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 mitochondrial adenine nucleotide translocator (ANT) plays the fundamental role of gatekeeper of cellular energy flow, carrying out the reversible exchange of ADP for ATP across the inner mitochondrial membrane. ADP enters the mitochondria where, through the oxidative phosphorylation process, it is the substrate of Fo-F1 ATP synthase, producing ATP that is dispatched from the mitochondrion to the cytoplasm of the host cell, where it can be used as energy currency for the metabolic needs of the cell that require energy. Long ago, we performed a method that allowed us to monitor the activity of ANT by continuously detecting the ATP gradually produced inside the mitochondria and exported in the extramitochondrial phase in exchange with externally added ADP, under conditions quite close to a physiological state, i.e., when oxidative phosphorylation takes place. More than 30 years after the development of the method, here we aim to put the spotlight on it and to emphasize its versatile applicability in the most varied pathophysiological conditions, reviewing all the studies, in which we were able to observe what really happened in the cell thanks to the use of the “ATP detecting system” allowing the functional activity of the ANT-mediated ADP/ATP exchange to be measured.
Thousands of reviews on the adenine nucleotide translocator (ANT) are available on PubMed®, the database (continuously updated) comprising more than 30 million citations for biomedical literature from MEDLINE, life science journals, and online books from the 1950s to today.
In particular, in the month of February 2021—the period in which we wrote this review—by typing as the keyword “adenine nucleotide translocator,” more than 18,000 original articles were traced by the system. We tried to narrow down the search by associating two other keys: “regulation” and “function.” The number of results obtained was greatly reduced, but it was always in the thousands. Of these, about 1500 were reviews.
All this is to say that this study of ours does not pretend to present yet another review on ANT to the scientific world—after all we do not have the necessary skills to embrace the subject from the point of view of genomics, proteomics, genetics, and molecular and cellular biology—but rather to tell how from our first entry in a biochemistry laboratory 35–40 years ago up to the present day, we have dealt with how this mitochondrial transport protein works.
To be precise, ours is a study dedicated to measuring the rate of appearance of ATP outside the mitochondria, under conditions close to a physiological environment that reflected what actually happens in the cell. Our main aim is to put the spotlight on the validity and versatility of this measurement method, which has not received adequate consideration, in our opinion, although it has been applied in the most varied pathophysiological conditions we have studied, as we will see later (Section 3).
We were still students when we collaborated in the tuning of the method that allows for the measuring of the ADP/ATP exchange by monitoring the extramitochondrial efflux of ATP gradually produced inside mitochondria for oxidative phosphorylation (OXPHOS) as a result of ADP addition and uptake into mitochondria. Even today we meet ANT in our study/research path in a great variety of diseases in which it is involved due to its pivotal role in cellular metabolism. Not only will we see in this roundup of research what the effect of laser light is on ANT activity and how it reacts following drug photosensitization of isolated mitochondria, but also how the antiviral drug 3′-Azido-3′-deoxythymidine (AZT) affects it, and even how its impairment leads to mitochondrial energy deficit in Down syndrome (DS), as well as its involvement in Alzheimer disease (AD) progression, in which its function intertwines with those of the two AD proteins, i.e., β-amyloid and TAU, and so on.
However, in order to facilitate reading, also addressing readers who deal with something else, but who are interested in knowing how the energy produced by mitochondria is transferred and made available in the cytosol, first we will briefly present mitochondria—recalling our first approach with them when we were students—and describe their functional mechanisms as well as the salient features of ANT, focusing essentially on those that will be taken into consideration in the subsequent paragraphs, postponing the reading to strictly specific studies. This will allow the reader to understand the experimental strategy adopted by the method used to monitor the transfer of energy in the form of ATP from the inside of the mitochondria to the outside, a method that, since its development more than 30 years ago, is still at the forefront, when the study of the cell state from a bioenergetic point of view is addressed under different pathophysiological conditions.
For more than a century, biologists have looked at mitochondria as bean-shaped organelles floating in the cytoplasm and working tirelessly and without respite to provide the energy required for life. However, that simple picture of mitochondria soon proved incredibly incomplete, especially since mitochondria do not have a stable shape—they stretch, shrink, blend, and divide all the time—and energy production is not their only role.
Before getting into the essential core topic of this review, we will provide some useful information about mitochondria in order to allow newbies to understand it.
