A Walk in the Memory, from the First Functional Approach up to Its Regulatory Role of Mitochondrial Bioenergetic Flow in Health and Disease: Focus on the Adenine Nucleotide Translocator

The hypothesis of ANT as a component of the mPTP was first questioned by Kokoszka et al. [85] because an mPT was detected after ablation of the genes encoding for ANT1 and ANT2 isoforms, expressed in mouse liver mitochondria, but recently Karch et al. [86] noted that the same mice overexpress the ANT4 isoform, which might have provided a permeabilization pathway. Readdressing the role of ANT in the mPT was obtained by results from experiments performed with ANT1/ANT2/ANT4 triple-knockout mice mitochondria: a PTP still existed, suggesting that the PTP forms in the absence of ANT.

However, further genetic ablation of the Ppif gene (which generates CyP-D null mitochondria) or treatment with CsA (which inhibits CyP-D) finally prevented any Ca2+-dependent permeabilization, suggesting that mitochondria possess at least two pathways for Ca2+-dependent permeabilization and that they are both inhibited by CsA [86]: one in the absence of ANT, which is not inhibited by BKA [83,87], and one in the presence of ANT, which is inhibited by BKA [87].

It seems that despite its role as the main energy-producing enzyme, it is precisely the ATP synthase that plays a role in the IMM depolarization caused by the channel activity of the mPTP. However, this aspect of the question concerning PTP, although of considerable importance, is a problem beyond our competence and therefore we refer the interested reader to specific readings [77,78,83,84,88].

Mitophagy is a particular form of autophagy, which is a quality control process [89] that cells have designed to sequester and remove damaged mitochondria and thus to defend against dysfunctional mitochondrial activities.

In fact, as the result of cellular senescence or pathological processes, mitochondria can damage cells both by failing to perform their critical functions (e.g., ATP generation, maintaining normal cell signaling) and by actively stimulating hazardous processes (e.g., release of toxic ROS, apoptosis, increasing cellular calcium levels) [90].

Briefly, mitophagy is a process triggered by a loss of mitochondrial membrane potential that then initiates the accumulation of two proteins, phosphatase and tensin homolog-induced kinase 1 (PINK1), and the E3 ubiquitin ligase Parkin on the outer surface of the damaged mitochondrion. In response to this event, the mitochondrion is wrapped in a membrane that isolates it from the rest of the cell, transforming it into an autophagosome; subsequently, the latter fuses with a lysosome, degrading the enclosed organelle [91]. It follows from this that mitophagy, far from being a deleterious process that destroys mitochondria, is a beneficial process; therefore, mutations that alter the function of the PINK1/Parkin proteins sometimes result in severe disease [90].

In healthy mitochondria, PINK1, a key component of the mitophagic machinery that marks dysfunctional mitochondria for degradation [92], is translocated via the TOM and TIM23 complex into the mitochondrial matrix and degraded. Since PINK1 translocation into the mitochondrial matrix via TIM23 requires Δψ, it follows that during mitochondrial dysfunction, which causes loss of Δψ, PINK1 cannot enter the matrix and then accumulates on the OMM, where it recruits the E3 ubiquitin ligase Parkin, thus initiating the mitophagic process [92].

Recently, Hoshino et al. carried out a sophisticated and meticulous work, published in Nature in 2019 [93], in which, using multiple mitophagy reporter systems and pro-mitophagy triggers, they identified numerous components of Parkin-dependent mitophagy, including, surprisingly, ANT. At the molecular level, ANT is required for inhibition of the presequence translocase TIM23, which leads to the stabilization of PINK1, in response to bioenergetics collapse.

If pharmacological inhibition of ADP/ATP exchange promotes mitophagy independently of its transport activity, genetic ablation of ANT paradoxically suppresses mitophagy in response to uncoupling or treatment with metabolic toxins, with consequent profound accumulation of aberrant mitochondria.

Ideally, ANT is a sophisticated sensor of bioenergetics failure from either proton gradient collapse or failure to carry out OXPHOS. It suppresses TIM23 translocation of PINK1, thus facilitating the stabilization of PINK1 on the OMM and activating mitophagy. The fact that PINK1 translocation continues to occur in uncoupled mitochondria lacking ANT proteins represents a substantial challenge not only to the understanding of mitophagy, but also to the current model of how proteins are transported into the mitochondrial matrix [94].

In conclusion, the findings show that the protein has two separate functions: ATP generation and quality control. In fact, although it is known that the mutations in the ANT gene cause diseases, they do not impact ANT’s ability to produce chemical energy, but rather cause defective quality control, an important piece of knowledge to start thinking about therapeutic approaches that may improve quality control.

As such, ANT proteins are likely the key integration sites for mitochondrial health, function, and cell death. Moving forward beyond 2020, further studies will be needed to define the regulatory mechanisms of these processes and to better characterize the different modes of action of ANT.

ANT kinetics have great physiological relevance since it is likely that the kinetic parameters of the carrier are a major determinant of the rate of cytosolic ATP utilization in situ. Most kinetic studies—in the late 1980s—employed isotopically labelled substrates of the carrier and measured the uptake/release of isotopes into/from the mitochondrial matrix. However, many problems were encountered when attempting to interpret data.

It soon became clear to everyone that direct accurate measurements of initial rate are almost impossible to achieve because the transport is rapid, the exchangeable pools are small, and the inhibitor stop method technique inhibits at variable rates dependent on conditions of energization and substrate concentration.

