Impact of 1,2-ethanedithiol treatment on luminescence and charge-transport characteristics in colloidal quantum-dot LEDs.

We report on a substantial increase in luminance and luminous efficiency of green-light emitting devices (LEDs) that use colloidal CdSe@ZnS quantum dots (QDs) as a light-emitting material in response to treatment with 1,2-ethanedithiol (EDT). The maximum luminance increased from 1146 to 8075 cd m-2, and luminous yield from 0.15 to 1.41 cd A-1 as a result of treating an incomplete device with drops of EDT right after spin-coating QDs onto a ZnO-nanoparticle layer. Based on systematic studies on substrate-dependent change in photoluminescence, and current-voltage and luminance-voltage characteristics, we propose that passivation of intra-gap defect states and relative shifts of energy levels relevant to the operation of QD LEDs are two main results of EDT treatment. In particular, we argue that energy-level shift without emission-color change can be attributed to surface-dipole effects.

Simultaneous quantification of tegoprazan and its major metabolite M1 in dog plasma using liquid chromatography-tandem mass spectrometry.

Tegoprazan is a novel potassium-competitive acid blocker (P-CAB) recently approved in Korea as a next-generation therapeutics for gastric acid-related diseases. In the present study, we demonstrate a simple bioanalytical liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of tegoprazan and its major metabolite (M1) in dog plasma. The developed method is based on protein precipitation and LC-MS/MS, validated according to the regulatory guidance for bioanalytical method validation. The calibration curves were linear in the concentration range of 50 ng/mL-50 µg/mL and 5 ng/mL-5 µg/mL for tegoprazan and M1, respectively. The inter- and intra-day precisions were evaluated with a coefficient of variation of <15%, and the mean accuracy ranged 92.6%-105%. The method exhibited good sensitivity and specificity. The stability of bench-top (for 8 h), freeze-thaw (3 cycles), and processed-samples (for 24 h at 4 °C) was acceptable. Tegoprazan was stable in dog plasma for 6 weeks at -70 °C. In conclusion, we successfully established a method for the simultaneous quantification of tegoprazan and M1 in dog plasma, and the method was validated for specificity, sensitivity, linearity, matrix effects, recovery, accuracy, precision, and stability. Finally, we show that the method was successfully applied to a pharmacokinetic study in dogs.

Effects of Tegoprazan, a Novel Potassium-Competitive Acid Blocker, on Rat Models of Gastric Acid-Related Disease.

Tegoprazan, a novel potassium-competitive acid blocker (P-CAB), is a next-generation therapeutics developed for the treatment of acid-related gastrointestinal diseases such as gastroesophageal reflux disease (GERD) and peptic ulcers. In the present study, the in vitro and in vivo pharmacological properties of tegoprazan were compared with those of esomeprazole, a representative proton pump inhibitor. In vitro enzyme assays were performed using ion-leaky vesicles containing gastric H+/K+-ATPases isolated from pigs. The in vivo efficacies of tegoprazan were evaluated in rat models of GERD and peptic ulcer. Tegoprazan inhibited the activity of porcine H+/K+-ATPase with an IC50 value of 0.53 µM in a reversible manner, whereas esomeprazole showed weak and irreversible inhibition with an IC50 value of 42.52 µM. In a GERD model, tegoprazan showed dose-dependent efficacy in inhibiting esophageal injury and gastric acid secretion with an ED50 of 2.0 mg/kg, which was 15-fold more potent than that of esomeprazole. In peptic ulcer models, tegoprazan exhibited superior antiulcer activity compared with esomeprazole. The ED50 of tegoprazan in the naproxen-, ethanol-, and water-immersion restraint stress-induced peptic ulcer models were 0.1, 1.4, and 0.1 mg/kg, respectively. In the acetic acid-induced peptic ulcer model, the curative ratio of tegoprazan at 10 mg/kg was higher than that of esomeprazole at 30 mg/kg (44.2% vs. 32.7%, respectively), after 5 days of repeated oral administration. Thus, tegoprazan is a novel P-CAB that shows potent and reversible inhibition of gastric H+/K+-ATPase and may provide stronger efficacy compared with previous proton pump inhibitors.

Effects of 1,2-ethanedithiol concentration on performance improvement of quantum-dot LEDs.

