Discrimination between two steps in the mitochondrial permeability transition process.
It is well known that a lag phase generally elapses between the addition of inducers of the mitochondrial permeability transition and the opening of the pore. To advance our present understanding as regards the significance of this phenomenon, we used experimental approaches which are sensitive to different aspects of the permeability transition process. The pore conformation was sensed by the fluorescence anisotropy changes of hematoporphyrin-labelled mitochondria. Membrane permeabilization was ascertained by following the matrix swelling consequent to external solute equilibration. We show that the anisotropy changes of mitochondria-bound hematoporphyrin precede both membrane depolarization (proton permeation) and matrix swelling (solute permeation), thus sensing a step of the permeability transition process that involves the pore in its closed state. We suggest that the opening of the pore is preceded by a structural remodelling of mitochondrial domains containing hematoporphyrin-near, pore-regulating histidines. Such a perturbation is strongly inhibited at acidic matrix pH and completely blocked by cyclosporin A. In sucrose-based media the opening of the pore can be strongly delayed, as compared to salt-based media, a fact which probably reflects perturbation of mitochondrial membranes by sugar. We conclude that the mitochondrial permeability transition could be described as an at least two-step process which is mainly regulated by conformational changes of the pore components.
Ricchelli
Fernanda
F
C.N.R. Institute of Biomedical Technologies/Padova Unit, Italy. rchielli@mail.bio.unipd.it
Jori
Giulio
G
Gobbo
Silvano
S
Nikolov
Peter
P
Petronilli
Valeria
V
D000818:Animals; D002118:Calcium / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology; D005454:Fluorescence Polarization; D006415:Hematoporphyrins / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007425:Intracellular Membranes / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials; D008930:Mitochondria, Liver / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D008933:Mitochondrial Swelling; D010539:Permeability; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar; D013395:Sucrose / Q000494:pharmacology
D006415:Hematoporphyrins; D007166:Immunosuppressive Agents; D006639:Histidine; D013395:Sucrose; D016572:Cyclosporine; D002118:Calcium