CALHM2 is a mitochondrial protein import channel that regulates fatty acid metabolism.

For mitochondrial metabolism to occur in the matrix, multiple proteins must be imported across the two (inner and outer) mitochondrial membranes. Classically, two protein import channels, TIM/TOM, are known to perform this function, but whether other protein import channels exist is not known. Here, using super-resolution microscopy, proteomics, and electrophysiological techniques, we identify CALHM2 as the import channel for the ECHA subunit of the mitochondrial trifunctional protein (mTFP), which catalyzes β-oxidation of fatty acids in the mitochondrial matrix.

Mitochondrial leak metabolism induces the Spemann-Mangold Organizer via Hif-1α in Xenopus.

An instructive role for metabolism in embryonic patterning is emerging, although a role for mitochondria is poorly defined. We demonstrate that mitochondrial oxidative metabolism establishes the embryonic patterning center, the Spemann-Mangold Organizer, via hypoxia-inducible factor 1α (Hif-1α) in Xenopus. Hypoxia or decoupling ATP production from oxygen consumption expands the Organizer by activating Hif-1α.

Identity, structure, and function of the mitochondrial permeability transition pore: controversies, consensus, recent advances, and future directions.

The mitochondrial permeability transition (mPT) describes a Ca-dependent and cyclophilin D (CypD)-facilitated increase of inner mitochondrial membrane permeability that allows diffusion of molecules up to 1.5 kDa in size. It is mediated by a non-selective channel, the mitochondrial permeability transition pore (mPTP).

Fluid shear stress enhances proliferation of breast cancer cells via downregulation of the c-subunit of the FF ATP synthase.

The presence of circulating cancer cells in the bloodstream is positively correlated with metastasis. We hypothesize that fluid shear stress (FSS) occurring during circulation alters mitochondrial function, enhancing metastatic behaviors of cancer cells. MCF7 and MDA-MB-231 human breast cancer cells subjected to FSS exponentially increased proliferation.

Mitochondrial ATP synthase c-subunit leak channel triggers cell death upon loss of its F subcomplex.

Mitochondrial ATP synthase is vital not only for cellular energy production but also for energy dissipation and cell death. ATP synthase c-ring was suggested to house the leak channel of mitochondrial permeability transition (mPT), which activates during excitotoxic ischemic insult. In this present study, we purified human c-ring from both eukaryotic and prokaryotic hosts to biophysically characterize its channel activity.

The nucleotide binding affinities of two critical conformations of Escherichia coli ATP synthase.

ATP synthase is essential in aerobic energy metabolism, and the rotary catalytic mechanism is one of the core concepts to understand the energetic functions of ATP synthase. Disulfide bonds formed by oxidizing a pair of cysteine mutations halted the rotation of the γ subunit in two critical conformations, the ATP-waiting dwell (αE284C/γQ274C) and the catalytic dwell (αE284C/γL276C). Tryptophan fluorescence was used to measure the nucleotide binding affinities for MgATP, MgADP and MgADP-AlF (a transition state analog) to wild-type and mutant F under reducing and oxidizing conditions.

ATP Synthase c-Subunit Leak Causes Aberrant Cellular Metabolism in Fragile X Syndrome.

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 mouse have a mitochondrial inner membrane leak contributing to a "leak metabolism." In human Fragile X syndrome (FXS) fibroblasts and in Fmr1 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.

The new role of FF ATP synthase in mitochondria-mediated neurodegeneration and neuroprotection.

The mitochondrial FF 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 (Δμ) 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 Δμ across the mitochondrial inner membrane.

ATP synthase c-subunit ring as the channel of mitochondrial permeability transition: Regulator of metabolism in development and degeneration.

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.

Alpha-Tocotrienol Prevents Oxidative Stress-Mediated Post-Translational Cleavage of Bcl-xL in Primary Hippocampal Neurons.

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.

A mitochondrial megachannel resides in monomeric FF ATP synthase.

Purified mitochondrial ATP synthase has been shown to form Ca-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 86 angle relative to the first.

Parkinson's disease protein DJ-1 regulates ATP synthase protein components to increase neuronal process outgrowth.

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 FF 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.

ATP Synthase C-Subunit-Deficient Mitochondria Have a Small Cyclosporine A-Sensitive Channel, but Lack the Permeability Transition Pore.

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.

Identification of two segments of the γ subunit of ATP synthase responsible for the different affinities of the catalytic nucleotide-binding sites.

