MAC inhibitors antagonize the pro-apoptotic effects of tBid and disassemble Bax / Bak oligomers.

Mitochondrial Apoptotic Channel inhibitors or iMACs are di-bromocarbazole derivatives with anti-apoptotic function which have been tested and validated in several mouse models of brain injury and neurodegeneration. Owing to the increased therapeutic potential of these compounds, we sought to expand our knowledge of their mechanism of action. We investigated the kinetics of MAC inhibition in mitochondria from wild type, Bak, and Bax knockout cell lines using patch clamp electrophysiology, fluorescence microscopy, ELISA, and semiquantitative western blot analyses. Our results show that iMACs work through at least two mechanisms: 1) by blocking relocation of the cytoplasmic Bax protein to mitochondria and 2) by disassembling Bax and Bak oligomers in the mitochondrial outer membrane. iMACs exert comparable effects on channel conductance of Bax or Bak and similarly affect cytochrome c release from Bax or Bak-containing mitochondria. Interestingly, wild type mitochondria were more susceptible to inhibition than the Bak or Bax knockouts. Western blot analysis showed that wild type mitochondria had lower steady state levels of Bak in the absence of apoptotic stimulation.

Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice.

A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane resists swelling and prevents organelle rupture to prevent cell death. DOI:http://dx.doi.org/10.7554/eLife.00772.001.

The therapeutic potential of mitochondrial channels in cancer, ischemia-reperfusion injury, and neurodegeneration.

Mitochondria communicate with the rest of the cell through channels located in their inner and outer membranes. Most of the time, the message is encoded by the flow of anions and cations e.g., through VDAC and PTP, respectively. However, proteins are also both imported and exported across the mitochondrial membranes e.g., through TOM and MAC, respectively. Transport through mitochondrial channels is exquisitely regulated and controls a myriad of processes; from energy production to cell death. Here, we examine the role of some of the mitochondrial channels involved in neurodegeneration, ischemia-reperfusion injury and cancer in the context of their potential as therapeutic targets.

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

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.

Mitochondrial apoptosis-induced channel (MAC) function triggers a Bax/Bak-dependent bystander effect.

Collateral spread of apoptosis to nearby cells is referred to as the bystander effect, a process that is integral to tissue homeostasis and a challenge to anticancer therapies. In many systems, apoptosis relies on permeabilization of the mitochondrial outer membrane to factors such as cytochrome c and Smac/DIABLO. This permeabilization occurs via formation of a mitochondrial apoptosis-induced channel (MAC) and was mimicked here by single-cell microinjection of cytochrome c into Xenopus laevis embryos. Waves of apoptosis were observed in vivo from the injected to the neighboring cells. This finding indicates that a death signal generated downstream of cytochrome c release diffused to neighboring cells and ultimately killed the animals. The role of MAC in bystander effects was then assessed in mouse embryonic fibroblasts that did or did not express its main components, Bax and/or Bak. Exogenous expression of green fluorescent protein-Bax triggered permeabilization of the outer membrane and apoptosis in these cells. Time-lapse videos showed that neighboring cells also underwent apoptosis, but expression of Bax and/or Bak was essential to this effect, because no bystanders were observed in cells lacking both of these MAC components. These results may guide development of novel therapeutic strategies to selectively eliminate tumors or minimize the size of tissue injury in degenerative or traumatic cell death.

Is mPTP the gatekeeper for necrosis, apoptosis, or both?

Permeabilization of the mitochondrial membranes is a crucial step in apoptosis and necrosis. This phenomenon allows the release of mitochondrial death factors, which trigger or facilitate different signaling cascades ultimately causing the execution of the cell. The mitochondrial permeability transition pore (mPTP) has long been known as one of the main regulators of mitochondria during cell death. mPTP opening can lead to matrix swelling, subsequent rupture of the outer membrane, and a nonspecific release of intermembrane space proteins into the cytosol. While mPTP was purportedly associated with early apoptosis, recent observations suggest that mitochondrial permeabilization mediated by mPTP is generally more closely linked to events of late apoptosis and necrosis. Mechanisms of mitochondrial membrane permeabilization during cell death, involving three different mitochondrial channels, have been postulated. These include the mPTP in the inner membrane, and the mitochondrial apoptosis-induced channel (MAC) and voltage-dependent anion-selective channel (VDAC) in the outer membrane. New developments on mPTP structure and function, and the involvement of mPTP, MAC, and VDAC in permeabilization of mitochondrial membranes during cell death are explored. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.

Role of mitochondrial ion channels in cell death.

