Purified mitochondrial ATP synthase has been shown to form Ca2+-activated, large conductance channel activity similar to that of mitochondrial megachannel (MMC) or mitochondrial permeability ...transition pore (mPTP) but the oligomeric state required for channel formation is being debated. We reconstitute purified monomeric ATP synthase from porcine heart mitochondria into small unilamellar vesicles (SUVs) with the lipid composition of mitochondrial inner membrane and analyze its oligomeric state by electron cryomicroscopy. The cryo-EM density map reveals the presence of a single ATP synthase monomer with no density seen for a second molecule tilted at an 86o angle relative to the first. We show that this preparation of SUV-reconstituted ATP synthase monomers, when fused into giant unilamellar vesicles (GUVs), forms voltage-gated and Ca2+-activated channels with the key features of mPTP. Based on our findings we conclude that the ATP synthase monomer is sufficient, and dimer formation is not required, for mPTP activity.
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.
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•Deletion of the c-subunit leads to loss of the mPTP channel•C-subunit KO mitochondria contain a CSA-sensitive channel•The c-subunit KO channel has lower conductance compared to mPTP•The c-subunit KO channel is sensitive to ANT inhibitors
Neginskaya et al. report that c-subunit-deficient mitochondria contain a CSA-sensitive channel. This channel is much smaller compared with the wild-type permeability transition pore and is sensitive to inhibitors of adenine nucleotide translocase. This work highlights the importance of the c-subunit in forming the permeability transition pore.
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 F
1
F
O
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.
Loss of the gene (Fmr1) encoding Fragile X mental retardation protein (FMRP) causes increased mRNA translation and aberrant synaptic development. We find neurons of the Fmr1−/y mouse have a ...mitochondrial inner membrane leak contributing to a “leak metabolism.” In human Fragile X syndrome (FXS) fibroblasts and in Fmr1−/y mouse neurons, closure of the ATP synthase leak channel by mild depletion of its c-subunit or pharmacological inhibition normalizes stimulus-induced and constitutive mRNA translation rate, decreases lactate and key glycolytic and tricarboxylic acid (TCA) cycle enzyme levels, and triggers synapse maturation. FMRP regulates leak closure in wild-type (WT), but not FX synapses, by stimulus-dependent ATP synthase β subunit translation; this increases the ratio of ATP synthase enzyme to its c-subunit, enhancing ATP production efficiency and synaptic growth. In contrast, in FXS, inability to close developmental c-subunit leak prevents stimulus-dependent synaptic maturation. Therefore, ATP synthase c-subunit leak closure encourages development and attenuates autistic behaviors.
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•ATP synthase c-subunit leak in Fragile X causes aberrant metabolism•Changes in ATP synthase component stoichiometry regulate protein synthesis rate•Inhibition of the leak normalizes synaptic spine morphology and Fragile X behavior
Lack of FMRP in Fragile X neurons is associated with a leak in the ATP synthase, the blockade of which normalizes cellular and behavioral disease phenotypes.
Familial Parkinson's disease (PD) protein DJ-1 mutations are linked to early onset PD. We have found that DJ-1 binds directly to the F
F
ATP synthase β subunit. DJ-1's interaction with the β subunit ...decreased mitochondrial uncoupling and enhanced ATP production efficiency while in contrast mutations in DJ-1 or DJ-1 knockout increased mitochondrial uncoupling, and depolarized neuronal mitochondria. In mesencephalic DJ-1 KO cultures, there was a progressive loss of neuronal process extension. This was ameliorated by a pharmacological reagent, dexpramipexole, that binds to ATP synthase, closing a mitochondrial inner membrane leak and enhancing ATP synthase efficiency. ATP synthase c-subunit can form an uncoupling channel; we measured, therefore, ATP synthase F
(β subunit) and c-subunit protein levels. We found that ATP synthase β subunit protein level in the DJ-1 KO neurons was approximately half that found in their wild-type counterparts, comprising a severe defect in ATP synthase stoichiometry and unmasking c-subunit. We suggest that DJ-1 enhances dopaminergic cell metabolism and growth by its regulation of ATP synthase protein components.
