Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

University of Rochester

Deregulation of the intracellular Ca2+ homeostasis by NMDA receptor activation leads to neuronal cell death. Induction of the mitochondrial permeability transition pore (MPT) by Ca2+ is a critical event in mediating cell death. In this study, we used fluorescent Ca2+ indicators to investigate the effect of high concentrations of NMDA on cytosolic and mitochondrial Ca2+ concentrations ([Ca2+]c and [Ca2+]m, respectively) in cultured striatal neurons. Exposure to NMDA resulted in an immediate, sustained increase in [Ca2+]c followed by a secondary increase in [Ca2+]c. This second increase of [Ca2+]c was prevented by pretreatment with N-methyl-valine-4-cyclosporin (NMV-Cys). Exposure of neurons to NMDA also resulted in an increase in [Ca2+]m that was followed by a precipitous decrease in the rhod-2 signal. This decrease followed the time frame of the secondary increase in [Ca2+]c. Preincubation of the neurons with NMV-Cys prevented the decrease in rhod-2 fluorescence. These dynamic changes in the rhod-2 signal and [Ca2+]m in response to NMDA were confirmed by using confocal microscopy. The presented results indicate that MPT can be detected in living neurons using fluorescent Ca2+ indicators, which would allow the study of the physiological role of MPT in cell death.

Mitochondrial permeability transition and calcium dynamics in striatal neurons upon intense NMDA receptor activation.

Mitochondria in a variety of cell types respond to physiological Ca(2+) oscillations in the cytosol dynamically with Ca(2+) uptakes. In heart cells, mitochondrial Ca(2+) uptakes occur by a ruthenium red-sensitive Ca(2+) uniporter (CaUP), a rapid mode of Ca(2+) uptake (RaM) and a ryanodine receptor (RyR) localized in the inner mitochondrial membrane (IMM). Three subtypes of RyRs have been described and cloned, however, the subtype identity of the mitochondrial ryanodine receptor (mRyR) is unknown. Using subtype specific antibodies, we characterized the mRyR in the IMM from rat heart as RyR1. These results are substantiated by the absence of RyR protein in heart mitochondria from RyR1 knockout mice. The bell-shape Ca(2+)-dependent [(3)H]ryanodine binding curve and its modulation by caffeine and adenylylmethylenediphosphonate (AMPPCP) give further evidence that mRyR functions pharmacologically like RyR1. Ryanodine prevents mitochondrial Ca(2+) uptake induced by raising extramitochondrial Ca(2+) to 10 microM. Similarly, ryanodine inhibits oxidative phosphorylation stimulated by 10 microM extramitochondrial Ca(2+). In summary, our results show that the mRyR in cardiac muscle has similar biochemical and pharmacological properties to the RyR1 in the sarcoplasmic reticulum (SR) of skeletal muscle. These results could also suggest an efficient mechanism by which mitochondria sequesters Ca(2+) via mRyR during excitation-contraction coupling to stimulate oxidative phosphorylation for ATP production to meet metabolic demands. Thus, the mRyR functions as a transducer for excitation-metabolism coupling.

Type 1 ryanodine receptor in cardiac mitochondria: transducer of excitation-metabolism coupling.

A protein discovered within inner mitochondrial membranes (IMM), designated as the mitochondrial ryanodine receptor (mRyR), has been recognized recently as a modulator of Ca(2+) fluxes in mitochondria. The present study provides fundamental pharmacological and electrophysiological properties of this mRyR. Rat cardiac IMM fused to lipid bilayers revealed the presence of a mitochondrial channel with gating characteristics similar to those of classical sarcoplasmic reticulum RyR (SR-RyR), but a variety of other mitochondrial channels obstructed clean recordings. Mitochondrial vesicles were thus solubilized and subjected to sucrose sedimentation to obtain mRyR-enriched fractions. Reconstitution of sucrose-purified fractions into lipid bilayers yielded Cs(+)-conducting, Ca(2+)-sensitive, large conductance (500-800 pS) channels with signature properties of SR-RyRs. Cytosolic Ca(2+) increased the bursting frequency and mean open time of the channel. Micromolar concentrations of ryanodine induced the appearance of subconductance states or inhibited channel activity altogether, while Imperatoxin A (IpTx(a)), a specific activator of RyRs, reversibly induced the appearance of distinct subconductance states. Remarkably, the cardiac mRyR displayed a Ca(2+) dependence of [(3)H]ryanodine binding curve similar to skeletal RyR (RyR1), not cardiac RyR (RyR2). Overall, the mRyR displayed elemental attributes that are present in single channel lipid bilayer recordings of SR-RyRs, although some exquisite differences were also noted. These results therefore provide the first direct evidence that a unique RyR occurs in mitochondrial membranes.

The mitochondrial ryanodine receptor in rat heart: a pharmaco-kinetic profile.

