Mitochondrial hyperpolarization during chronic complex I inhibition is sustained by low activity of complex II, III, IV and V.
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.
Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.
Marleen
M
Forkink
Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.
Ganesh R
GR
Manjeri
Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.
Dania C
DC
Liemburg-Apers
Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.
Esther
E
Nibbeling
Department of Cell Physiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.
Maxime
M
Blanchard
Nencki Institute of Experimental Biology, Warsaw, Poland.
Aleksandra
A
Wojtala
Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders, Radboud University Medical Centre, Nijmegen, The Netherlands.
Jan A M
JA
Smeitink
Nencki Institute of Experimental Biology, Warsaw, Poland.
Mariusz R
MR
Wieckowski
Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.
Peter H G M
PH
Willems
Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands. Electronic address: w.koopman@ncmls.ru.nl.
Werner J H
WJ
Koopman
D019069:Cell Respiration / Q000235:genetics*; D042967:Electron Transport Complex I / Q000737:chemistry / Q000235:genetics*; D042963:Electron Transport Complex II / Q000737:chemistry / Q000235:genetics; D014450:Electron Transport Complex III / Q000737:chemistry / Q000235:genetics; D003576:Electron Transport Complex IV / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry / Q000235:genetics; D051336:Mitochondrial Membranes / Q000737:chemistry / Q000378:metabolism*; D010085:Oxidative Phosphorylation
C478189:respiratory complex II; D000226:Mitochondrial ADP, ATP Translocases; D042963:Electron Transport Complex II; D003576:Electron Transport Complex IV; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III
ATeam; Live-cell microscopy; Respirometry; SypHer; TMRM