Mitochondrial F-ATP Synthase Co-Migrating Proteins and Ca2+-Dependent Formation of Large Channels.

Monomers, dimers, and individual FOF1-ATP synthase subunits are, presumably, involved in the formation of the mitochondrial permeability transition pore (PTP), whose molecular structure, however, is still unknown. We hypothesized that, during the Ca2+-dependent assembly of a PTP complex, the F-ATP synthase (subunits) recruits mitochondrial proteins that do not interact or weakly interact with the F-ATP synthase under normal conditions. Therefore, we examined whether the PTP opening in mitochondria before the separation of supercomplexes via BN-PAGE will increase the channel stability and channel-forming capacity of isolated F-ATP synthase dimers and monomers in planar lipid membranes. Additionally, we studied the specific activity and the protein composition of F-ATP synthase dimers and monomers from rat liver and heart mitochondria before and after PTP opening. Against our expectations, preliminary PTP opening dramatically suppressed the high-conductance channel activity of F-ATP synthase dimers and monomers and decreased their specific "in-gel" activity. The decline in the channel-forming activity correlated with the reduced levels of as few as two proteins in the bands: methylmalonate-semialdehyde dehydrogenase and prohibitin 2. These results indicate that proteins co-migrating with the F-ATP synthase may be important players in PTP formation and stabilization.

Regulation of Mitochondrial Permeability Transition Pore Opening by Monovalent Cations in Liver Mitochondria.

The opening of the permeability transition pore (PTP) in mitochondria is a key event in the initiation of cell death in various pathologic states, including ischemia/reperfusion. The activation of K+ transport into mitochondria protects cells from ischemia/reperfusion. However, the role of K+ transport in PTP regulation is unclear. Here, we studied the role of K+ and other monovalent cations in the regulation of the PTP opening in an in vitro model. The registration of the PTP opening, membrane potential, Ca2+-retention capacity, matrix pH, and K+ transport was performed using standard spectral and electrode techniques. We found that the presence of all cations tested in the medium (K+, Na+, choline+, and Li+) strongly stimulated the PTP opening compared with sucrose. Several possible reasons for this were examined: the effect of ionic strength, the influx of cations through selective and non-selective channels and exchangers, the suppression of Ca2+/H+ exchange, and the influx of anions. The data obtained indicate that the mechanism of PTP stimulation by cations includes the suppression of K+/H+ exchange and acidification of the matrix, which facilitates the influx of phosphate. Thus, the K+/H+ exchanger and the phosphate carrier together with selective K+ channels compose a PTP regulatory triad, which might operate in vivo.

Pioglitazone Is a Mild Carrier-Dependent Uncoupler of Oxidative Phosphorylation and a Modulator of Mitochondrial Permeability Transition.

Pioglitazone (PIO) is an insulin-sensitizing antidiabetic drug, which normalizes glucose and lipid metabolism but may provoke heart and liver failure and chronic kidney diseases. Both therapeutic and adverse effects of PIO can be accomplished through mitochondrial targets. Here, we explored the capability of PIO to modulate the mitochondrial membrane potential (ΔΨm) and the permeability transition pore (mPTP) opening in different models in vitro. ΔΨm was measured using tetraphenylphosphonium and the fluorescent dye rhodamine 123. The coupling of oxidative phosphorylation was estimated polarographically. The transport of ions and solutes across membranes was registered by potentiometric and spectral techniques. We found that PIO decreased ΔΨm in isolated mitochondria and intact thymocytes and the efficiency of ADP phosphorylation, particularly after the addition of Ca2+. The presence of the cytosolic fraction mitigated mitochondrial depolarization but made it sustained. Carboxyatractyloside diminished the PIO-dependent depolarization. PIO activated proton transport in deenergized mitochondria but not in artificial phospholipid vesicles. PIO had no effect on K+ and Ca2+ inward transport but drastically decreased the mitochondrial Ca2+-retention capacity and protective effects of adenine nucleotides against mPTP opening. Thus, PIO is a mild, partly ATP/ADP-translocase-dependent, uncoupler and a modulator of ATP production and mPTP sensitivity to Ca2+ and adenine nucleotides. These properties contribute to both therapeutic and adverse effects of PIO.

Dynamics of the permeability transition pore size in isolated mitochondria and mitoplasts.

