Computational modeling of mitochondrial K⁺- and H⁺-driven ATP synthesis

Highlights

• A computational model of H⁺ and K⁺ driven F₁F_o ATP synthase was formulated.

• Model simulations corroborate energetic advantage conferred by K⁺ transport through F₁F_o.

• Integrated model of mitochondrial function simulates experimental data during energetic transitions.

• Simulations predict key role for the K⁺/H⁺ exchanger in mitochondrial energetic performance.

Abstract

ATP synthase (F₁F_o) is a rotary molecular engine that harnesses energy from electrochemical-gradients across the inner mitochondrial membrane for ATP synthesis. Despite the accepted tenet that F₁F_o transports exclusively H⁺, our laboratory has demonstrated that, in addition to H⁺, F₁F_o ATP synthase transports a significant fraction of ΔΨ_m-driven charge as K⁺ to synthesize ATP. Herein, we utilize a computational modeling approach as a proof of principle of the feasibility of the core mechanism underlying the enhanced ATP synthesis, and to explore its bioenergetic consequences. A minimal model comprising the ‘core’ mechanism constituted by ATP synthase, driven by both proton (PMF) and potassium motive force (KMF), respiratory chain, adenine nucleotide translocator, Pi carrier, and K⁺/H⁺ exchanger (KHEmito) was able to simulate enhanced ATP synthesis and respiratory fluxes determined experimentally with isolated heart mitochondria. This capacity of F₁F_o ATP synthase confers mitochondria with a significant energetic advantage compared to K⁺ transport through a channel not linked to oxidative phosphorylation (OxPhos). The K⁺-cycling mechanism requires a KHE_mito that exchanges matrix K⁺ for intermembrane space H⁺, leaving PMF as the overall driving energy of OxPhos, in full agreement with the standard chemiosmotic mechanism. Experimental data of state 4➔3 energetic transitions, mimicking low to high energy demand, could be reproduced by an integrated computational model of mitochondrial function that incorporates the ‘core’ mechanism. Model simulations display similar behavior compared to the experimentally observed changes in ΔΨ_m, mitochondrial K⁺ uptake, matrix volume, respiration, and ATP synthesis during the energetic transitions at physiological pH and K⁺ concentration. The model also explores the role played by KHE_mito in modulating the energetic performance of mitochondria. The results obtained support the available experimental evidence on ATP synthesis driven by K⁺ and H⁺ transport through the F₁F_o ATP synthase.

Introduction

ATP synthesis in E. coli, mitochondria, and chloroplasts is catalyzed by F₁F_o ATP synthase that transduces phosphorylation and redox potentials into electrochemical (ion gradients) and chemical (ATP) energy [1,2]. Foundationally, bioenergetics rests on the principle of F₁F_o ATP synthase harnessing the energy stored in H⁺ gradients to generate ATP.

Recently, our laboratory has described that a large part of the ATP synthesis flux is driven by K⁺ [3,4]. In spite of the large F₁F_o selectivity for H⁺ over K⁺ (or Na⁺) (~10⁶-fold with regard to both alkali metal ions), the large abundance of cytoplasmic K⁺ over H⁺ (>10⁶-fold excess) in mammalian cells, reveals that, in principle, a non-trivial flux of K⁺ could actually be driving the F₁F_o ATP synthase in its normal operation. The portion of the H⁺ gradient not directly dissipated by the ATP synthase activity would be available to drive the efflux of K⁺ through the K⁺/H⁺ exchanger (KHE) restoring the osmotic balance. In this way, the K⁺- and H⁺- transporting function of ATP synthase remains fully compatible with Mitchell's chemiosmotic mechanism [1,5].

The K⁺ transport mechanism allows mitochondrial volume regulation of respiration since, unlike H⁺, K⁺ is, functionally, osmotically active owing to its 6 orders-of-magnitude greater concentration than H⁺. Purified F₁F_o ATP synthase reconstituted into proteoliposomes can harness energy from K⁺ flux to generate ATP, when subjected to a transmembrane K⁺ gradient [3,4]. Pure K⁺ current-driven ATP synthase activity was accompanied by ATP generation recorded as an increase in the photon rate over background that, in turn, was significantly inhibited by specific F_o blockers venturicidin/oligomycin [3,4].

Isolated rat heart mitochondria show that, compared to absence of K⁺, at constant osmolality (260 mOsm), physiological pH and K⁺ concentration drive significantly enhanced fluxes of ATP synthesis and respiration [3,6]. Under the same conditions, and using radioactive tracers, we quantified the mitochondrial PMF, its individual components, ΔΨ_m and ΔpH, and volume, under both states 4 and 3 respiration, in the presence or absence of K⁺ [3,6].

In the conventional view of cation flux cycles in mitochondria, a H⁺ gradient is driving solely H⁺ through F₁F_o to make ATP whereas K⁺ only enters the matrix through an “ordinary” channel, dissipating the energy change of this K⁺ movement as heat (see Fig. 1A). In the new mechanism studied herein, in addition to that of H⁺, a significant fraction of the energy of ΔΨ_m-driven charge moves as K⁺ whose flux is directly harnessed by F₁F_o to make ATP (see Fig. 1B) rather than being wasted as heat in an ordinary channel, while the remainder of the H⁺ gradient energy is utilized to remove K⁺ through KHE_mito. The bioenergetic advantage in this mechanism over the conventional one 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 would generate heat but not ATP.

The mechanism of K⁺- and H⁺-driven ATP synthesis remains fully compatible with Mitchell's chemiosmotic mechanism (reviewed in [5]) and the present work computationally tests some of its major mechanistic and bioenergetic implications. We utilize a computational modeling approach that accounts for the mechanism of K⁺- and H⁺-driven ATP synthesis to address its feasibility to predict integrated biological outcomes. We explore the model's ability to simulate experimental data obtained with isolated mitochondria that, in turn, had validated the proteoliposomal data [3]. We also seek to analyze the bioenergetic impact produced by K⁺-and H⁺-driven ATP synthesis as well as put forward testable predictions emerging from the model results. Herein, the mechanistic role(s) of K⁺ are highlighted. Overall, the results obtained support the experimental evidence available on ATP synthesis driven by K⁺ and H⁺ transport through the F₁F_o ATP synthase [3,4,6].