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

Abstract

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.

Keywords: ATP synthesis; mitochondrial K+ transport; mitochondrial KATP channel; proteoliposomes; single molecule bioenergetics; unitary K+ currents.

Graphical Abstract

ATP Synthase K⁺- and H⁺-Fluxes Drive ATP Synthesis and Enable Mitochondrial K⁺-“Uniporter” Function: I. Characterization of Ion Fluxes

Figure 1.

(A) Scheme of the proteoliposome (PL) system; ionic composition of the internal and external buffer, used in K⁺ transport experiments. (B) Kinetics of K⁺ flux into PL; effect of IF₁ depletion. The KCO, Dz, significantly enhanced the rate of K⁺ flux into PL; this effect was blocked both by the F_o inhibitor, Vent, and the mK_ATP blocker, 5-HD, and was absent in IF₁-depleted F₁F_o. *P < 0.05. (C) Ratiometric fluorescence measurements of ΔΨ generated in F₁F_o-reconstituted proteoliposomes by a K⁺ gradient performed with oxonol VI (excitation 560, emission 640/615 nm). PL-reconstituted F₁F_o develops a stable, non-zero K⁺ diffusion-potential (ΔΨ ∼55 mV; orange and light blue traces) in a K⁺ concentration gradient (200 mM K⁺ _out/0.5 mM K⁺ _in; other conditions as in panel A except PBFI omitted) that was completely dissipated by 1 µM FCCP (orange trace). Addition of 10 nM valinomycin (instead of FCCP) resulted in a maximal K⁺ permeability and increase of the oxonol VI fluorescence ratio representing a membrane potential of ∼150 mV (light blue trace). F_o inhibitors DCCD and Oligo (*PL were preincubated with each drug for 15 min; green and dark blue traces) prevented the development of ΔΨ by the K⁺ gradient; thus, ATP synthase (and not some other contaminating K⁺ conductance/channel) is responsible for ΔΨ under control conditions because it is K⁺-permeable. PL without a K⁺-gradient do not develop ΔΨ (yellow trace). The Nernst potential-calibrated values of ΔΨ were set by different K⁺ gradients using 10 nM valinomycin (right Y-axis). (D-G) Planar lipid bilayer experiments. (D) Unitary K⁺ currents from purified F₁F_o and (E) from conventional mitochondrial membrane preparation (at −40 mV), reconstituted into lipid bilayers; pre-intervention baseline recordings are on the left, and the effect of the various compounds are shown (2 mM MgATP, 30 µM Dz, 4 µM Vent, 50 µM Glib). KCOs reverse ATP-inhibited permeation of F₁F_o by K⁺ that can be blocked by Vent and Glib. (F, G) Unitary K⁺ currents elicited in response to a voltage ramp (14.1 mV/sec) distinguish multiple conductance levels represented by O₁-O₇ (C-closed state). The 216 pS conductance (O₅) is predominantly active in the recording shown in panel (F), while the 293 pS conductance (O₆) is active during the recording in panel (G) (highlighted by red lines).

Figure 2.

(A) Scheme of the PL and reaction conditions to measure K⁺ transport-coupled ATP synthesis (and hydrolysis). Δμ_H = 0 for ATP synthesis and ΔΨ=0 by using FCCP; n.b., omitting FCCP eliminates ATP synthesis ruling out the presence of an unsuspected, underlying K⁺/H⁺ antiport mechanism. (B, C) K⁺-gradient-driven ATP production in PL is activated by Dz and (D) pinacidil, and (B–D) attenuated by inhibitors of F₁F_o, inhibitors of the mK_ATP and BK_Ca channels, and by K⁺ gradient dissipation (Val or No K⁺ grad). (E) Na⁺-gradient-driven ATP production in PL is activated by Dz and pinacidil and attenuated by inhibitors of complex V and the mK_ATP (performed as in panel (A) except equimolar concentration of Na⁺ substituted for K⁺). These experiments prove that the entity activated by KCOs is the F₁F_o. *P<0.05.

Figure 3.