ATP synthase uses a rotary mechanism to couple transmembrane proton translocation to ATP synthesis and hydrolysis, which occur at the catalytic sites in the β subunits. In the presence of Mg, the three catalytic sites of ATP synthase have vastly different affinities for nucleotides, and the position of the central γ subunit determines which site has high, medium, or low affinity. Affinity differences and their changes as rotation progresses underpin the ATP synthase catalytic mechanism.

Inhibition of Bcl-xL prevents pro-death actions of ΔN-Bcl-xL at the mitochondrial inner membrane during glutamate excitotoxicity.

ABT-737 is a pharmacological inhibitor of the anti-apoptotic activity of B-cell lymphoma-extra large (Bcl-xL) protein; it promotes apoptosis of cancer cells by occupying the BH3-binding pocket. We have shown previously that ABT-737 lowers cell metabolic efficiency by inhibiting ATP synthase activity. However, we also found that ABT-737 protects rodent brain from ischemic injury in vivo by inhibiting formation of the pro-apoptotic, cleaved form of Bcl-xL, ΔN-Bcl-xL.

Direct interaction of the resistance to inhibitors of cholinesterase type 3 protein with the serotonin receptor type 3A intracellular domain.

Pentameric ligand-gated ion channels (pLGIC) are expressed in both excitable and non-excitable cells that are targeted by numerous clinically used drugs. Assembly from five identical or homologous subunits yields homo- or heteromeric pentamers, respectively. The protein known as Resistance to Inhibitors of Cholinesterase (RIC-3) was identified to interfere with assembly and functional maturation of pLGICs.

Physiological roles of the mitochondrial permeability transition pore.

Neurons experience high metabolic demand during such processes as synaptic vesicle recycling, membrane potential maintenance and Ca 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 FF ATP synthase, a multi-protein enzyme that contains a membrane-inserted portion, an extra-membranous enzymatic portion and an extensive regulatory complex.

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

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.

Functional Chimeras of GLIC Obtained by Adding the Intracellular Domain of Anion- and Cation-Conducting Cys-Loop Receptors.

Pentameric ligand-gated ion channels (pLGICs), also called Cys-loop receptors in eukaryotic superfamily members, play diverse roles in neurotransmission and serve as primary targets for many therapeutic drugs. Structural studies of full-length eukaryotic pLGICs have been challenging because of glycosylation, large size, pentameric assembly, and hydrophobicity. X-ray structures of prokaryotic pLGICs, including the Gloeobacter violaceus LGIC (GLIC) and the Erwinia chrysanthemi LGIC (ELIC), and truncated eukaryotic pLGICs have significantly improved and complemented the understanding of structural details previously obtained with acetylcholine-binding protein and Torpedo nicotinic acetylcholine receptors.

Experimental determination of the vertical alignment between the second and third transmembrane segments of muscle nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChR) are members of the Cys-loop ligand-gated ion channel superfamily. Muscle nAChR are heteropentamers that assemble from two α, and one each of β, γ, and δ subunits. Each subunit is composed of three domains, extracellular, transmembrane and intracellular.

The beta subunit loop that couples catalysis and rotation in ATP synthase has a critical length.

ATP synthase uses a unique rotational mechanism to convert chemical energy into mechanical energy and back into chemical energy. The helix-turn-helix structure in the C-terminal domain of the β subunit containing the conserved DELSEED motif, termed "DELSEED-loop," was suggested to be involved in coupling between catalysis and rotation. If this is indeed the role of the loop, it must have a critical length, the minimum length required to sustain its function.

ATP synthase with its gamma subunit reduced to the N-terminal helix can still catalyze ATP synthesis.

ATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. As part of the synthesis mechanism, the torque of the rotor has to be converted into conformational rearrangements of the catalytic binding sites on the stator to allow synthesis and release of ATP. The gamma subunit of the rotor, which plays a central role in the energy conversion, consists of two long helices inside the central cavity of the stator cylinder plus a globular portion outside the cylinder.

The role of the betaDELSEED-loop of ATP synthase.

ATP synthase uses a unique rotational mechanism to convert chemical energy into mechanical energy and back into chemical energy. The helix-turn-helix motif, termed "DELSEED-loop," in the C-terminal domain of the beta subunit was suggested to be involved in coupling between catalysis and rotation. Here, the role of the DELSEED-loop was investigated by functional analysis of mutants of Bacillus PS3 ATP synthase that had 3-7 amino acids within the loop deleted.