An uncoupling channel within the c-subunit ring of the F1FO ATP synthase is the mitochondrial permeability transition pore.
ABSTRACT: Mitochondria maintain tight regulation of inner mitochondrial membrane (IMM) permeability to sustain ATP production. Stressful events cause cellular calcium (Ca(2+)) dysregulation followed by rapid loss of IMM potential known as permeability transition (PT), which produces osmotic shifts, metabolic dysfunction, and cell death. The molecular identity of the mitochondrial PT pore (mPTP) was previously unknown. We show that the purified reconstituted c-subunit ring of the FO of the F1FO ATP synthase forms a voltage-sensitive channel, the persistent opening of which leads to rapid and uncontrolled depolarization of the IMM in cells. Prolonged high matrix Ca(2+) enlarges the c-subunit ring and unhooks it from cyclophilin D/cyclosporine A binding sites in the ATP synthase F1, providing a mechanism for mPTP opening. In contrast, recombinant F1 beta-subunit applied exogenously to the purified c-subunit enhances the probability of pore closure. Depletion of the c-subunit attenuates Ca(2+)-induced IMM depolarization and inhibits Ca(2+) and reactive oxygen species-induced cell death whereas increasing the expression or single-channel conductance of the c-subunit sensitizes to death. We conclude that a highly regulated c-subunit leak channel is a candidate for the mPTP. Beyond cell death, these findings also imply that increasing the probability of c-subunit channel closure in a healthy cell will enhance IMM coupling and increase cellular metabolic efficiency.
Project description:Permeability transition (PT) is an increase in mitochondrial inner membrane permeability that can lead to a disruption of mitochondrial function and cell death. PT is responsible for tissue damage in stroke and myocardial infarction. It is caused by the opening of a large conductance (?1.5 nS) channel, the mitochondrial PT pore (mPTP). We directly tested the role of the c-subunit of ATP synthase in mPTP formation by measuring channel activity in c-subunit knockout mitochondria. We found that the classic mPTP conductance was lacking in c-subunit knockout mitochondria, but channels sensitive to the PT inhibitor cyclosporine A could be recorded. These channels had a significantly lower conductance compared with the cyclosporine A-sensitive channels detected in parental cells and were sensitive to the ATP/ADP translocase inhibitor bongkrekic acid. We propose that, in the absence of the c-subunit, mPTP cannot be formed, and a distinct cyclosporine A-sensitive low-conductance channel emerges.
Project description:Mitochondrial permeability transition pore (mPTP) opening allows free movement of ions and small molecules leading to mitochondrial membrane depolarization and ATP depletion that triggers cell death. A multi-protein complex of the mitochondrial ATP synthase has an essential role in mPTP. However, the molecular identity of the central 'pore' part of mPTP complex is not known. A highly purified fraction of mammalian mitochondria containing C-subunit of ATPase (C-subunit), calcium, inorganic polyphosphate (polyP) and polyhydroxybutyrate (PHB) forms ion channels with properties that resemble the native mPTP. We demonstrate here that amount of this channel-forming complex dramatically increases in intact mitochondria during mPTP activation. This increase is inhibited by both Cyclosporine A, an inhibitor of mPTP and Ruthenium Red, an inhibitor of the Mitochondrial Calcium Uniporter. Similar increases in the amount of complex formation occurs in areas of mouse brain damaged by ischemia-reperfusion injury. These findings suggest that calcium-induced mPTP is associated with de novo assembly of a channel comprising C-subunit, polyP and PHB.
Project description:The mitochondrial permeability transition pore (mPTP) has long been known to have a role in mitochondrial calcium (Ca(2+)) homeostasis under pathological conditions as a mediator of the mitochondrial permeability transition and the activation of the consequent cell death mechanism. However, its role in the context of mitochondrial Ca(2+) homeostasis is not yet clear. Several studies that were based on PPIF inhibition or knock out suggested that mPTP is involved in the Ca(2+) efflux mechanism, while other observations have revealed the opposite result. The c subunit of the mitochondrial F1/FO ATP synthase has been recently found to be a fundamental component of the mPTP. In this work, we focused on the contribution of the mPTP in the Ca(2+) efflux mechanism by modulating the expression of the c subunit. We observed that forcing mPTP opening or closing did not impair mitochondrial Ca(2+) efflux. Therefore, our results strongly suggest that the mPTP does not participate in mitochondrial Ca(2+) homeostasis in a physiological context in HeLa cells.