Effect of DCCD on lipid bilayer-reconstituted F₁F_o activity during conduction of pure H⁺ (at +28 mV, green traces) and K⁺ (at 0 mV, orange traces) currents (cis: 150 mEq/L K⁺, pH 7.2/trans: 50 mEq/L K⁺, pH 7.2; 1 kHz-recorded data for multiple-minute experiments are plotted as 6-sec averages). (A) Continuous recording of unitary H⁺ current (phase “I”; green trace); H⁺ current is completely blocked by DCCD with t_½∼6 min (phase “II”; exposure to DCCD is indicated by the black horizontal bar). (B) Baseline performance of F₁F_o initially driven by H⁺ current (phase I; green), then switched to K⁺ current (phase “III,” orange), followed by addition of DCCD (black bar, initiated at “0” time) which fails to block K⁺ current-driven activity after 20 min (phase “IV,” orange), then switched back to H⁺ current (at 20 min DCCD exposure, phase “II,” green) leading to rapid inhibition of activity, then back to K⁺ current (at 50 min DCCD, phase “V,” orange) displaying persistence of inhibition. (C, Panels I-V) Mechanistic schema of “phases” of F₁F_o driven by K⁺ (orange) or H⁺ (green) currents corresponding to those depicted in panels A, B. (C_I) H⁺-driven rotation at the reversal potential for K⁺ (E_K+); (C_II) inhibition of H⁺-driven rotation after DCCD reaction with protonated E58; (C_III) K⁺-driven rotation at the reversal potential for H⁺ (E_H+); (C_IV) inability of DCCD to inhibit K⁺-driven rotation due to occupancy of E58 by K⁺ (rather than by a H⁺ required for DCCD reaction and formation of the acylurea adduct); (C_V) once the enzyme is deactivated by DCCD during prior H⁺ current passage (and formation of a stable acylurea adduct on E58), subsequent K⁺ flux is also blocked. Together, these results serve as another line of proof that mammalian F₁F_o can operate utilizing K⁺ flux, and that both K⁺ and H⁺ travel the same route within the complex on the c-ring.

Figure 4.

Direct, “single molecule bioenergetics” demonstration of K⁺-driven ATP synthesis by F₁F_o using simultaneous measurements of K⁺ currents and single-photon detection of ATP. (A) Experimental scheme for simultaneous measurements of unitary K⁺ currents (by voltage clamp) and low light level detection of ATP synthesis activity (luciferase/luciferin bioluminescence) from reconstituted single molecules of F₁F_o in a lipid bilayer formed on a 30-50 µm glass pipette (cis: 150 mEq/L K⁺, pH 7.2/trans: 50 mEq/L K⁺, pH 7.2). (B) Representative experiment performed as described in (A). Top traces: K⁺ current in control (left) and that after F₁F_o inhibition by Vent + Oligo (right), together with corresponding measurements with F₁F_o activity stalled at E_rev. Bottom panels: Frequency histograms of detected bioluminescence photons from ATP generated by K⁺-driven F₁F_o (background-subtracted photon counts measured during K⁺ current (0mV) and at the E_rev (18mV);  Control experiment (left) vs Vent + Oligo block (right)). Note that the Control photon-rate frequency histogram during active K⁺ currents (at 0 mV, E_H+) shows the production of ∼50 photons/100 ms above that when stalled (no currents at +18 mV, E_rev; n = 1000 observations each histogram, P<<0.0001 for paired comparison), and that both the 0 mV current and associated photon production are abolished with + Vent/Oligo (E_H+ vs E_rev; n = 1000 observations each histogram, P = ns). (C) Concatenated experimental results: K⁺-driven photon production in control (0 mV vs stalled at +18mV,  n = 11 paired observations, *P<<0.0001 by Fisher's combined probability test) and that after F₁F_o inhibition by Vent + Oligo (paired observations with control, n = 5, **P<<0.0001 by Fisher's combined probability test); measured photon production rates from K⁺-driven ATP synthesis appeared to be quantized in relation to the number of incorporated F₁F_o (“Possible number of ATP synthases” on right y-axis; see text for further details). (D) Analysis of apparent quantal nature of K⁺-driven photon (ATP) production rate vs the proposed number of bilayer-incorporated ATP synthase molecules (from experimental results, panel (C); regression analysis slope of ∼25 photons/100 ms/ATP synthase, R² = 0.99. (E) Evidence for F₁F_o lipid bilayer insertions as functional dimers. Time course of the cumulative K⁺ current-time integral recording (running ATP synthesis index) from a representative F₁F_o experiment in control (blue trace) and during Oligo inhibition (yellow and orange traces); arrow (yellow trace, 5 min Oligo) indicates abrupt change of the current-time integral slope (to half of the control value) consistent with the initial activity of an F₁F_o dimer being reduced by one functional unit to the equivalent of a monomer; after 15 min Oligo (orange trace), the activity of the remaining F₁F_o (monomer) becomes fully inhibited (n.b., slight deviation from a zero-slope is a typical baseline artifact of cumulative electrical drifts in 4 + minute electrical recordings, and because it occurs in lipid bilayer recordings without protein incorporation, does not represent residual ATP synthase activity). (F) Relationship between ATP synthesis rates calculated from single photon vs K⁺ current measurements (see text for details). The number of F₁F_o molecules is shown in brackets for several independent experiments; the average ATP synthesis rate is ∼1000 ATP/sec/F₁F_o driven solely by K⁺ current in the nominal absence of ATP (thus without significant counter-torque on F₁, in contrast to physiological conditions). ATP synthesis rates estimated independently from single photon vs K⁺ current measurements are in excellent agreement, deviating from unity by < 18%,  R² = 0.98.