In order to circumvent the problems inherent in measuring ANT kinetics in intact mitochondria, Krämer and Klingenberg [13,95,96,97,98] measured ANT kinetics in a reconstituted system composed of artificial liposomes and the isolated carrier. Unfortunately, even in this case, various problems related to time-dependent variations, to K+ gradient used to assess, and so on showed up.

Several other methods beyond the isotopic one (method 1) for determining the ATP–ADP steady-state exchange rate mediated by ANT have been described in the past.

Some of these directly measure ADP and/or ATP by (2) thin-layer chromatography [99] or (3) high-performance liquid chromatography [100]. Another employ coupled reactions that yield an end-product that can be detected (4) luminometrically, detecting chemiluminescence upon ATP-dependent oxidation of luciferin, catalyzed by firefly luciferase [101]. Furthermore, there are techniques that employ (5) fluorescent derivatives of nucleotides in order to estimate the rate of release of the fluorescent molecule upon ANT-mediated exchange for ADP [102].

Among the aforementioned methodologies, the radioactive method, a very sensitive assay that is already operational with small amounts of mitochondrial protein, requires the handling of radioactive material, and methods 2 and 3 require expertise in using specialized equipment. Above all, they are essentially end-point assays.

Only methods 4 and perhaps 5 could be considered on-line measurements. However, for its part, method 5 relies on materials that are not commercially available and are therefore not appealing to the broader scientific community. Method 4 (the luciferase method), if used as an on-line method, suffers from several drawbacks: First, there is a lack of constant proportionality between ATP concentration and luminescence caused by product inhibition of the luciferase reaction by oxyluciferin; second, for the reliable estimation of the kinetics of ATP formation, the endogenous ATP concentration prior to the assay needs to be measured; third, the required use of a low ionic strength medium and room temperature restricts experiments to conditions that are nowhere near physiological. Although some of the disadvantages of this method have been overcome by various modifications [103], it is certainly not a suitable method for the purpose.

Overall, for a comprehensive appraisal of methods to measure ATP–ADP exchange rates in mitochondria and reconstituted systems, the reader is referred to the review by Martin Klingenberg [22].

Taking a cue from measurements and kinetic determinations of other transporters [16] for which a variety of metabolite detecting systems (consisting of enzyme/s and cofactor/s designed to selectively ascertain outside mitochondria the appearance of molecules derived from the mitochondrial metabolism of the taken-up substrate) have been developed, the idea of applying the same criterion to ANT tickled us a lot and proved to be successful.

Obviously, this carrier plays a primary role in cellular metabolism because the function of the mitochondrion is—by definition—to supply the cell with ATP, which is also the universal energy signal of metabolism, being generated or consumed in every metabolic pathway. Therefore, the carrier is important for the regulation of cellular metabolism and hence—as we will see—it is involved in various pathological states (see below, Section 4).

Of course, in this case, unlike the other mitochondrial carriers, we were faced with a “complex” case: if a correct study of the permeability properties of the mitochondria could not disregard their integrity and functionality, making it essential to verify the mitochondrial coupling before each experiment, in this case the question was scorching. It was possible to follow the ADP/ATP antiport exchange by using exclusively mitochondria in which OXPHOS occurred. Therefore, we were in conditions where the proton gradient would be consumed both to operate the carrier and for the OXPHOS.

The appearance of ATP in the extramitochondrial phase, revealed by a specific detecting system, is the result of a process consisting of several stages: (1) uptake of ADP via ANT in exchange with endogenous ATP; (2) synthesis of ATP via ATP synthase, which makes use of the energy resulting from the oxidation of endogenous substrates; and (3) export of the newly synthesized ATP in exchange with ADP.

Using rat liver mitochondria (RLM), a method was developed to continuously monitor the ATP efflux from mitochondria, which occurs by adding ADP to the mitochondrial suspension incubated in the presence of glucose, NADP+, hexokinase (HK), and glucose-6-phosphate dehydrogenase (G6PDH) by revealing the appearance of ATP in the extramitochondrial phase photometrically as an increase of absorbance at 340 nm due to NADP+ reduction [104], taking into account that there is a stoichiometric ratio of 1:1 between effluxed ATP and formed NADPH (Figure 1).

Really, the experimental strategy is complex. In fact, ATP production by mitochondria incubated with ADP can occur both via ATP synthase activity in OXPHOS and via adenylate kinase (ADK), which is localized in the intermembrane space. Thus, in order to separate the contribution of these two pathways to ATP production and appearance outside mitochondria, we used CAT, oligomycin (OLIGO), and Ap5A to inhibit ANT, ATP synthase, and ADK, respectively.

We observed that in the presence of Ap5A, the rate of ATP appearance was completely inhibited by adding either CAT or OLIGO. Instead, when the same experiment was carried out photometrically in the presence of CAT (5 μM) and OLIGO (2.5 μM), thus monitoring only the ADK-dependent ATP production, ATP was formed as a result of ADP addition. As expected, the addition of Ap5A (20 μM) completely blocked the rate of NADPH formation.

The rate of NADP+ reduction showed a hyperbolic dependence on ADP concentration and proved to be a measure of the activity of the ADP/ATP translocator in the presence of Ap5A, used to inhibit the adenylate kinase [104,105], as established by the determination of limiting step of the global process (see below).

Importantly, for the first time, the ADP/ATP exchange was studied by using respiring and phosphorylating mitochondria, i.e., under roughly “physiological” conditions, since the intramitochondrial ATP concentration is not enhanced by a loading procedure, but is derived only from the uptake and phosphorylation of externally added ADP.

Furthermore, assuming that the available energy is not limiting, the flow rate of ATP will depend on either the rate of the antiporter or that of the ATP synthase.