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 Ca 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 Ca 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.