Although the nuclear processes responsible for genomic DNA replication and repair are well characterized, the pathways involved in mitochondrial DNA (mtDNA) replication and repair remain unclear. DNA repair has been identified as being particularly important within the mitochondrial compartment due to the organelle's high propensity to accumulate oxidative DNA damage. It has been postulated that continual accumulation of mtDNA damage and subsequent mutagenesis may function in cellular aging. Mitochondrial base excision repair (mtBER) plays a major role in combating mtDNA oxidative damage; however, the proteins involved in mtBER have yet to be fully characterized. It has been established that during nuclear long-patch (LP) BER, FEN1 is responsible for cleavage of 5' flap structures generated during DNA synthesis. Furthermore, removal of 5' flaps has been observed in mitochondrial extracts of mammalian cell lines; yet, the mitochondrial localization of FEN1 has not been clearly demonstrated. In this study, we analyzed the effects of deleting the yeast FEN1 homolog, RAD27, on mtDNA stability in Saccharomyces cerevisiae. Our findings demonstrate that Rad27p/FEN1 is localized in the mitochondrial compartment of both yeast and mice and that Rad27p has a significant role in maintaining mtDNA integrity.

Evidence for a role of FEN1 in maintaining mitochondrial DNA integrity.

Ca(2+) channels that underlie mitochondrial Ca(2+) transport first reported decades ago have now just recently been precisely characterized electrophysiologically. Numerous data indicate that mitochondrial Ca(2+) uptake via these channels regulates multiple intracellular processes by shaping cytosolic and mitochondrial Ca(2+) transients, as well as altering the cellular metabolic and redox state. On the other hand, mitochondrial Ca(2+) overload also initiates a cascade of events that leads to cell death. Thus, characterization of mitochondrial Ca(2+) channels is central to a comprehensive understanding of cell signaling. Here, we discuss recent progresses in the biophysical and electrophysiological characterization of several distinct mitochondrial Ca(2+) channels.

Mitochondrial ryanodine receptors and other mitochondrial Ca2+ permeable channels.

Heart mitochondria utilize multiple Ca(2+) transport mechanisms. Among them, the mitochondrial ryanodine receptor provides a fast Ca(2+) uptake pathway across the inner membrane to control "excitation and metabolism coupling." In the present study, we identified a novel ryanodine-sensitive channel in the native inner membrane of heart mitochondria and characterized its pharmacological and biophysical properties by directly patch clamping mitoplasts. Four distinct channel conductances of ∼100, ∼225, ∼700, and ∼1,000 picosiemens (pS) in symmetrical 150 mm CsCl were observed. The 225 pS cation-selective channel exhibited multiple subconductance states and was blocked by high concentrations of ryanodine and ruthenium red, known inhibitors of ryanodine receptors. Ryanodine exhibited a concentration-dependent modulation of this channel, with low concentrations stabilizing a subconductance state and high concentrations abolishing activity. The 100, 700, and 1,000 pS conductances exhibited different channel characteristics and were not inhibited by ryanodine. Taken together, these findings identified a novel 225 pS channel as the native mitochondrial ryanodine receptor channel activity in heart mitoplasts with biophysical and pharmacological properties that distinguish it from previously identified mitochondrial ion channels.

Single channel characterization of the mitochondrial ryanodine receptor in heart mitoplasts.

ADP is not only a key substrate for ATP generation, but also a potent inhibitor of mitochondrial permeability transition pore (mPTP). In this study, we assessed how oxidative stress affects the potency of ADP as an mPTP inhibitor and whether its reduction of reactive oxygen species (ROS) production might be involved. We determined quantitatively the effects of ADP on mitochondrial Ca(2+) retention capacity (CRC) until the induction of mPTP in normal and stressed isolated cardiac mitochondria. We used two models of chronic oxidative stress (old and diabetic mice) and two models of acute oxidative stress (ischemia reperfusion (IR) and tert-butyl hydroperoxide (t-BH)). In control mitochondria, the CRC was 344 ± 32 nmol/mg protein. 500 μmol/L ADP increased CRC to 774 ± 65 nmol/mg protein. This effect of ADP seemed to relate to its concentration as 50 μmol/L had a significantly smaller effect. Also, oligomycin, which inhibits the conversion of ADP to ATP by F0F1ATPase, significantly increased the effect of 50 μmol/L ADP. Chronic oxidative stress did not affect CRC or the effect of 500 μmol/L ADP. After IR or t-BH exposure, CRC was drastically reduced to 1 ± 0.2 and 32 ± 4 nmol/mg protein, respectively. Surprisingly, ADP increased the CRC to 447 ± 105 and 514 ± 103 nmol/mg protein in IR and t-BH, respectively. Thus, it increased CRC by the same amount as in control. In control mitochondria, ADP decreased both substrate and Ca(2+)-induced increase of ROS. However, in t-BH mitochondria the effect of ADP on ROS was relatively small. We conclude that ADP potently restores CRC capacity in severely stressed mitochondria. This effect is most likely not related to a reduction in ROS production. As the effect of ADP relates to its concentration, increased ADP as occurs in the pathophysiological situation may protect mitochondrial integrity and function.