Ion channels located in the outer and inner mitochondrial membranes are key regulators of cellular signaling for life and death. Permeabilization of mitochondrial membranes is one of the most critical steps in the progression of several cell death pathways. The mitochondrial apoptosis-induced channel (MAC) and the mitochondrial permeability transition pore (mPTP) play major roles in these processes. Here, the most recent progress and current perspectives about the roles of MAC and mPTP in mitochondrial membrane permeabilization during cell death are presented. The crosstalk signaling of MAC and mPTP formation/activation mediated by cytosolic Ca(2+) signaling, Bcl-2 family proteins, and other mitochondrial ion channels is also discussed. Understanding the mechanisms that regulate opening and closing of MAC and mPTP has revealed new therapeutic targets that potentially could control cell death in pathologies such as cancer, ischemia/reperfusion injuries, and neurodegenerative diseases.

Mitochondrial ion channels as therapeutic targets.

The study of mitochondrial ion channels changed our perception of these double-wrapped organelles from being just the power house of a cell to the guardian of a cell's fate. Mitochondria communicate with the cell through these special channels. Most of the time, the message is encoded by ion flow across the mitochondrial outer and inner membranes. Potassium, sodium, calcium, protons, nucleotides, and proteins traverse the mitochondrial membranes in an exquisitely regulated manner to control a myriad of processes, from respiration and mitochondrial morphology to cell proliferation and cell death. This review is an update on both well established and putative mitochondrial channels regarding their composition, function, regulation, and therapeutic potential.

Mitochondrial ryanodine receptors and other mitochondrial Ca2+ permeable channels.

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.

MAC and Bcl-2 family proteins conspire in a deadly plot.

Apoptosis is an elemental form of programmed cell death; it is fundamental to higher eukaryotes and essential to mechanisms controlling tissue homeostasis. Apoptosis is also involved in many pathologies including cancer, neurodegenerative diseases, aging, and infarcts. This cell death program is tightly regulated by Bcl-2 family proteins by controlling the formation of the mitochondrial apoptosis-induced channel or MAC. Assembly of MAC corresponds to permeabilization of the mitochondrial outer membrane, which is the so called commitment step of apoptosis. MAC provides the pathway through the mitochondrial outer membrane for the release of cytochrome c and other pro-apoptotic factors from the intermembrane space. While overexpression of anti-apoptotic Bcl-2 eliminates MAC activity, oligomers of the pro-apoptotic members Bax and/or Bak are essential structural component(s) of MAC. Assembly of MAC from Bax or Bak was monitored in real time by directly patch-clamping mitochondria with micropipettes containing the sentinel tBid, a direct activator of Bax and Bak. Herein, a variety of high affinity inhibitors of MAC (iMAC) that may prove to be crucial tools in mechanistic studies have recently been identified. This review focuses on characterization of MAC activity, its regulation by Bcl-2 family proteins, and a discussion of how MAC can be pharmacologically turned on or off depending on the pathology to be treated.

Extracellular ATP and P2Y2 receptors mediate intercellular Ca(2+) waves induced by mechanical stimulation in submandibular gland cells: Role of mitochondrial regulation of store operated Ca(2+) entry.

Coordination of Ca(2+) signaling among cells contributes to synchronization of salivary gland cell function. However, mechanisms that underlie this signaling remain elusive. Here, intercellular Ca(2+) waves (ICW) in submandibular gland cells were investigated using Fura-2 fluorescence imaging. Mechanical stimulation of single cells induced ICW propagation from the stimulated cells through approximately 7 layers of cells or approximately 120microm. Our findings indicate that an extracellular ATP-dependent pathway is involved because the purinergic receptor antagonist suramin and the ATP hydrolyzing enzyme apyrase blocked ICW propagation. However, the gap junction uncoupler oleamide had no effect. ATP is released from mechanically stimulated cells possibly through opening of mechanosensitive maxi-anion channels, and does not appear to be directly linked to cytosolic Ca(2+). The ICW is propagated by diffusing ATP, which activates purinergic receptors in neighboring cells. This purinergic signaling induces a Ca(2+) transient that is dependent on Ca(2+) release via IP(3) receptors in the ER and store operated Ca(2+) entry (SOCE). Finally, inhibition of mitochondrial Ca(2+) uptake modified ICW indicating an important role of these organelles in this phenomenon. These studies increase our understanding of purinergic receptor signaling in salivary gland cells, and its role as a coordination mechanism of Ca(2+) signals induced by mechanical stimulation.

Mitochondrial apoptosis is amplified through gap junctions.

