molecular identity remains unknown. Mitochondrial calcium uniporter (MCU) was recently identified as the likely ion-conducting pore19, 20. In addition, MICU1 was identified as a mitochondrial regulator of uniporter-mediated Ca2+ uptake in HeLa cells 21. Here we identified CCDC90A, hereafter referred to as MCUR1 (Mitochondrial Calcium Uniporter Regulator 1), an integral membrane protein required for MCU-dependent mitochondrial Ca2+ uptake. MCUR1 binds to MCU and regulates ruthenium red-sensitive MCU-dependent Ca2+ uptake. MCUR1 knockdown does not alter MCU localization, but abrogates Ca2+ uptake by energized mitochondria in intact and permeabilized cells. Ablation of MCUR1 disrupts oxidative phosphorylation, lowers cellular ATP, and activates AMP kinase-dependent pro-survival autophagy. Thus, MCUR1 is a critical component of a mitochondrial uniporter channel complex required for mitochondrial Ca2+ uptake and maintenance of normal cellular bioenergetics.

During sepsis, acute lung injury (ALI) results from activation of innate immune cells and endothelial cells by endotoxins, leading to systemic inflammation through proinflammatory cytokine overproduction, oxidative stress, and intracellular Ca 2+ overload. Despite considerable investigation, the underlying molecular mechanism(s) leading to LPS-induced ALI remain elusive. To determine whether stromal interaction molecule 1-dependent (STIM1-dependent) signaling drives endothelial dysfunction in response to LPS, we investigated oxidative and STIM1 signaling of EC-specific Stim1-knockout mice. Here we report that LPSmediated Ca 2+ oscillations are ablated in ECs deficient in Nox2, Stim1, and type II inositol triphosphate receptor (Itpr2). LPS-induced nuclear factor of activated T cells (NFAT) nuclear accumulation was abrogated by either antioxidant supplementation or Ca 2+ chelation. Moreover, ECs lacking either Nox2 or Stim1 failed to trigger store-operated Ca 2+ entry (SOCe) and NFAT nuclear accumulation. LPS-induced vascular permeability changes were reduced in EC-specific Stim1 -/-mice, despite elevation of systemic cytokine levels. Additionally, inhibition of STIM1 signaling prevented receptor-interacting protein 3-dependent (RIP3-dependent) EC death. Remarkably, BTP2, a small-molecule calcium release-activated calcium (CRAC) channel blocker administered after insult, halted LPS-induced vascular leakage and pulmonary edema. These results indicate that ROS-driven Ca 2+ signaling promotes vascular barrier dysfunction and that the SOCe machinery may provide crucial therapeutic targets to limit sepsis-induced ALI.

SUMMARY Resting mitochondrial matrix Ca2+ is maintained through a MICU1-established threshold inhibition of MCU activity. It is not known how MICU1 interacts with MCU to establish this Ca2+ threshold for mitochondrial Ca2+ uptake and MCU activity. Here, we show that MICU1 localizes to the mitochondrial matrix side of the inner mitochondrial membrane and MICU1/MCU binding is determined by a MICU1 N-terminal polybasic domain and two interacting coiled-coil domains of MCU. Further investigation reveals MICU1 forms homo-oligomers, and this oligomerization is independent of the polybasic region. However the polybasic region confers MICU1 oligomeric binding to MCU and controls mitochondrial Ca2+ current (IMCU). Moreover, MICU1 EF-hands regulate MCU channel activity but do not determine MCU binding. Loss of MICU1 promotes MCU activation leading to oxidative burden and a halt to cell migration. These studies establish a molecular mechanism for MICU1 control of MCU-mediated mitochondrial Ca2+ accumulation, and dysregulation of this mechanism likely enhances vascular dysfunction.

Dysregulation of mitochondrial Ca 2؉ -dependent bioenergetics has been implicated in various pathophysiological settings, including neurodegeneration and myocardial infarction. Although mitochondrial Ca 2؉ transport has been characterized, and several molecules, including LETM1, have been identified, the functional role of LETM1-mediated Ca 2؉ transport remains unresolved. This study examines LETM1-mediated mitochondrial Ca 2؉ transport and bioenergetics in multiple cell types, including fibroblasts derived from patients with Wolf-Hirschhorn syndrome (WHS). The results show that both mitochondrial Ca 2؉ influx and efflux rates are impaired in LETM1 knockdown, and similar phenotypes were observed in ⌬EF hand, D676A D688K

In dystrophic muscle, an increase in reactive oxygen species (ROS) production and sarcolemmal calcium (Ca 2þ) influx contributes to stretch-induced muscle damage however mechanistic insights into the activation of these pathways is lacking. In mdx myofibers (murine Duchenne muscular dystrophy), we have demonstrated that with mechanical stretch, the microtubule (MT) cytoskeleton is a critical mechano-transduction element for the activation of NADPH oxi-dase2 (Nox2) derived ROS production; a pathway we term X-ROS signaling [1]. Downstream, we showed that X-ROS sensitized stretch activated channels (SACs) to increase sarcolemmal Ca 2þ influx during stretch. The significance of the MT cytoskeleton activation of X-ROS in mdx was revealed when the acute targeting of MT density proffered protection from contraction induced damage. In mammalian cells, the MT network is a dynamic structure in which MT density is determined by the stability of MT filaments. Our initial studies used acute pharmacological stabilization (taxol) or destabilization (colchicine) to establish MT network density as critical for the mechano-activation of X-ROS. We now interrogate critical upstream pathways and use new pharmacological and molecular approaches to explore the role of endogenous modulators of MT stability and how they may contribute to the enhanced X-ROS in dystrophic skeletal muscle.

Necroptosis represents a form of alternative programmed cell death that is dependent on the kinase RIP1. RIP1-dependent necroptotic death manifests as increased reactive oxygen species (ROS) production in mitochondria and is accompanied by loss of ATP biogenesis and eventual dissipation of mitochondrial membrane potential. Here, we show that tumor necrosis factor alpha (TNF-␣)-induced necroptosis requires the adaptor proteins FADD and NEMO. FADD was found to mediate formation of the TNF-␣-induced pronecrotic RIP1-RIP3 kinase complex, whereas the IB Kinase (IKK) subunit NEMO appears to function downstream of RIP1-RIP3. Interestingly, loss of RelA potentiated TNF-␣-dependent necroptosis, indicating that NEMO regulates necroptosis independently of NF-B. Using both pharmacologic and genetic approaches, we demonstrate that the overexpression of antioxidants alleviates ROS elevation and necroptosis. Finally, elimination of BAX and BAK or overexpression of Bcl-x L protects cells from necroptosis at a later step. These findings provide evidence that mitochondria play an amplifying role in inflammation-induced necroptosis.

Knockdown of SLC25A23 decreases mitochondrial Ca2+ uptake, and SLC25A23 interacts with MCU and MICU1, components of mitochondrial Ca2+ uniporter. Expression of SLC25A23 EF-hand-domain mutants has a dominant-negative phenotype of reduced mitochondrial Ca2+ uptake. It also attenuates basal ROS and oxidant-induced ATP decline and cell death.