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.

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.