Project description:Mitochondrial permeability transition is a phenomenon in which the mitochondrial permeability transition pore (PTP) abruptly opens, resulting in mitochondrial membrane potential (??m) dissipation, loss of ATP production, and cell death. Several genetic candidates have been proposed to form the PTP complex, however, the core component is unknown. We identified a necessary and conserved role for spastic paraplegia 7 (SPG7) in Ca(2+)- and ROS-induced PTP opening using RNAi-based screening. Loss of SPG7 resulted in higher mitochondrial Ca(2+) retention, similar to cyclophilin D (CypD, PPIF) knockdown with sustained ??m during both Ca(2+) and ROS stress. Biochemical analyses revealed that the PTP is a heterooligomeric complex composed of VDAC, SPG7, and CypD. Silencing or disruption of SPG7-CypD binding prevented Ca(2+)- and ROS-induced ??m depolarization and cell death. This study identifies an ubiquitously expressed IMM integral protein, SPG7, as a core component of the PTP at the OMM and IMM contact site.
Project description:Ion transport across the mitochondrial inner and outer membranes is central to mitochondrial function, including regulation of oxidative phosphorylation and cell death. Although essential for ATP production by mitochondria, recent findings have confirmed that the c-subunit of the ATP synthase also houses a large conductance uncoupling channel, the mitochondrial permeability transition pore (mPTP), the persistent opening of which produces osmotic dysregulation of the inner mitochondrial membrane and cell death. This review will discuss recent advances in understanding the molecular components of mPTP, its regulatory mechanisms and how these contribute directly to its physiological as well as pathological roles.
Project description:Healthy mitochondria use an electrochemical gradient across the inner mitochondrial membrane (IMM) to generate energy in the form of ATP. A variety of endogenous and exogenous factors can lead to transient or sustained depolarization of the IMM, including mitochondrial fission events, expression of uncoupling proteins, electron transport chain (ETC) inhibitors, or chemical uncouplers. This depolarization in turn leads to a variety of physiological responses, ranging from selective mitochondrial clearance (mitophagy) to cell death. How cells recognize and ultimately respond to depolarized mitochondria remains incompletely understood. Here we show that the small GTPases RalA and RalB both relocalize to mitochondria following depolarization in a process dependent on clathrin-mediated endocytosis (CME). Furthermore, both genetic and pharmacologic inhibition of RalA and RalB leads to an increase in the activity of the atypical I?B kinase TBK1 both basally and in response to mitochondrial depolarization. This phenotype was also observed following inhibition of Ral relocalization. Collectively, these data suggest a model in which RalA and RalB inhibit TBK1 and that relocalization of Ral to depolarized mitochondria facilitates TBK1 activation through release of this inhibition.
Project description:Mitochondrial Ca(2+) controls numerous cell functions, such as energy metabolism, reactive oxygen species generation, spatiotemporal dynamics of Ca(2+) signaling, cell growth and death in various cell types including neurons. Mitochondrial Ca(2+) accumulation is mainly mediated by the mitochondrial Ca(2+) uniporter (MCU), but recent reports also indicate that mitochondrial Ca(2+)-influx mechanisms are regulated not only by MCU, but also by multiple channels/transporters. We previously reported that ryanodine receptor (RyR), which is a one of the main Ca(2+)-release channels at endoplasmic/sarcoplasmic reticulum (SR/ER) in excitable cells, is expressed at the mitochondrial inner membrane (IMM) and serves as a part of the Ca(2+) uptake mechanism in cardiomyocytes. Although RyR is also expressed in neuronal cells and works as a Ca(2+)-release channel at ER, it has not been well investigated whether neuronal mitochondria possess RyR and, if so, whether this mitochondrial RyR has physiological functions in neuronal cells. Here we show that neuronal mitochondria express RyR at IMM and accumulate Ca(2+) through this channel in response to cytosolic Ca(2+) elevation, which is similar to what we observed in another excitable cell-type, cardiomyocytes. In addition, the RyR blockers dantrolene or ryanodine significantly inhibits mitochondrial Ca(2+) uptake in permeabilized striatal neurons. Taken together, we identify RyR as an additional mitochondrial Ca(2+) uptake mechanism in response to the elevation of [Ca(2+)]c in neurons, suggesting that this channel may play a critical role in mitochondrial Ca(2+)-mediated functions such as energy metabolism.