B-cell lymphoma-extra large (Bcl-xL) is an anti-apoptotic member of the Bcl2 family of proteins, which supports neurite outgrowth and neurotransmission by improving mitochondrial function. During ...excitotoxic stimulation, however, Bcl-xL undergoes post-translational cleavage to ∆N-Bcl-xL, and accumulation of ∆N-Bcl-xL causes mitochondrial dysfunction and neuronal death. In this study, we hypothesized that the generation of reactive oxygen species (ROS) during excitotoxicity leads to formation of ∆N-Bcl-xL. We further proposed that the application of an antioxidant with neuroprotective properties such as α-tocotrienol (TCT) will prevent ∆N-Bcl-xL-induced mitochondrial dysfunction via its antioxidant properties. Primary hippocampal neurons were treated with α-TCT, glutamate, or a combination of both. Glutamate challenge significantly increased cytosolic and mitochondrial ROS and ∆N-Bcl-xL levels. ∆N-Bcl-xL accumulation was accompanied by intracellular ATP depletion, loss of mitochondrial membrane potential, and cell death. α-TCT prevented loss of mitochondrial membrane potential in hippocampal neurons overexpressing ∆N-Bcl-xL, suggesting that ∆N-Bcl-xL caused the loss of mitochondrial function under excitotoxic conditions. Our data suggest that production of ROS is an important cause of ∆N-Bcl-xL formation and that preventing ROS production may be an effective strategy to prevent ∆N-Bcl-xL-mediated mitochondrial dysfunction and thus promote neuronal survival.
The mitochondrial permeability transition pore (mPTP) or mitochondrial megachannel is arguably one of the most mysterious phenomena in biology today. mPTP has been at the center of ongoing extensive ...scientific research for the last several decades. In this review we will discuss recent advances in the field that enhance our understanding of the molecular composition of mPTP, its regulatory mechanisms and its pathophysiological role. We will describe our recent findings on the role of ATP synthase c-subunit ring as a central player in mitochondrial permeability transition and as an important metabolic regulator during development and in degenerative diseases.
•ATP synthase c-subunit is the leak channel of mitochondrial permeability transition.•c-subunit channel is the master regulator of cell metabolism in embryonic and synaptic development.•F1/FO stoichiometry change is critical for c-subunit regulatory function.•Non-reversible dissociation of ATP synthase F1 from FO triggers age-related ischemic and degenerative diseases.•We discuss models for ATP synthase channel gating in physiological and pathological conditions.
The mitochondrial F1Fo ATP synthase is one of the most abundant proteins of the mitochondrial inner membrane, which catalyzes the final step of oxidative phosphorylation to synthesize ATP from ADP ...and Pi. ATP synthase uses the electrochemical gradient of protons (ΔμH+) across the mitochondrial inner membrane to synthesize ATP. Under certain pathophysiological conditions, ATP synthase can run in reverse to hydrolyze ATP and build the necessary ΔμH+ across the mitochondrial inner membrane. Tight coupling between these two processes, proton translocation and ATP synthesis, is achieved by the unique rotational mechanism of ATP synthase and is necessary for efficient cellular metabolism and cell survival. The uncoupling of these processes, dissipation of mitochondrial inner membrane potential, elevated levels of ROS, low matrix content of ATP in combination with other cellular malfunction trigger the opening of the mitochondrial permeability transition pore in the mitochondrial inner membrane. In this review we will discuss the new role of ATP synthase beyond oxidative phosphorylation. We will highlight its function as a unique regulator of cell life and death and as a key target in mitochondria-mediated neurodegeneration and neuroprotection.
The hyc operon of Escherichia coli encodes the H2‐evolving hydrogenase 3 (Hyd‐3) complex that, in conjunction with formate dehydrogenase H (Fdh‐H), constitutes a membrane‐associated formate ...hydrogenlyase (FHL) catalyzing the disproportionation of formate to CO2 and H2 during fermentative growth at low pH. Recently, an operon (hyf) encoding a potential second H2‐evolving hydrogenase (Hyd‐4) was identified in E. coli. In this study the roles of the hyc‐ and hyf‐encoded systems in formate‐dependent H2 production and Fdh‐H activity have been investigated. In cells grown on glucose under fermentative conditions at slightly acidic pH the production of H2 was mostly Hyd‐3‐ and Fdh‐H‐dependent, and Fdh‐H activity was also mainly Hyd‐3‐dependent. However, at slightly alkaline pH, H2 production was found to be largely Hyd‐4, Fdh‐H and F0F1‐ATPase‐dependent, and Fdh‐H activity was partially dependent on Hyd‐4 and F0F1‐ATPase. These results suggest that, at slightly alkaline pH, H2 production and Fdh‐H activity are dependent on both the F0F1‐ATPase and a novel FHL, designated FHL‐2, which is composed of Hyd‐4 and Fdh‐H, and is driven by a proton gradient established by the F0F1‐ATPase.
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