Mitochondrial hyperpolarization during chronic complex I inhibition is sustained by low activity of complex II, III, IV and V

Research output: Contribution to journalArticlepeer-review


  • Marleen Forkink
  • Dania C Liemburg-Apers
  • Esther Nibbeling
  • Maxime Blanchard
  • Aleksandra Wojtala
  • Jan A M Smeitink
  • Mariusz R Wieckowski
  • Peter H G M Willems
  • Werner J H Koopman

Colleges, School and Institutes


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 (24h) 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.


Original languageEnglish
Pages (from-to)1247-56
Number of pages10
JournalBiochimica et Biophysica Acta
Issue number8
Early online date24 Apr 2014
Publication statusPublished - Aug 2014


  • Cell Respiration, Electron Transport Complex I, Electron Transport Complex II, Electron Transport Complex III, Electron Transport Complex IV, HEK293 Cells, Humans, Mitochondria, Mitochondrial ADP, ATP Translocases, Mitochondrial Membranes, Oxidative Phosphorylation, ATeam, Live-cell microscopy, Respirometry, TMRM, SypHer