Figure 5.

Respiration of isolated rat heart mitochondria. (A) Seahorse-instrument oxygen consumption rate (OCR) under state 3 was determined in KCl- or sucrose-based assay medium in the presence of 5/5mM G/M and 0.5mM ADP. The oligomycin-sensitive OCR was obtained by subtracting from state 3 OCR the state 4 OCR measured in the presence of 10μM oligomycin; n = 60/3 experiments, P<0.0001. (B) P/O ratio was measured using high resolution respirometry as described in Methods under similar conditions as those described in panel G; n = 9/3 experiments, P = ns. (C) The ATP synthesis flux was calculated as the product of the oligomycin-sensitive OCR times the P/O ratio in K⁺- or sucrose-based assay medium; n = 60/3 experiments, P < 0.0001. (D) The K⁺/H⁺ stoichiometry was calculated from the difference between the ATP synthesis flux in the presence of KCl vs in the presence of sucrose, in ratio to the ATP flux in sucrose; n = 46/3 experiments. (E) Respiratory control ratio (RCR) was obtained from the OCR ratio state 3/state 4; n = 60/3 experiments, P = ns. (F) The proton motive force (PMF) was determined according to: PMF = ΔΨ_m—Z∙ΔpH using ΔΨ_m (panel (G); n = 8/3 experiments; P<0.0001) and Z∙ΔpH (panel (H); n = 8/3 experiments; P<0.05) obtained under the indicated conditions with Z = 2.303∙RT/F (see also Supplemental Experimental Procedures). (I) Summary scheme describing the ion fluxes and their respective driving forces, ΔΨ_m and ΔpH, determined under state 3 respiration in K⁺- or sucrose-based medium (see also Supplemental Experimental Procedures). *P<0.05; ^****P<0.0001

Figure 6.

Scheme of the H⁺ and K⁺ transport across the inner mitochondrial membrane. From the energetic standpoint, all the energy available to perform work and execute the ionic movements derives from the original H⁺ gradient established by proton pumps in the respiratory chain. A central point is the obligatory preservation of charge and mass balance under the steady state circuits. In the “original view of cation flux cycles” (A), a certain (majority) of the H⁺ gradient is being harnessed by F₁F_o directly to make ATP, whereas a certain amount of K⁺ is entering the matrix through an ordinary K⁺ channel mechanism (a “mK_ATP-uniporter” channel), driven by ΔΨ, and extruded via KHE utilizing the energy remaining in the fraction of the H⁺ gradient not harnessed by F₁F_o. The equivalent energy of this fraction being used to extrude K⁺, and a large fraction of that non-ATP-producing energy would essentially be dissipated as heat in the constant cycle of K⁺ recirculation (red circuit in A). In the new mechanism (B) the same amount of energy available in the original H⁺ gradient but largely lost as heat is entirely available to produce ATP, simply by having the mK_ATP-uniporter mechanism reside inside, and as natural part of, F₁F_o with the traffic of H⁺ or K⁺ contributing its energy to producing ATP. The remainder of the H⁺ gradient energy is now utilized to remove all the K⁺ that entered via F₁F_o (n.b., this exchange process of extruding K⁺, restoring Δμ_K, is the way that the H⁺ gradient energy is still the original, entire driving force for ATP production). However, the gain is that more ATP is produced for the same input energy by not wasting some of that energy on maintaining what was originally thought to be a separate K⁺ cycle that does not/cannot generate any ATP. Engineered this way, it is a better, tightly coupled system of energy supply-demand matching through the K⁺ cycle utilizing F₁F_o because the matrix influx of K⁺ is truly directly proportional to ATP synthesis.