Mitochondria possess two highly specialized membranes that together create two separate mitochondrial compartments, namely, the intermembrane space and the matrix.
The inner mitochondrial membrane (IMM), unlike the external one that envelopes the organelle, is sub-divided into two compartments, the inner boundary membrane (IBM) and the cristae membranes (CM) [1], with a surface 1.5–2-fold larger than the IBM part [2]. Both parts are connected via so-called cristae junctions that are often slit-like, as electron tomography has revealed [3]. Although by and large they constitute a continuous membrane, the sub-compartments achieve different tasks and thus have a distinct protein composition [2]. For example, the IBM is the site of protein import and thus displays a reliable presence of transport proteins, called the translocase of the inner membrane, TIM [4]. It also hosts other carriers and channel proteins. On the contrary, the cristae are the bioenergetic units harboring the five mitochondrial respiratory complexes (mRC), NADH:ubiquinone oxidoredutase (NADH-dehydrogenase, complex I, CI), succinate dehydrogenase (SDH, complex II, CII), Cytochrome c-reductase (bc1 complex, complex III, CIII), cytochrome c oxidase (complex IV, CIV), and ATP synthase (complex V, CV). All together, they form the OXPHOS system, which is not the only process of the cell’s energy metabolism involving mitochondria.
The inner compartment of the matrix is moderately dense and hosts strands of DNA and ribosomes, molecular tools through which the mitochondria are able to code for part of their proteins. The matrix contains the complete catalytic kit of enzymes that take part in terminal oxidative metabolism, such as the Krebs cycle, the beta-oxidation of fatty acids and the urea cycle. The metabolism of amino acids, lipids, cholesterol, steroids, and nucleotides also takes place in this mitochondrial district.
As for the outer mitochondrial membrane (OMM), which is essentially smooth and elastic, it is characterized by a high lipid/protein ratio and contains a channel for anions, whose activity is dependent on the electric potential, the voltage dependent anion channel (VDAC), also called “porin,” together with devices used both for communication with other cellular structures and for the recognition and import of mitochondrial proteins encoded by the nucleus [5]. It seems that VDAC controls the passage of metabolites between the cytosol and mitochondrial intermembrane space via the modulation of different open and closed states of the protein [6,7]. In this regard, we refer the reader to Section 4.2.4.1, in which VDAC1 activity decreases in concomitance of its physical interaction with hexokinase (HK) [8]. In addition, through its location in the OMM and association with various ligands and proteins, it serves as a convergence point for a variety of cell survival and death signals, linking energy, redox, and signaling pathways in mitochondria and other cellular compartments. Regarding this, it is interesting to note, for example, that HK and glycerol kinase interact with the porins of the external surface of the OMM [9] in such a way as to have preferential access to the ATP generated in the mitochondria; in addition, the binding of HK with mitochondria has been considered the basis for energy metabolism in cell proliferation [10].
The regulation operated by the VDAC causes a concentration of substrate different (or higher) than that of the extramitochondrial phase similar to cytosol, to establish itself in the intermembrane space due to the phenomenon of micro-compartmentation. Although we will return to this topic soon, now just a digression about the concept of micro-compartmentation presupposes that (i) in the mitochondria microenvironments are created with a composition of substrates different from that of the surrounding environment and that (ii) the mutual interaction of proteins can lead to a sort of “channeling” in which the products become reagents for enzymes that are physically “close/bound together,” more or less a situation similar to mRC supercomplexes (see Section 3.6.1).
We first heard the term “bioenergetics” when, as internal students, we entered the renowned Institute of Biological Chemistry at the University of Bari. In our lab everything contributed to creating a bioenergetics atmosphere: The electrons came and went along the mRC, the Gilson oxygraph marked the notches on the paper of the recorder following the addition of respiratory substrates causing oxygen consumption, and discussions of how mitochondria generate energy and how metabolites enter and/or leave the mitochondria and so on were on the agenda. One thing is certain: our research has always covered basic aspects of mitochondrial bioenergetics and, in the last few years, it has ranged in the field of the regulation of energy metabolism in health and disease.
