ATP Synthase K⁺- and H⁺-Fluxes Drive ATP Synthesis and Enable Mitochondrial K⁺-"Uniporter" Function: I....

ATP synthase (F₁F_o) synthesizes daily our body's weight in ATP, whose production-rate can be transiently increased several-fold to meet changes in energy utilization. Using purified mammalian F₁F_o-reconstituted proteoliposomes and isolated mitochondria, we show F₁F_o can utilize both ΔΨ_m-driven H⁺- and K⁺-transport to synthesize ATP under physiological pH = 7.2 and K⁺ = 140 mEq/L conditions. Purely K⁺-driven ATP synthesis from single F₁F_o molecules measured by bioluminescence photon detection could be directly demonstrated along with simultaneous measurements of unitary K⁺ currents by voltage clamp, both blocked by specific F_o inhibitors. In the presence of K⁺, compared to osmotically-matched conditions in which this cation is absent, isolated mitochondria display 3.5-fold higher rates of ATP synthesis, at the expense of 2.6-fold higher rates of oxygen consumption, these fluxes being driven by a 2.7:1 K⁺: H⁺ stoichiometry. The excellent agreement between the functional data obtained from purified F₁F_o single molecule experiments and ATP synthase studied in the intact mitochondrion under unaltered OxPhos coupling by K⁺ presence, is entirely consistent with K⁺ transport through the ATP synthase driving the observed increase in ATP synthesis. Thus, both K⁺ (harnessing ΔΨ_m) and H⁺ (harnessing its chemical potential energy, Δμ_H) drive ATP generation during normal physiology.