Oligomycin-induced proton uncoupling

Oligomycin is a classical mitochondrial reagent that binds to the proton channel on the F_o component of ATP synthase. As a result, oligomycin blocks mitochondrial ATP synthesis, proton translocation, and O₂ uptake. Here we show that oligomycin induces proton uncoupling subsequent to inhibition of ATP synthesis, as evidenced by recovery of O₂ uptake to near baseline levels. Uncoupling is uniquely rapid and readily observed in HepG2 cells but is also observed at longer times in the unrelated H1299 cell line. Proton fluxes plateau at oligomycin concentrations in the region 0.25-5 μM. At the plateau, fluxes are lower than expected for the classical mitochondrial permeability transition pore, although in H1229 cells, fluxes increase to levels consistent with pore opening at higher oligomycin concentrations. Uncoupling is observed in cells metabolizing either pyruvate or lactate and reversed by addition of glucose to restore ATP synthesis. Uncoupling is not sensitive to cyclosporin A and is not reversed by the ANT inhibitor bongkrekic acid. However, bongkrekic acid inhibits uncoupling if added before oligomycin, which we interpret in terms of maintenance of mitochondrial ATP levels.