Mitochondrial hyperpolarization during chronic complex I inhibition is sustained by low activity of complex II,III, IV and V |
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Authors: | Marleen Forkink Ganesh R Manjeri Dania C Liemburg-Apers Esther Nibbeling Maxime Blanchard Aleksandra Wojtala Jan AM Smeitink Mariusz R Wieckowski Peter HGM Willems Werner JH Koopman |
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Institution: | 1. Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands;2. Department of Cell Physiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands;3. Nencki Institute of Experimental Biology, Warsaw, Poland;4. Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders, Radboud University Medical Centre, Nijmegen, The Netherlands |
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Abstract: | The mitochondrial oxidative phosphorylation (OXPHOS) system consists of four electron transport chain (ETC) complexes (CI–CIV) and the FoF1-ATP synthase (CV), which sustain ATP generation via chemiosmotic coupling. The latter requires an inward-directed proton-motive force (PMF) across the mitochondrial inner membrane (MIM) consisting of a proton (ΔpH) and electrical charge (Δψ) gradient. CI actively participates in sustaining these gradients via trans-MIM proton pumping. Enigmatically, at the cellular level genetic or inhibitor-induced CI dysfunction has been associated with Δψ depolarization or hyperpolarization. The cellular mechanism of the latter is still incompletely understood. Here we demonstrate that chronic (24 h) CI inhibition in HEK293 cells induces a proton-based Δψ hyperpolarization in HEK293 cells without triggering reverse-mode action of CV or the adenine nucleotide translocase (ANT). Hyperpolarization was associated with low levels of CII-driven O2 consumption and prevented by co-inhibition of CII, CIII or CIV activity. In contrast, chronic CIII inhibition triggered CV reverse-mode action and induced Δψ depolarization. CI- and CIII-inhibition similarly reduced free matrix ATP levels and increased the cell's dependence on extracellular glucose to maintain cytosolic free ATP. Our findings support a model in which Δψ hyperpolarization in CI-inhibited cells results from low activity of CII, CIII and CIV, combined with reduced forward action of CV and ANT. |
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Keywords: | ΔpH pH gradient across the mitochondrial inner membrane Δψ mitochondrial membrane potential AA antimycin A ANT adenine nucleotide translocase BA bongkrekic acid CI&ndash CV complex I&ndash complex V CoQ coenzyme Q cyt c cytochrome c ETC electron transport chain FCCP carbonyl cyanide-4-trifluoromethoxyphenyl-hydrazone KCN potassium cyanide MALO tert-butyl methyl malonate MEFs mouse embryonic fibroblasts MIM mitochondrial inner membrane MOM mitochondrial outer membrane OLI oligomycin A OXPHOS oxidative phosphorylation PMF proton motive force PA piericidin A PM plasma membrane ROT rotenone RU ratio units TCA tricarboxylic acid TMRM tetramethylrhodamine methyl ester |
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