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We demonstrated that ATP synthase serves the functions of a primary mitochondrial K+ “uniporter,” i.e., the primary way for K+ to enter mitochondria. This K+ entry is proportional to ATP synthesis, regulating matrix volume and energy supply-vs-demand matching. We show that ATP synthase can be upregulated by endogenous survival-related proteins via IF1. We identified a conserved BH3-like domain of IF1 which overlaps its “minimal inhibitory domain” that binds to the β-subunit of F1. Bcl-xL and Mcl-1 possess a BH3-binding-groove that can engage IF1 and exert effects, requiring this interaction, comparable to diazoxide to augment ATP synthase's H+ and K+ flux and ATP synthesis. Bcl-xL and Mcl-1, but not Bcl-2, serve as endogenous regulatory ligands of ATP synthase via interaction with IF1 at this BH3-like domain, to increase its chemo-mechanical efficiency, enabling its function as the recruitable mitochondrial KATP-channel that can limit ischemia-reperfusion injury. Using Bayesian phylogenetic analysis to examine potential bacterial IF1-progenitors, we found that IF1 is likely an ancient (∼2 Gya) Bcl-family member that evolved from primordial bacteria resident in eukaryotes, corresponding to their putative emergence as symbiotic mitochondria, and functioning to prevent their parasitic ATP consumption inside the host cell.

ATP synthase (F1Fo) 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 F1Fo-reconstituted proteoliposomes and isolated mitochondria, we show F1Fo can utilize both ΔΨm-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 F1Fo 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 Fo 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 F1Fo 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 ΔΨm) and H+ (harnessing its chemical potential energy, ΔμH) drive ATP generation during normal physiology.

ATP synthase (F(1)F(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 F(1)F(o)-reconstituted proteoliposomes and isolated mitochondria, we show F(1)F(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 F(1)F(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 F(1)F(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.