Those were years of discovery and great ferment in the scientific world of bioenergetics and isolated mitochondria were the chief playground for the “experts in bioenergetics.” In Bari, Ernesto Quagliariello carried out an intense scientific and organizational activity dotted with international events. For about 20 years prior, in fact, annual meetings on mitochondria—organized by researchers from the laboratory of biochemistry at the University of Bari, in collaboration with their colleagues from other Italian and foreign universities—were held in Bari or in the villages around Bari. These meetings were a tradition. They saw the participation of leading biochemists and molecular biologists, including Albert Lehninger; Sir Hans Krebs, Nobel Prize in Chemistry in 1953; Bill Slater and Britton Chance; Vladi Skulachev, friend and regular visitor to Bari; and John Walker, Nobel Prize in Chemistry in 1997. It was in one of these meetings that Peter Mitchell presented an articulated speech on chemiosmotic theory, a topic that in 1978 earned him the Nobel Prize in Chemistry. It was also at one of the Bari meetings, where all new aspects of mitochondrial processes were debated, that the term “bioenergetics” was launched, having becoming more popular after Lehninger’s little book “Bioenergetics” was first published in 1964.
Among the great scholars hosted, we absolutely cannot forget the German biochemist Martin Klingenberg, who carried out research mainly on mitochondria, in particular on the ADP–ATP transporter.
It is worth mentioning that Martin Klingenberg and his co-workers were among the first to fully recognize the implications of Mitchell’s work on metabolite transport across the IMM. In further pioneering studies, his laboratory staff purified the ADP/ATP carrier [11] and reconstituted that protein into proteoliposomes, thus allowing detailed characterization of its transport properties [11,12,13].
At that time, no textbook would have even nearly covered the broad range of topics addressed in “Bari Meetings” lectures. The proceedings of the symposia of the “Bari Meetings”—published in books, whose quotations can be found in the literature—provided year after year a comprehensive and timely record of the biochemical aspects of mitochondrial transport, with great strides towards the understanding of its molecular mechanism. Multiple transport systems were defined in terms of their catalysts, pumping systems and carriers. Therefore, right in the period in which the research on these carrier systems was fervent and competitive, upon our arrival in that laboratory and after becoming familiar with the organelle, we could not help but collaborate in this research that led to the discovery of other new carriers in mitochondria of various origins, also depending on the physiological role of the organ.
At the time of our internship as students—and also afterwards, during the period of learning as trainees, or in any case during doctorate school (for D.V.)—before each experiment, having an exact design goal, the isolation of mitochondria followed by specific tests of functionality, was our daily bread. Thus, we learned not only to treat this precious but delicate organelle with care and skill, but also to grasp the substantial differences between organs, either due to the type of tissue—just consider the heart, whose tissue is tenacious, so the shredding is more cumbersome and the homogenization is repeated longer to allow the walls to break and the mitochondria to escape—or due to the mitochondrial yield. This type of approach was useful for us to gain experience and grow, to be able to face and cope with the challenges we are now involved in.
Without a shadow of a doubt, doing bioenergetics in those days was an art more than a science, as we did not have the highly elaborate and refined techniques of today. Using those of that time, a successful preparation of a mitochondrial suspension was an indispensable prerequisite for the “success” of the type of experiments on “mitochondrial transport.” In fact, the measurements to be made, those concerning transport, could not ignore the fact that the mitochondria were “respiring and phosphorylating.” Thus, no type of experiment began without conducting an oxygraph check.
Working with mitochondria functioning from a bioenergetic point of view, i.e., mitochondria capable of (i) respiring, i.e., consuming molecular oxygen, (ii) creating membrane potential useful for OXPHOS and other processes, and (iii) synthesizing ATP has always been our flagship. All three parameters are essential for mitochondria to participate in cellular metabolism due to the presence of transport proteins on the IMM.
Already at that time, the understanding of mitochondrial transport was changing. In fact, the concept that (i) the transporters mediating the uptake/export of substrates across mitochondria and (ii) the enzymes that metabolize them were subject to a sort of micro-compartmentation (see above, Section 2.1), which seemed to favor the occurrence of reactions in which transporters and enzymes were involved, was reaching an ever-increasing consensus.
In light of the considerations made above on the role of the mitochondrial membrane in controlling the flow of metabolites and for the presence of the micro-compartmentation occurring in IMS, and also because mitochondrial transport is related to and sometimes regulated by energy metabolism, we consider in this paper the studies on isolated mitochondria or mitochondria present in a cellular homogenate from tissue or cells—both respiring and phosphorylating—and not those, even valuable ones, that have as their object artificial systems consisting of isolated transport proteins and liposomes. This is because the closer the experimental system is to “nature,” the more reliable the conclusions are that research can allow us to draw. We verified this at our own expense.