The death of one cell can precipitate the death of nearby cells in a process referred to as the bystander effect. We investigated whether mitochondrial apoptosis generated a bystander effect and, if so, by which pathway. Microinjection with cytochrome c mimicked function of the mitochondrial apoptosis-induced channel MAC and caused apoptosis of both target and nearby osteoblasts. This effect was suppressed by inhibiting gap junction intercellular communication. A bystander effect was also observed after exogenous expression of tBid, which facilitates MAC formation and cytochrome c release. Interestingly, in connexin-43 deficient osteoblasts, microinjection of cytochrome c induced apoptosis only in the target cell. These findings indicate that a death signal was generated downstream of MAC function and was transmitted through gap junctions to amplify apoptosis in neighboring cells. This concept may have implications in development of new therapeutic approaches.

MAC inhibitors suppress mitochondrial apoptosis.

MAC (mitochondrial apoptosis-induced channel) forms in the mitochondrial outer membrane and unleashes cytochrome c to orchestrate the execution of the cell. MAC opening is the commitment step of intrinsic apoptosis. Hence closure of MAC may prevent apoptosis. Compounds that blocked the release of fluorescein from liposomes by recombinant Bax were tested for their ability to directly close MAC and suppress apoptosis in FL5.12 cells. Low doses of these compounds (IC50 values ranged from 19 to 966 nM) irreversibly closed MAC. These compounds also blocked cytochrome c release and halted the onset of apoptotic markers normally induced by IL-3 (interleukin-3) deprivation or staurosporine. Our results reveal the tight link among MAC activity, cytochrome c release and apoptotic death, and indicate this mitochondrial channel is a promising therapeutic target.

Assembly of the mitochondrial apoptosis-induced channel, MAC.

Although Bcl-2 family proteins control intrinsic apoptosis, the mechanisms underlying this regulation are incompletely understood. Patch clamp studies of mitochondria isolated from cells deficient in one or both of the pro-apoptotic proteins Bax and Bak show that at least one of the proteins must be present for formation of the cytochrome c-translocating channel, mitochondrial apoptosis-induced channel (MAC), and that the single channel behaviors of MACs containing exclusively Bax or Bak are similar. Truncated Bid catalyzes MAC formation in isolated mitochondria containing Bax and/or Bak with a time course of minutes and does not require VDAC1 or VDAC3. Mathematical analysis of the stepwise changes in conductance associated with MAC formation is consistent with pore assembly by a barrel-stave model. Assuming the staves are two transmembrane alpha-helices in Bax and Bak, mature MAC pores would typically contain approximately 9 monomers and have diameters of 5.5-6 nm. The mitochondrial permeability data are inconsistent with formation of lipidic pores capable of transporting megadalton-sized macromolecules as observed with recombinant Bax in liposomes.

Reflections on VDAC as a voltage-gated channel and a mitochondrial regulator.

There is excellent agreement between the electrophysiological properties and the structure of the mitochondrial outer membrane protein, VDAC, ex vivo. However, the inference that the well-defined canonical "open" state of the VDAC pore is the normal physiological state of the channel in vivo is being challenged by several lines of evidence. Knowing the atomic structure of the detergent solubilized protein, a long sought after goal, will not be sufficient to understand the functioning of this channel protein. In addition, detailed information about VDAC's topology in the outer membrane of intact mitochondria, and the structural changes that it undergoes in response to different stimuli in the cell will be needed to define its physiological functions and regulation.

A tale of two mitochondrial channels, MAC and PTP, in apoptosis.

The crucial step in the intrinsic, or mitochondrial, apoptotic pathway is permeabilization of the mitochondrial outer membrane. Permeabilization triggers release of apoptogenic factors, such as cytochrome c, from the mitochondrial intermembrane space into the cytosol where these factors ensure propagation of the apoptotic cascade and execution of cell death. However, the mechanism(s) underlying permeabilization of the outer membrane remain controversial. Two mechanisms, involving opening of two different mitochondrial channels, have been proposed to be responsible for the permeabilization; the permeability transition pore (PTP) in the inner membrane and the mitochondrial apoptosis-induced channel (MAC) in the outer membrane. Opening of PTP would lead to matrix swelling, subsequent rupture of the outer membrane, and an unspecific release of intermembrane proteins into the cytosol. However, many believe PTP opening is a consequence of apoptosis and this channel is thought to principally play a role in necrosis, not apoptosis. Activation of MAC is exquisitely regulated by Bcl-2 family proteins, which are the sentinels of apoptosis. MAC provides specific pores in the outer membrane for the passage of intermembrane proteins, in particular cytochrome c, to the cytosol. The electrophysiological characteristics of MAC are very similar to Bax channels and depletion of Bax significantly diminishes MAC activity, suggesting that Bax is an essential constituent of MAC in some systems. The characteristics of various mitochondrial channels and Bax are compared. The involvement of MAC and PTP activities in apoptosis of disease and their pharmacology are discussed.