Project description:In the heart, augmented Ca(2+) fluxing drives contractility and ATP generation through mitochondrial Ca(2+) loading. Pathologic mitochondrial Ca(2+) overload with ischemic injury triggers mitochondrial permeability transition pore (MPTP) opening and cardiomyocyte death. Mitochondrial Ca(2+) uptake is primarily mediated by the mitochondrial Ca(2+) uniporter (MCU). Here, we generated mice with adult and cardiomyocyte-specific deletion of Mcu, which produced mitochondria refractory to acute Ca(2+) uptake, with impaired ATP production, and inhibited MPTP opening upon acute Ca(2+) challenge. Mice lacking Mcu in the adult heart were also protected from acute ischemia-reperfusion injury. However, resting/basal mitochondrial Ca(2+) levels were normal in hearts of Mcu-deleted mice, and mitochondria lacking MCU eventually loaded with Ca(2+) after stress stimulation. Indeed, Mcu-deleted mice were unable to immediately sprint on a treadmill unless warmed up for 30 min. Hence, MCU is a dedicated regulator of short-term mitochondrial Ca(2+) loading underlying a "fight-or-flight" response that acutely matches cardiac workload with ATP production.
Project description:Diazoxide, a well-known opener of the mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, has been demonstrated to exert cardioprotective effect against ischemic injury through the mitoK(ATP) channel and protein kinase C (PKC). We aimed to clarify the role of PKC isoforms and the relationship between the PKC isoforms and the mitoK(ATP) channel in diazoxide-induced cardioprotection.In H9c2 cells and neonatal rat cardiomyocytes, PKC-epsilon activation was examined by Western blotting and kinase assay. Flavoprotein fluorescence, mitochondrial Ca(2+) and mitochondrial membrane potential were measured by confocal microscopy. Cell death was determined by TUNEL assay.Diazoxide (100 microM) induced translocation of PKC-epsilon from the cytosolic to the mitochondrial fraction. Specific blockade of PKC-epsilon by either epsilonV1-2 or dominant negative mutant PKC-epsilon (PKC-epsilon KR) abolished the anti-apoptotic effect of diazoxide. Diazoxide-induced flavoprotein oxidation was inhibited by either epsilonV1-2 or PKC-epsilon KR transfection. Treatment with 5-hydroxydecanoate (5-HD) did not affect translocation and activation of PKC-epsilon induced by diazoxide. Transfection with wild type PKC-epsilon mimicked the flavoprotein-oxidizing effect of diazoxide, and this effect was completely blocked by epsilonV1-2 or 5-HD. Diazoxide prevented the increase in mitochondrial Ca(2+), mitochondrial depolarization and cytochrome c release induced by hypoxia and all these effects of diazoxide were blocked by epsilonV1-2 or 5-HD.Diazoxide induced isoform-specific translocation of PKC-epsilon as an upstream signaling molecule for the mitoK(ATP) channel, rendering cardiomyocytes resistant to hypoxic injury through inhibition of the mitochondrial death pathway.
Project description:Mitochondrial Ca<sup>2+</sup> uptake has a key role in cellular Ca<sup>2+</sup> homeostasis. Excessive matrix Ca<sup>2+</sup> concentrations, especially when coincident with oxidative stress, precipitate opening of an inner mitochondrial membrane, high-conductance channel: the mitochondrial permeability transition pore (mPTP). mPTP opening has been implicated as a final cell death pathway in numerous diseases and therefore understanding conditions dictating mPTP opening is crucial for developing targeted therapies. Here, we have investigated the impact of mitochondrial metabolic state on the probability and consequences of mPTP opening. Isolated mitochondria were energised using NADH- or FADH<sub>2</sub>-linked substrates. The functional consequences of Ca<sup>2+</sup>-induced mPTP opening were assessed by Ca<sup>2+</sup> retention capacity, using fluorescence-based analysis, and simultaneous measurements of mitochondrial Ca<sup>2+</sup> handling, membrane potential, respiratory rate and production of reactive oxygen species (ROS). Succinate-induced, membrane potential-dependent reverse electron transfer sensitised mitochondria to mPTP opening. mPTP-induced depolarisation under succinate subsequently inhibited reverse electron transfer. Complex I-driven respiration was reduced after mPTP opening but sustained in the presence of complex II-linked substrates, consistent with inhibition of complex I-supported respiration by leakage of matrix NADH. Additionally, ROS generated at complex III did not sensitise mitochondria to mPTP opening. Thus, cellular metabolic fluxes and metabolic environment dictate mitochondrial functional response to Ca<sup>2+</sup> overload.