The size of the permeability transition pore (PTP) is accepted to be ≤1.5 kDa. However, different authors reported values from 650 to 4000 Da. The present study is focused on the variability of the average PTP size in and between mitochondrial samples, its reasons and relations with PTP dynamics. Measurement of PTP size by the standard method revealed its 500 Da-range variability between mitochondrial samples. Sequential measurements in the same sample showed that the PTP size tends to grow with time and Ca2+ concentration. Selective damage to the mitochondrial outer membrane (MOM) reduced the apparent PTP size by ~200-300 Da. Hypotonic and hypertonic osmotic shock and partial removal of the MOM with the preservation of the mitochondrial inner membrane intactness decreased the apparent PTP size by ~50%. We developed an approach to continuous monitoring of the PTP size that revealed the existence of stable PTP states with different pore sizes (~700, 900-1000, ~1350, 1700-1800, and 2100-2200 Da) and transitions between them. The transitions were accelerated by elevating the Ca2+ concentration, temperature, and osmotic pressure, which demonstrates an increased capability of PTP to accommodate to large molecules (plasticity). Cyclosporin A inhibited the transitions between states. The analysis of PTP size dynamics in osmotically shocked mitochondria and mitoplasts confirmed the importance of the MOM for the stabilization of PTP structure. Thus, this approach provides a new tool for PTP studies and the opportunity to reconcile data on the PTP size and mitochondrial megachannel conductance.

Regulation of permeability transition pore opening in mitochondria by external NAD(H).

BACKGROUND: The opening of the permeability transition pore (PTP) in mitochondria plays a critical role in the pathogenesis of numerous diseases. Mitochondrial matrix pyridine nucleotides are potent regulators of the PTP, but the role of extramitochondrial nucleotides is unclear. METHODS: The PTP opening was explored in isolated mitochondria and mitochondria in permeabilized differentiated and undifferentiated cells in the presence of added NAD(P)(H) in combination with Mg2+, adenine nucleotides (AN), and the inhibitors of AN translocase (ANT), voltage-dependent anion channel (VDAC), and cyclophilin D. RESULTS: Added NAD(H) and AN, but not NADP(H), inhibited the PTP opening with comparable potency. PTP suppression required neither NAD(H) oxidation nor reduction. The protective effects of NAD(H) and cyclosporin A were synergistic, and the effects of NAD(H) and millimolar AN were additive. The conformation-specific ANT inhibitors were unable to cancel the protective effect of NADH even under total ANT inhibition. Besides, NAD(H) activated the efflux of mitochondrial AN via ANT. VDAC ligand (Mg2+) and blockers (G3139 and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid) potentiated and attenuated the protective effect of NAD(H), respectively. However, in embryonic and cancer (undifferentiated) cells, in contrast to isolated differentiated hepatocytes and cardiocytes, the suppression of PTP opening by NADH was negligible though all cells tested possessed a full set of VDAC isoforms. CONCLUSIONS: The study revealed a novel mechanism of PTP regulation by external (cytosolic) NAD(H) through the allosteric site in the OM or the intermembrane space. GENERAL SIGNIFICANCE: The mechanism might contribute to the resistance of differentiated cells under different pathological conditions including ischemia/reperfusion.

Pyridine nucleotides regulate the superoxide anion flash upon permeabilization of mitochondrial membranes: An MCLA-based study.

The permeabilization of mitochondrial membranes via permeability transition pore opening or by the pore-forming peptide alamethicin causes a flash of superoxide anion (SA) and hydrogen peroxide production and the inhibition of matrix aconitase. It was shown using the SA probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA) that the substrates of NAD-dependent dehydrogenases, inhibitors of the respiratory chain, and NAD(P)H at millimolar concentrations suppressed or delayed SA flashes. In the presence of added NADH and NADPH, SA flashes were observed only after considerable oxidation of pyridine nucleotides. The production of SA was maximal at NADPH and NADH redox potentials from -315 to -295 mV and from -325 to -270 mV, respectively, depending on NAD(P)H concentration. SA generation supported by NADPH was severalfold greater than that supported by NADH. In intact mitochondria, NADPH- and NADH-dependent SA generation was negligible. Respiratory substrates at physiological or lower concentrations were incapable of suppressing the NADPH-supported SA flash. These data indicate that, in conditions close to pathophysiological, matrix NADPH oxidoreductase(s), presumably, an adrenodoxin reductase in complex with adrenodoxin, can essentially contribute to SA flashes associated with transient or irreversible permeability transition pore opening or membrane permeabilization by another mechanism.