We report systematic efficiency variations of green-emitting CdSe@ZnS quantum-dot (QD) LEDs (QLEDs) in response to in situ treatments with 1,2-ethanedithiol (EDT) solutions at various concentrations. The main effect of in situ EDT treatment on a QD layer spin-coated onto a ZnO layer was vacuum-level shift due to dipole moments on the surface of the QD layer and at the interface between QD and ZnO layers. Competing contributions of these dipole moments were responsible for changes in energy level configurations and, accordingly, electron and hole barriers that resulted in discrepancies in electron- and hole-current variations. QLED efficiency was best when treated with an EDT solution of 4 mM, attributable to the largest increase in the hole- to electron current ratio. The maximum luminous yield of the 4 mM EDT-treated QLED was 5.43 cd A-1, which is 10 times higher than that of an untreated device. Furthermore, the luminous yield of this treated device remained as high as 2.56 cd A-1 at a luminance of 500 cd m-2.

Ca(2+) clearance by plasmalemmal NCLX, Li(+)-permeable Na(+)/Ca(2+) exchanger, is required for the sustained exocytosis in rat insulinoma INS-1 cells.

Na(+)/Ca(2+) exchangers are key players for Ca(2+) clearance in pancreatic ß-cells, but their molecular determinants and roles in insulin secretion are not fully understood. In the present study, we newly discovered that the Li(+)-permeable Na(+)/Ca(2+) exchangers (NCLX), which were known as mitochondrial Na(+)/Ca(2+) exchangers, contributed to the Na(+)-dependent Ca(2+) movement across the plasma membrane in rat INS-1 insulinoma cells. Na(+)/Ca(2+) exchange activity by NCLX was comparable to that by the Na(+)/Ca(2+) exchanger, NCX. We also confirmed the presence of NCLX proteins on the plasma membrane using immunocytochemistry and cell surface biotinylation experiments. We further investigated the role of NCLX on exocytosis function by measuring the capacitance increase in response to repetitive depolarization. Small interfering (si)RNA-mediated downregulation of NCLX did not affect the initial exocytosis, but significantly suppressed sustained exocytosis and recovery of exocytosis. XIP (NCX inhibitory peptide) or Na(+) replacement for inhibiting Na(+)-dependent Ca(2+) clearance also selectively suppressed sustained exocytosis. Consistent with the idea that sustained exocytosis requires ATP-dependent vesicle recruitment, mitochondrial function, assessed by mitochondrial membrane potential (ΔΨ), was impaired by siNCLX or XIP. However, depolarization-induced exocytosis was hardly affected by changes in intracellular Na(+) concentration, suggesting a negligible contribution of mitochondrial Na(+)/Ca(2+) exchanger. Taken together, our data indicate that Na(+)/Ca(2+) exchanger-mediated Ca(2+) clearance mediated by NCLX and NCX is crucial for optimizing mitochondrial function, which in turn contributes to vesicle recruitment for sustained exocytosis in pancreatic ß-cells.

Universal Efficiency Improvement in Organic Solar Cells Based on a Poly(3-hexylthiophene) Donor and an Indene-C60 Bisadduct Acceptor with Additional Donor Nanowires.

With poly(3-hexylthiophene) (P3HT) nanowire (NW) inclusion in active layers (ALs), organic solar cells (OSCs) based on P3HT donor and indene-C60 bisadduct (ICBA) acceptor showed power conversion efficiency (PCE) improvements for both bulk heterojunction (BHJ)- and bilayer (BL)-structure AL devices. The PCE increase was approximately 14 % for both types of P3HT:ICBA OSCs. However, improvements in short-circuit current density (Jsc ) were about 4.4 and 6.4 % for BHJ- and BL-type AL devices, respectively. A systematic study showed that the addition of P3HT NWs did not result in enhanced internal quantum efficiencies for either type of device. However, the difference in light-harvesting efficiency was important in accounting for Jsc variations. Interestingly, there was no correlation between Jsc and PCE variations, whereas the open-circuit voltage (Voc ) and fill factor (FF) showed correlations with the PCE. The variation in FF is discussed in terms of Voc and equivalent-circuit parameters based on a nonideal diode model.

Acredinones A and B, voltage-dependent potassium channel inhibitors from the sponge-derived fungus Acremonium sp. F9A015.

Two new benzophenones, acredinones A (1) and B (2), were isolated from a marine-sponge-associated Acremonium sp. fungus. Their chemical structures were elucidated on the interpretation of spectroscopic data. The structure of 1 was confirmed by palladium-catalyzed hydrogenation, followed by spectroscopic data analysis. Acredinones A (1) and B (2) inhibited the outward K(+) currents of the insulin secreting cell line INS-1 with IC50 values of 0.59 and 1.0 µM, respectively.

Hydrogen peroxide induces vasorelaxation by enhancing 4-aminopyridine-sensitive Kv currents through S-glutathionylation.

Hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor. Since opposing vasoactive effects have been reported for H2O2 depending on the vascular bed and experimental conditions, this study was performed to assess whether H2O2 acts as a vasodilator in the rat mesenteric artery and, if so, to determine the underlying mechanisms. H2O2 elicited concentration-dependent relaxation in mesenteric arteries precontracted with norepinephrine. The vasodilatory effect of H2O2 was reversed by treatment with dithiothreitol. H2O2-elicited vasodilation was significantly reduced by blocking 4-aminopyridine (4-AP)-sensitive Kv channels, but it was resistant to blockers of big-conductance Ca(2+)-activated K(+) channels and inward rectifier K(+) channels. A patch-clamp study in mesenteric arterial smooth muscle cells (MASMCs) showed that H2O2 increased Kv currents in a concentration-dependent manner. H2O2 speeded up Kv channel activation and shifted steady state activation to hyperpolarizing potentials. Similar channel activation was seen with oxidized glutathione (GSSG). The H2O2-mediated channel activation was prevented by glutathione reductase. Consistent with S-glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester showed incorporation of glutathione (GSH) in the Kv channel proteins in the presence of H2O2. Interestingly, conditions of increased oxidative stress within MASMCs impaired the capacity of H2O2 to stimulate Kv channels. Not only was the H2O2 stimulatory effect much weaker, but the inhibitory effect of H2O2 was unmasked. These data suggest that H2O2 activates 4-AP-sensitive Kv channels, possibly through S-glutathionylation, which elicits smooth muscle relaxation in rat mesenteric arteries. Furthermore, our results support the idea that the basal redox status of MASMCs determines the response of Kv currents to H2O2.

Mitochondrial ion channels/transporters as sensors and regulators of cellular redox signaling.

SIGNIFICANCE: Mitochondrial ion channels/transporters and the electron transport chain (ETC) serve as key sensors and regulators for cellular redox signaling, the production of reactive oxygen species (ROS) and nitrogen species (RNS) in mitochondria, and balancing cell survival and death. Although the functional and pharmacological characteristics of mitochondrial ion transport mechanisms have been extensively studied for several decades, the majority of the molecular identities that are responsible for these channels/transporters have remained a mystery until very recently. RECENT ADVANCES: Recent breakthrough studies uncovered the molecular identities of the diverse array of major mitochondrial ion channels/transporters, including the mitochondrial Ca2+ uniporter pore, mitochondrial permeability transition pore, and mitochondrial ATP-sensitive K+ channel. This new information enables us to form detailed molecular and functional characterizations of mitochondrial ion channels/transporters and their roles in mitochondrial redox signaling. CRITICAL ISSUES: Redox-mediated post-translational modifications of mitochondrial ion channels/transporters and ETC serve as key mechanisms for the spatiotemporal control of mitochondrial ROS/RNS generation. FUTURE DIRECTIONS: Identification of detailed molecular mechanisms for redox-mediated regulation of mitochondrial ion channels will enable us to find novel therapeutic targets for many diseases that are associated with cellular redox signaling and mitochondrial ion channels/transporters.

Leptin promotes K(ATP) channel trafficking by AMPK signaling in pancreatic ß-cells.

Leptin is a pivotal regulator of energy and glucose homeostasis, and defects in leptin signaling result in obesity and diabetes. The ATP-sensitive potassium (K(ATP)) channels couple glucose metabolism to insulin secretion in pancreatic ß-cells. In this study, we provide evidence that leptin modulates pancreatic ß-cell functions by promoting K(ATP) channel translocation to the plasma membrane via AMP-activated protein kinase (AMPK) signaling. K(ATP) channels were localized mostly to intracellular compartments of pancreatic ß-cells in the fed state and translocated to the plasma membrane in the fasted state. This process was defective in leptin-deficient ob/ob mice, but restored by leptin treatment. We discovered that the molecular mechanism of leptin-induced AMPK activation involves canonical transient receptor potential 4 and calcium/calmodulin-dependent protein kinase kinase ß. AMPK activation was dependent on both leptin and glucose concentrations, so at optimal concentrations of leptin, AMPK was activated sufficiently to induce K(ATP) channel trafficking and hyperpolarization of pancreatic ß-cells in a physiological range of fasting glucose levels. There was a close correlation between phospho-AMPK levels and ß-cell membrane potentials, suggesting that AMPK-dependent K(ATP) channel trafficking is a key mechanism for regulating ß-cell membrane potentials. Our results present a signaling pathway whereby leptin regulates glucose homeostasis by modulating ß-cell excitability.

