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.

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 β-cells. In this study, we provide evidence that leptin modulates pancreatic β-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 β-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/calmodulindependent protein kinase kinase β. 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 β-cells in a physiological range of fasting glucose levels. There was a close correlation between phospho-AMPK levels and β-cell membrane potentials, suggesting that AMPK-dependent K ATP channel trafficking is a key mechanism for regulating β-cell membrane potentials. Our results present a signaling pathway whereby leptin regulates glucose homeostasis by modulating β-cell excitability.T he K ATP channel, an inwardly rectifying K + channel that consists of pore-forming Kir6.2 and regulatory sulfonylurea receptor 1 (SUR1) subunits (1), functions as an energy sensor: its gating is regulated mainly by the intracellular concentrations of ATP and ADP. In pancreatic β-cells, K ATP channels are inhibited or activated in response to the rise or fall in blood glucose levels, leading to changes in membrane excitability and insulin secretion (2, 3). Thus, K ATP channel gating has been considered an important mechanism in coupling blood glucose levels to insulin secretion. Recently, trafficking of K ATP channels to the plasma membrane was highlighted as another important mechanism for regulating K ATP channel activity (4-6).AMP-activated protein kinase (AMPK) is a key enzyme regulating energy homeostasis (7). We recently demonstrated that K ATP channels are recruited to the plasma membrane in glucosedeprived conditions via AMPK signaling in pancreatic β-cells (6). Inhibition of AMPK signaling significantly reduces K ATP currents, even after complete wash-out of intracellular ATP (6). Given these results, we proposed a model that recruitment of K ATP channels to the plasma membrane via AMPK signaling is crucial for K ATP channel activation in low-glucose conditions. However, the physiological relevance of this model remains unclear because pancreatic β-cells had to be incubated in media containing less than 3 mM glucose to recruit a sufficient number of K ATP channels to the plasma membrane (6). We thus hypothesized that there should be an e...

Stretch‐activated channels (SACs) were studied in isolated rat atrial myocytes using the whole‐cell and single‐channel patch clamp techniques. Longitudinal stretch was applied by using two patch electrodes. In current clamp configuration, mechanical stretch of 20 % of resting cell length depolarised the resting membrane potential (RMP) from ‐63·6 ± 0·58 mV (n= 19) to ‐54·6 ± 2·4 mV (n= 13) and prolonged the action potential duration (APD) by 32·2 ± 8·8 ms (n= 7). Depolarisation, if strong enough, triggered spontaneous APs. In the voltage clamp configuration, stretch increased membrane conductance in a progressive manner. The current‐voltage (I–V) relationship of the stretch‐activated current (ISAC) was linear and reversed at ‐6·1 ± 3·7 mV (n= 7). The inward component of ISAC was abolished by the replacement of Na+ with NMDG+, but ISAC was hardly altered by the Cl− channel blocker DIDS or removal of external Cl−. The permeability ratio for various cations (PCs:PNa:PLi= 1·05:1:0·98) indicated that the SAC current was a non‐selective cation current (ISAC,NC). The background current was also found to be non‐selective to cations (INSC,b); the permeability ratio (PCs:PNa:PLi= 1·49:1:0·70) was different from that of ISAC,NC. Gadolinium (Gd3+) acted on INSC,b and ISAC,NC differently. Gd3+ inhibited INSC,b in a concentration‐dependent manner with an IC50 value of 46·2 ± 0·8 μM (n= 5). Consistent with this effect, Gd3+ hyperpolarised the resting membrane potential (‐71·1 ± 0·26 mV, n= 9). In the presence of Gd3+ (0·1 mM), stretch still induced ISAC,NC and diastolic depolarisation. Single‐channel activities were recorded in isotonic Na+ and Cs+ solutions using the inside‐out configuration. In NMDG+ solution, outward currents were abolished. Gd3+ (100 μM) strongly inhibited channel opening both from the inside and outside. In the presence of Gd3+ (100 μM) in the pipette solution, an increase in pipette pressure induced an increase in channel opening (21·27 ± 0·24 pS; n= 7), which was distinct from background activity. We concluded from the above results that longitudinal stretch in rat atrial myocytes induces the activation of non‐selective cation channels that can be distinguished from background channels by their different electrophysiology and pharmacology.

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 Ca 2ϩ 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 Ca 2ϩ handling in dentate granule cells was impaired as early as the second postnatal month, and this Ca 2ϩ dysregulation caused an impairment of post-tetanic potentiation in mossy fiber-CA3 synapses. The alteration of cellular Ca 2ϩ clearance in Tg2576 mice is region-specific within hippocampus because in another region, CA1 pyramidal neuron, no significant difference in Ca 2ϩ clearance was detected between wild-type and Tg2576 mice at this early stage. Impairment of mitochondrial Ca 2ϩ 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.

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 ϳ100, ϳ225, ϳ700, and ϳ1,000 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 Ca2ϩ transport plays critical roles in the regulation of mitochondrial function under both physiological and pathological conditions (1-3). An increase of matrix Ca 2ϩ within the physiological range facilitates ATP production (4) by stimulating the activity of Ca 2ϩ -dependent dehydrogenases (5, 6) in the tricarboxylic acid cycle and the ATP synthase (7). Dysregulation of mitochondrial Ca 2ϩ handling and subsequent alterations in bioenergetics contribute to contractile dysfunction in heart failure (8, 9). Moreover, mitochondrial Ca 2ϩ overload during ischemia/reperfusion induces opening of the mitochondrial permeability transition pore (mPTP), 2 mitochondrial swelling, and an increase of reactive oxygen species generation, all of which lead to cardiac cell death (10, 11). Several Ca 2ϩ transport pathways have been identified. The mitochondrial Ca 2ϩ uniporter (MCU) is a large capacity, low affinity system that provides a slow Ca 2ϩ uptake mechanism (12). A highly Ca 2ϩ selective, inwardly rectifying conductance with biophysical properties similar to those expected of the MCU was characterized in elegant patch clamp studies of mitoplasts isolated from Cos-7 cells (13) and human cardiomyocytes (14). Recently, MICU1, a gene encoding an EF hand protein that regulates MCU, was identified (15). However, heart mitochondria are also known to exhibit rapid Ca 2ϩ uptake and efflux pathways that possess Ca 2ϩ sensitivity and pharmacology different from those of the MCU (3).We previously reported that a ryanodine-sensitive, fast Ca 2ϩ uptake pathway co-exists with MCU in heart mitochondria (16, 17). High affinity binding of [ 3 H]ryanodine, immunogold staining, and Western blot analysis revealed th...

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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.

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.