The nuclear receptor interacting factor-3 transcriptional coregulator mediates rapid apoptosis in breast cancer cells through direct and bystander-mediated events.

We previously reported that amino acids 20 to 50 of nuclear receptor interacting factor-3 mediates rapid apoptosis in breast cancer cell lines but not in cells derived from other tissues. We refer to this short region as death domain-1 (DD1). Small interfering RNA studies indicated that DD1-mediated apoptosis is caspase-2 dependent. In this study, we examined DD1-mediated apoptosis in more detail and generated stable caspase-2 knockdown breast cancer cells. These cells are resistant to DD1-mediated apoptosis. Time-lapse movies suggested that DD1-mediated apoptosis also leads to a "bystander effect." We found that within 5 h of DD1 expression, breast cancer cells release a factor(s) into the medium that leads to apoptosis of naive breast cancer cells or DD1-resistant cells (e.g., HeLa). The DD1-expressing caspase-2 knockdown cells also release a factor(s) that kills other cells, indicating that this effect is not dependent on the apoptogenic process. The bystander effect seems dependent on the production of reactive oxygen species (ROS). These and other studies indicate that DD1 expression in breast cancer cells leads to at least two death signals: one involving the rapid production of ROS and/or other soluble factors that directly or indirectly leads to a bystander effect and a second caspase-2-dependent process that leads to apoptosis in cells in which DD1 is expressed.

A fluorescence assay for peptide translocation into mitochondria.

Translocation of the presequence is an early event in import of preproteins across the mitochondrial inner membrane by the TIM23 complex. Import of signal peptides, whose sequences mimic mitochondrial import presequences, was measured using a novel, qualitative, fluorescence assay in about 1h. This peptide assay was used in conjunction with classical protein import analyses and electrophysiological approaches to examine the mechanisms underlying the functional effects of depleting two TIM23 complex components. Tim23p forms, at least in part, the pore of this complex while Tim44p forms part of the translocation motor. Depletion of Tim23p eliminates TIM23 channel activity, which interferes with both peptide and preprotein translocation. In contrast, depletion of Tim44p disrupts preprotein but not peptide translocation, which has no effect on TIM23 channel activity. Two conclusions were made. First, this fluorescence peptide assay was validated as two different mutants were accurately identified. Hence, this assay could provide a rapid means of screening mutants to identify those that fail an initial step in import, i.e., translocation of the presequence. Second, translocation of signal peptides required normal channel activity and disruption of the presequence translocase-associated motor complex did not modify TIM23 channel activity nor prevent presequence translocation.

Tim17p regulates the twin pore structure and voltage gating of the mitochondrial protein import complex TIM23.

The TIM23 complex mediates import of preproteins into mitochondria, but little is known of the mechanistic properties of this translocase. Here patch clamping reconstituted inner membranes allowed for first time insights into the structure and function of the preprotein translocase. Our findings indicate that the TIM23 channel has "twin pores" (two equal sized pores that cooperatively gate) thereby strikingly resembling TOM, the translocase of the outer membrane. Tim17p and Tim23p are homologues, but their functions differ. Tim23p acts as receptor for preproteins and may largely constitute the preprotein-conducting passageway. Conversely depletion of Tim17p induces a collapse of the twin pores into a single pore, whereas N terminus deletion or C terminus truncation results in variable sized pores that cooperatively gate. Further analysis of Tim17p mutants indicates that the N terminus is vital for both voltage sensing and protein sorting. These results suggest that although Tim23p is the main structural unit of the pore Tim17p is required for twin pore structure and provides the voltage gate for the TIM23 channel.

Regulation of the mitochondrial apoptosis-induced channel, MAC, by BCL-2 family proteins.

Programmed cell death or apoptosis is central to many physiological processes and pathological conditions such as organogenesis, tissue homeostasis, cancer, and neurodegenerative diseases. Bcl-2 family proteins tightly control this cell death program by regulating the permeabilization of the mitochondrial outer membrane and, hence, the release of cytochrome c and other pro-apoptotic factors. Control of the formation of the mitochondrial apoptosis-induced channel, or MAC, is central to the regulation of apoptosis by Bcl-2 family proteins. MAC is detected early in apoptosis by patch clamping the mitochondrial outer membrane. The focus of this review is on the regulation of MAC activity by Bcl-2 family proteins. The role of MAC as the putative cytochrome c release channel during early apoptosis and insights concerning its molecular composition are also discussed.