Impaired short-term plasticity in mossy fiber synapses caused by mitochondrial dysfunction of dentate granule cells is the earliest synaptic deficit in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) in the early stages is characterized by memory impairment, which may be attributable to synaptic dysfunction. Oxidative stress, mitochondrial dysfunction, and Ca²âº dysregulation are key factors in the pathogenesis of AD, but the causal relationship between these factors and synaptic dysfunction is not clearly understood. We found that in the hippocampus of an AD mouse model (Tg2576), mitochondrial Ca²âº handling in dentate granule cells was impaired as early as the second postnatal month, and this Ca²âº dysregulation caused an impairment of post-tetanic potentiation in mossy fiber-CA3 synapses. The alteration of cellular Ca²âº clearance in Tg2576 mice is region-specific within hippocampus because in another region, CA1 pyramidal neuron, no significant difference in Ca²âº clearance was detected between wild-type and Tg2576 mice at this early stage. Impairment of mitochondrial Ca²âº uptake was associated with increased mitochondrial reactive oxygen species and depolarization of mitochondrial membrane potential. Mitochondrial dysfunctions in dentate granule cells and impairment of post-tetanic potentiation in mossy fiber-CA3 synapses were fully restored when brain slices obtained from Tg2576 were pretreated with antioxidant, suggesting that mitochondrial oxidative stress initiates other dysfunctions. Reversibility of early dysfunctions by antioxidants at the preclinical stage of AD highlights the importance of early diagnosis and antioxidant therapy to delay or prevent the disease processes.

Sustained CaMKII activity mediates transient oxidative stress-induced long-term facilitation of L-type Ca(2+) current in cardiomyocytes.

Oxidative stress remodels Ca(2+) signaling in cardiomyocytes, which promotes altered heart function in various heart diseases. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) was shown to be activated by oxidation, but whether and how CaMKII links oxidative stress to pathophysiological long-term changes in Ca(2+) signaling remain unknown. Here, we present evidence demonstrating the role of CaMKII in transient oxidative stress-induced long-term facilitation (LTF) of L-type Ca(2+) current (I(Ca,L)) in rat cardiomyocytes. A 5-min exposure of 1mM H(2)O(2) induced an increase in I(Ca,L), and this increase was sustained for ~1h. The CaMKII inhibitor KN-93 fully reversed H(2)O(2)-induced LTF of I(Ca,L), indicating that sustained CaMKII activity underlies this oxidative stress-induced memory. Simultaneous inhibition of oxidation and autophosphorylation of CaMKII prevented the maintenance of LTF, suggesting that both mechanisms contribute to sustained CaMKII activity. We further found that sarcoplasmic reticulum Ca(2+) release and mitochondrial ROS generation have critical roles in sustaining CaMKII activity via autophosphorylation- and oxidation-dependent mechanisms. Finally, we show that long-term remodeling of the cardiac action potential is induced by H(2)O(2) via CaMKII. In conclusion, CaMKII and mitochondria confer oxidative stress-induced pathological cellular memory that leads to cardiac arrhythmia.

Distinctive characteristics and functions of multiple mitochondrial Ca2+ influx mechanisms.

Intracellular Ca(2+) is vital for cell physiology. Disruption of Ca(2+) homeostasis contributes to human diseases such as heart failure, neuron-degeneration, and diabetes. To ensure an effective intracellular Ca(2+) dynamics, various Ca(2+) transport proteins localized in different cellular regions have to work in coordination. The central role of mitochondrial Ca(2+) transport mechanisms in responding to physiological Ca(2+) pulses in cytosol is to take up Ca(2+) for regulating energy production and shaping the amplitude and duration of Ca(2+) transients in various micro-domains. Since the discovery that isolated mitochondria can take up large quantities of Ca(2+) approximately 5 decades ago, extensive studies have been focused on the functional characterization and implication of ion channels that dictate Ca(2+) transport across the inner mitochondrial membrane. The mitochondrial Ca(2+) uptake sensitive to non-specific inhibitors ruthenium red and Ru360 has long been considered as the activity of mitochondrial Ca(2+) uniporter (MCU). The general consensus is that MCU is dominantly or exclusively responsible for the mitochondrial Ca(2+) influx. Since multiple Ca(2+) influx mechanisms (e.g. L-, T-, and N-type Ca(2+) channel) have their unique functions in the plasma membrane, it is plausible that mitochondrial inner membrane has more than just MCU to decode complex intracellular Ca(2+) signaling in various cell types. During the last decade, four molecular identities related to mitochondrial Ca(2+) influx mechanisms have been identified. These are mitochondrial ryanodine receptor, mitochondrial uncoupling proteins, LETM1 (Ca(2+)/H(+) exchanger), and MCU and its Ca(2+) sensing regulatory subunit MICU1. Here, we briefly review recent progress in these and other reported mitochondrial Ca(2+) influx pathways and their differences in kinetics, Ca(2+) dependence, and pharmacological characteristics. Their potential physiological and pathological implications are also discussed.

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.