Affiliations: Centro per lo Studio delle Biomembrane, CNR, Università di Padova, Via G. Colombo 3, 35121 Padova, Italy | Dipartimento di Chimica Biologica, Università di Padova, Via G. Colombo 3, 35121 Padova, Italy | Dipartimento di Scienze Biomediche, Università di Padova, Via G. Colombo 3, 35121 Padova, Italy
Note: [] Correspondence to: Prof. Fabio Di Lisa, Dipartimento di Chimica Biologica, Via G. Colombo 3, 35121 Padova, Italy. Fax: +39 049 8073310; E‐mail: [email protected].
Abstract: The involvement of mitochondrial permeability transition pore (MTP) in cellular processes is generally investigated by indirect means, such as changes in mitochondrial membrane potential or pharmacological inhibition. However, such effects could not be related univocally to MTP. In addition, source of errors could be represented by the increased retention of membrane potential probes induced by cyclosporin A (CsA) and the interactions between fluorescent probes. We developed a direct technique for monitoring MTP. Cells were co‐loaded with calcein‐AM and CoCl_2, resulting in the quenching of the cytosolic signal without affecting the mitochondrial fluorescence. MTP inducers caused a rapid decrease in mitochondrial calcein fluorescence which, however, was not completely prevented by CsA. Besides the large and rapid efflux of calcein induced by MTP agonists, we also observed a constant and spontaneous decrease of mitochondrial calcein which was completely prevented by CsA. Thus, MTP likely fluctuates between open and closed states in intact cells.
