Evaluation of mitochondrial membrane potential using a computerized device with a tetraphenylphosphonium-selective electrode.
Mitochondrial membrane potential (Deltapsi(m)) plays important roles in the normal function of cells and in pathobiochemical situations. The application of ion-selective electrodes for the measurement of Deltapsi(m) is important for studying normal biological reactions and pathways and mitochondrial diseases. We constructed and optimized a computerized device for real-time monitoring of the Deltapsi(m), which included modification of tetraphenylphosphonium (TPP(+))-selective membrane that improved reproducibility of the TPP(+)-selective electrode. Application of MATLAB software increased the sensitivity of the system. We tested our improved device for membrane potential measurements of isolated mitochondria (in absolute scale of millivolts). In addition, we assessed relative changes of Deltapsi(m) (as changes in TPP(+) concentration) of digitonin-permeabilized cells (hepatocytes, control transmitochondrial cybrids, HeLa G and BSC-40) after addition of substrates, inhibitors, and uncoupler of respiratory chain. Our system can be successfully used for studies of many aspects of the regulation of mitochondrial bioenergetics and as a diagnostic tool for mitochondrial oxidative phosphorylation disorders.
Labajova
Anna
A
Institute of Pathophysiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic. Anna.Labajova@lfl.cuni.cz
Vojtiskova
Alena
A
Krivakova
Pavla
P
Kofranek
Jiri
J
Drahota
Zdenek
Z
Houstek
Josef
J
D000818:Animals; D016287:Computer Peripherals; D003199:Computer Systems; D004594:Electrophysiology / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D017736:Ion-Selective Electrodes; D008297:Male; D008564:Membrane Potentials / Q000502:physiology*; D008930:Mitochondria, Liver / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000378:metabolism*; D009861:Onium Compounds / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
D007202:Indicators and Reagents; D009861:Onium Compounds; D009943:Organophosphorus Compounds; C013289:tetraphenylphosphonium