Thus, in the 1980s, when LaNoue and Schoolwerth [14,15] wrote a sublime work collecting all the evidence of the existence of mitochondrial carriers discovered up to then, we began our research activity, essentially adopting the use of spectroscopic techniques (fluorescence and photometry) with which the reactions could be monitored both inside the mitochondria and in the extramitochondrial phase. To do this, isolated and coupled mitochondria from rat liver, heart and left ventricle, kidney, brain, and from cerebellar granule cells as well as from tumor cells and even from plants (durum wheat, potato tuber, Jerusalem artichoke), yeast (Saccharomyces cerevisiae), and others were used in order to investigate new aspects of metabolism and their permeability properties.
As mitochondria represent closed spaces in the cell, for energy metabolism to take place it is necessary that there be metabolite “traffic” through the mitochondrial membrane: some from the cytosol will enter the mitochondria to be degraded for energy purposes and/or to give to other metabolites; others will come out of the mitochondria and become substrates for some reactions in the cytosol. Obviously, the presence of enzymes in specific compartments is crucial and their catalytic activity is a function of known parameters distinctive of enzymatic reactions. Over the years, the discovery of the existence of multiple translocators involved in different metabolic pathways, made in light of the location of the enzymes that participate in them, have given answers to unsolved questions that remained unanswered in the study of energy metabolism and/or in didactic texts. Furthermore, since most of the translocated substrates or the newly synthesized metabolites produced by their intramitochondrial metabolism can be oxidized in the mitochondrial matrix, their transport can be monitored fluorimetrically as a change in the intramitochondrial cofactor red/ox state [16]. Similarly, as an additional tool for studying transport, a variety of “metabolite detecting systems” were developed, consisting of enzyme/s and cofactor/s designed to photometrically ascertain the appearance outside mitochondria of molecules derived from the mitochondrial metabolism of the substrate taken up. Given that the components of the detection system are mostly present in the cytosol and the mitochondrial metabolism is, at least in part, active, we believe that this approach is quite close to the physiological situation.
An important aspect to consider is the fact that each cell has its own specific metabolism with a specific role for its mitochondria. For example, if we consider the urea cycle, it takes place exclusively in the liver and involves enzymes located in two cellular compartments, i.e., carbamoyl phosphate synthetase, ornithine transcarbamylase, and glutamate dehydrogenase are mitochondrial proteins, whereas all the others are present exclusively in the cytosol. Therefore, it is necessary for ornithine (ORN) and glutamate to enter the mitochondria and for citrulline to come out. In the early 1970s, the existence of a carrier for ORN operating in uniport was demonstrated [17]; subsequently, following the “physiological” logic, the existence of another carrier that works in antiport with citrulline was proposed [18].
Since each organ has its own specific enzymatic kit and therefore its own biochemical specificity, the situation is supposed to be different in other mitochondria. However, unexpectedly, following the swelling of kidney mitochondria in ORN solutions, we observed a collapse of absorption at 546 nm, an index of mitochondrial swelling and evidence of the entry of the amino acid in question into mitochondria. There is a carrier for ORN in kidney mitochondria that is absent in the liver, which exchanges ORN with Pi with 1:1 stoichiometry [19,20]. Obviously, this is an electrophoretic exchange process, with the membrane potential being the energy used. In the kidney, the transport of ORN is physiologically correlated to its catabolism in the renal cell where the ornithine aminotransferase enzyme is responsible for its degradation.
In the light of the example reported, it can be deduced that it is not correct to transfer the specific characteristics of a metabolite transport occurring in mitochondria isolated from an organ or tissue to other mitochondria, i.e., of different origins. However, the hypothesis that what happens in the mitochondria of a certain origin may also occur in others of different origin, and therefore with different characteristics, is plausible.
Only some carriers are exempt from this limitation: those that are ubiquitous. Among them, besides the carrier of the Pi, surely the ANT mediating the ADP/ATP exchange is the first to be mentioned, in accordance with the fact that the primary function of mitochondria in the cell is the synthesis of ATP, the molecule working as the energy currency for cells.