ADP protects cardiac mitochondria under severe oxidative stress.

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.

Molecular and functional identification of a mitochondrial ryanodine receptor in neurons.

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.

An uncoupling channel within the c-subunit ring of the F1FO ATP synthase is the mitochondrial permeability transition pore.

Mitochondria provide energy in form of ATP in eukaryotic cells. However, it is not known when, during embryonic cardiac development, mitochondria become able to fulfill this function. To assess this, we measured mitochondrial oxygen consumption and the activity of the complexes (Cx) 1 and 2 of the electron transport chain (ETC) and used immunoprecipitation to follow the generation of mitochondrial supercomplexes. We show that in the heart of mouse embryos at embryonic day (E) 9.5, mitochondrial ETC activity and oxidative phosphorylation (OXPHOS) are not coupled, even though the complexes are present. We show that Cx-1 of the ETC is able to accept electrons from the Krebs cycle, but enzyme assays that specifically measure electron flow to ubiquinone or Cx-3 show no activity at this early embryonic stage. At E11.5, mitochondria appear functionally more mature; ETC activity and OXPHOS are coupled and respond to ETC inhibitors. In addition, the assembly of highly efficient respiratory supercomplexes containing Cx-1, -3, and -4, ubiquinone, and cytochrome c begins at E11.5, the exact time when Cx-1 becomes functional activated. At E13.5, ETC activity and OXPHOS of embryonic heart mitochondria are indistinguishable from adult mitochondria. In summary, our data suggest that between E9.5 and E11.5 dramatic changes occur in the mitochondria of the embryonic heart, which result in an increase in OXPHOS due to the activation of complex 1 and the formation of supercomplexes.

Initiation of electron transport chain activity in the embryonic heart coincides with the activation of mitochondrial complex 1 and the formation of supercomplexes.

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.

Cell death disguised: The mitochondrial permeability transition pore as the c-subunit of the F(1)F(O) ATP synthase.

Fenretinide is a chemotherapeutic agent in clinical trials for the treatment of neuroblastoma, among the most common and most deadly cancers of childhood. Fenretinide induces apoptosis in neuroblastoma cells through accumulation of mitochondrial reactive oxygen species released from Complex II. The neurotrophin receptor, p75NTR, potentiates this effect. The signaling activity of p75NTR is dependent upon its cleavage to its intracellular domain, p75ICD, trafficking of p75ICD to the nucleus, and functioning of p75ICD as a transcription factor. Mitochondrial Complex II comprises 4 subunits, all of which are encoded by nuclear DNA. We therefore hypothesized that the fenretinide-potentiating effects of p75NTR are the result of transcriptional enrichment of Complex II by p75ICD. However, the present studies demonstrate that neither induced expression of p75ICD or its active fragments nor overexpression of p75NTR results in altered expression or activity of Complex II.

Induction of Expression of p75 Neurotrophin Receptor Intracellular Domain Does Not Induce Expression or Enhance Activity of Mitochondrial Complex II.

Neurons experience high metabolic demand during such processes as synaptic vesicle recycling, membrane potential maintenance and Ca(2+) exchange/extrusion. The energy needs of these events are met in large part by mitochondrial production of ATP through the process of oxidative phosphorylation. The job of ATP production by the mitochondria is performed by the F1FO ATP synthase, a multi-protein enzyme that contains a membrane-inserted portion, an extra-membranous enzymatic portion and an extensive regulatory complex. Although required for ATP production by mitochondria, recent findings have confirmed that the membrane-confined portion of 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, uncoupling of oxidative phosphorylation and cell death. Recent advances in understanding the molecular components of mPTP and its regulatory mechanisms have determined that decreased uncoupling occurs in states of enhanced mitochondrial efficiency; relative closure of mPTP therefore contributes to cellular functions as diverse as cardiac development and synaptic efficacy.

Physiological roles of the mitochondrial permeability transition pore.

Mitochondrial ATP generation by oxidative phosphorylation combines the stepwise oxidation by the electron transport chain (ETC) of the reducing equivalents NADH and FADH2 with the generation of ATP by the ATP synthase. Recent studies show that the ATP synthase is not only essential for the generation of ATP but may also contribute to the formation of the mitochondrial permeability transition pore (PTP). We present a model, in which the PTP is located within the c-subunit ring in the Fo subunit of the ATP synthase. Opening of the PTP was long associated with uncoupling of the ETC and the initiation of programmed cell death. More recently, it was shown that PTP opening may serve a physiologic role: it can transiently open to regulate mitochondrial signaling in mature cells, and it is open in the embryonic mouse heart. This review will discuss how the ATP synthase paradoxically lies at the center of both ATP generation and cell death.

Gisela Beutner

Department of Pediatrics-Division Cardiology

University of Rochester

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

Gisela Beutner

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pediatrics-Division Cardiology

University of Rochester

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

Department of Pediatrics-Division Cardiology