Involvement of cyclophilin D in mitochondrial permeability transition induction in intact cells

The mitochondrial permeability transition (MPT) is involved in both Ca2+ signaling and cell death. The present study aimed to clarify the involvement of cyclophilin D, a peptidyl prolyl cis–trans isomerase (PPIase), in MPT induction in intact cells. To achieve this, we used C6 cells overexpressing wild-type or PPIase-deficient cyclophilin D, and measured the inner mitochondrial membrane permeability to calcein, a 623-Da hydrophilic fluorescent molecule, to evaluate MPT induction. In vector control cells, the percentage of MPT induction by ionomycin increased as the Ca2+ concentration in the extracellular medium increased. This result indicates that the present method is valid for numerical evaluation of MPT induction. In C6 cells expressing the PPIase-deficient mutant, the percentage of MPT induction was significantly decreased compared with wild-type CypD-overexpressing cells or vector control cells. These results suggest that cyclophilin D is involved in MPT induction by Ca2+ in intact cells.