Chloromethyltetramethylrosamine (Mitotracker Orange (TM)) induces the mitochondrial permeability transition and inhibits respiratory complex I - Implications for the mechanism of cytochrome c release

We have investigated the interactions with isolated mitochondria and intact cells of chloromethyltetramethylrosamine (CMTMRos), a probe (Mitotracker O range(TM)) that is increasingly used to monitor the mitochondrial membrane potential (Delta psi(m)) in situ. CMTRIRos binds to isolated mitochondria a nd undergoes a large fluorescence quenching. Most of the binding is energy- independent and can be substantially reduced by sulfhydryl reagents. A smal ler fraction of the probe is able to redistribute across the inner membrane in response to a membrane potential, with further fluorescence quenching. Within minutes, however, this energy-dependent fluorescence quenching spont aneously reverts to the same level obtained by treating mitochondria with t he uncoupler carbonylcyanide-p-trifluoromethoxyphenyl hydrazone, We show th at this event depends on inhibition of the mitochondrial respiratory chain at complex I and on induction of the permeability transition pore by CMTMRo s, with concomitant depolarization, swelling, and release of cytochrome c, After staining cells with CMTMRos, depolarization of mitochondria in situ w ith protonophores is accompanied by changes of CMTMRos fluorescence that ra nge between small and undetectable, depending on the probe concentration. A lasting decrease of cellular CMTMRos fluorescence associated with mitochon dria only results from treatment with thiol reagents, suggesting that CMTMR os binding to mitochondria in living cells largely occurs at SH groups via the probe chloromethyl moiety irrespective of the magnitude of Delta psi(m) . Induction of the permeability transition precludes the use of CMTMRos as a reliable probe of Delta psi(m) in situ and demands a reassessment of the conclusion that cytochrome c release can occur without membrane depolarizat ion and/or onset of the permeability transition.