Cysteine 203 of Cyclophilin D Is Critical for Cyclophilin D Activation of the Mitochondrial Permeability Transition Pore*

Background: Cyclophilin D, a known mitochondrial permeability transition pore (mPTP) regulator, is associated with cellular protection. Results: Mutation of cysteine 203 of cyclophilin D inhibits mPTP opening and improves cell viability. Conclusion: Cysteine 203 of cyclophilin D is a critical residue for mPTP activation. Significance: This work provides novel mechanistic insights into mPTP regulation. The mitochondrial permeability transition pore (mPTP) opening plays a critical role in mediating cell death during ischemia/reperfusion (I/R) injury. Our previous studies have shown that cysteine 203 of cyclophilin D (CypD), a critical mPTP mediator, undergoes protein S-nitrosylation (SNO). To investigate the role of cysteine 203 in mPTP activation, we mutated cysteine 203 of CypD to a serine residue (C203S) and determined its effect on mPTP opening. Treatment of WT mouse embryonic fibroblasts (MEFs) with H2O2 resulted in an 50% loss of the mitochondrial calcein fluorescence, suggesting substantial activation of the mPTP. Consistent with the reported role of CypD in mPTP activation, CypD null (CypD−/−) MEFs exhibited significantly less mPTP opening. Addition of a nitric oxide donor, GSNO, to WT but not CypD−/− MEFs prior to H2O2 attenuated mPTP opening. To test whether Cys-203 is required for this protection, we infected CypD−/− MEFs with a C203S-CypD vector. Surprisingly, C203S-CypD reconstituted MEFs were resistant to mPTP opening in the presence or absence of GSNO, suggesting a crucial role for Cys-203 in mPTP activation. To determine whether mutation of C203S-CypD would alter mPTP in vivo, we injected a recombinant adenovirus encoding C203S-CypD or WT CypD into CypD−/− mice via tail vein. Mitochondria isolated from livers of CypD−/− mice or mice expressing C203S-CypD were resistant to Ca2+-induced swelling as compared with WT CypD-reconstituted mice. Our results indicate that the Cys-203 residue of CypD is necessary for redox stress-induced activation of mPTP.