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
Having all this information available and making use of our skills, our goal was to measure ANT activity in conditions very close to physiological ones.
The challenge in measuring transport was essentially represented by the method to be used to follow the entry of a metabolite into the mitochondria or its efflux from them.
It is intuitive that the undisputed guarantee of entry was provided by isotopic methods, for which the radioactivity of the marked metabolite was traced: These were the direct methods. In this regard, the old review by Palmieri and Klingenberg, published in Methods in Enzymology in 1979 [21], is exhaustive and treated in the smallest detail. These methods allowed us to study the uptake of a radioactive substrate added outside the mitochondria and/or the efflux of a marked substrate (with which the mitochondria had been loaded) induced by the addition of an unlabeled substrate to the mitochondrial suspension. The method itself, apart from being expensive, requires special laboratories with equipment dedicated only to isotope studies. In using this method, it is crucial to ensure that you measure the substrate that has actually entered the mitochondria, distinguishing it from what remains in the medium and/or is possibly non-specifically bound to the mitochondria. This risk was counteracted with the subsequent introduction of the inhibitor stop technique (for refs see [22]). A very important note characterizing this method: the inhibitor used must be impermeable to the mitochondria, act quickly, and not be harmful to the mitochondria.
We were young at that time and two technicians, Nicola and Andrea Cataldo, supported us with the technique. They, more than the laboratory technicians, were real miniature architects: Using Plexiglas, a square, and a chisel they were able to build an ad hoc system that included a Plexiglas tray in which—at 0 °C, i.e., in the presence of water and ice—aluminum blocks with 12 housings for Eppendorf tubes were immersed.
The uptake reaction took place following the simultaneous addition of the radioactive substrate and/or the inhibitor through 12 metal rods on which the drop of solution to be added was placed, supported by a wooden bracket. That was art!
The direct methods were contrasted by the “indirect” ones that allowed us to follow the transport of a metabolite through a reaction associated with the appearance of the same in the mitochondria or in the extramitochondrial phase. The substantial difference between the two methods, apart from the fact that the indirect method is a “continuous” method because the measurement is performed by continuously recording the substrate uptake by absorbance or fluorescence variations, consists of the fact that in the first case, i.e., the direct one, any mitochondrial activity is blocked to prevent metabolism of the substrate entering the mitochondria; in the second case, the indirect one, metabolism is largely possible.
About this very important note, for many years, fumarate was considered a substrate unable to enter mitochondria. Yet fumarase has mitochondrial localization. What was the cause of this inconsistency? Chappell and Haarhoff [23] came to the conclusion that fumarate is a non-penetrant metabolite, as they did not observed an accumulation of radioactivity in the mitochondria. This was caused by the fact that in the matrix, the labeled fumarate is transformed—via fumarase—into malate, which, in turn, exits from mitochondria in exchange with fumarate itself [24], thus preventing its accumulation [22].
Other advantages of the indirect method include the speed of execution of the measurement, low costs, and also the requirement of small amounts of mitochondria (even lower than 0.1 mg protein). Taking advantage of these “advantages,” we were able, in the following years, to miniaturize the working system by carrying out measurements on mitochondria present in a homogenate of cells or tissue.
However, it is imperative that if you want to measure the transport rate using spectrophotometric or fluorimetric techniques, the transport (not the reaction (s) dependent on it) must limit the rate with which the observed parameter changes. In fact, in a typical experiment, the measured parameter can depend on three steps: (i) transport, (ii) an intramitochondrial reaction, and (iii) an extramitochondrial reaction. It is therefore essential that the step that regulates the rate—that is, limits it—is transport.
Sometimes detergent, which breaks down the permeability barrier, causing an increase in the process rate, gives us information that the limiting step is actually the transport through the membrane. However, it is not always possible to apply this ploy. Indeed, sometimes the solubilization of the membrane operated by the detergent causes inactivation of enzymes involved in the process and/or, for example, in the case of measuring the ADP/ATP exchange rate, the collapse of the ΔΨ on which the carrier’s activity depends. Thus, in order to ascertain which step limits the rate of appearance of ATP as synthesized via OXPHOS, i.e., to distinguish between the rate of ADP/ATP exchange via ANT and the rate of ATP synthesis via ATP synthase, a control flux analysis (the control strength criterion) may be applied, as in [25].
According this “control strength criterion” (see [14,15])—based on the sensitivity of the global process to a transport inhibitor—if the reciprocal of the measured rate is plotted as a function of the non-penetrant inhibitor concentration, the resulting Dixon plot extrapolated to zero concentration provides a measure of transport in the absence of an inhibitor. Thus, the coincidence of the experimental point measured in the absence of an inhibitor with the intercept shows that you are measuring the rate of the transport (see [25]). Anyway, it must be ensured that the detecting system activity does not limit the measured rate.
It was said above that mitochondria participate in various cellular processes and that all this is made possible by the different localization of the enzymes, inside or outside mitochondria. The fundamental reason explains the movement of metabolites back and forth within mitochondria where they are needed. It follows that the cytoplasm and the internal compartments of the mitochondria are put into communication through carrier-mediated transport processes.
The discovery in the 1960s of the existence of protein-mediated transport processes in the mitochondria was due to the English biochemist Brian Chappell. His truly groundbreaking work on the permeability properties of isolated mitochondria, performed formerly in Cambridge and later in Bristol, was highly original and widely influential in the field, earning him an international reputation. His group was able to make continuous indirect measurements of the entry of metabolic substrates into isolated mitochondria by observing the osmotic behavior of mitochondria in a solution containing the specific ion. This work was complemented by direct measurements of substrate and ion transport using radiolabeled substrates and also ion-selective electrodes. The result was the discovery and characterization of some eight specific substrate transport systems in the mitochondrial membrane, a concept that was completely new at the time and had great implications for the understanding of cell metabolism and ATP synthesis. The main carrier systems were first characterized in Chappell’s lab, the earliest ones being the phosphate/OH− exchange system (the phosphate transporter) and the ADP/ATP-exchange system, i.e., ANT. Both these two carrier systems were soon recognized by Mitchell as an essential component of the chemiosmotic circuit.
In the first, the enzyme catalyzes the net transfer of H3PO4 across the membrane in a neutral process: Since the predominant ionic species present at neutral pH is H2PO4−, the mechanism must involve either the exchange of H2PO4− for OH− or the cotransport of H2PO4− with H+ [26].
In the other, i.e., ANT, which is highly abundant, constituting about 14% of the proteins in the IMM, the carrier mediates the exchange of ATP for ADP. Most importantly, the flows of ATP and ADP are coupled. It is a wasteful exchange from an energy point of view: about one-fourth of the energy produced by the flow of reducing equivalents along the mRC is consumed to regenerate the continuous membrane potential modified by the exchange of adenine nucleotides. In fact, at physiological pH, ADP has three negative charges and ATP four. The exchange reaction is therefore electrogenic and the directionality of transport is imposed by the concentration gradients of the substrates and by the electrical component (ΔΨ) of the proton gradient so that the entry of ADP and exit of ATP are favored. The freshly coined ATP is then dispatched from the mitochondrion to the host cell to maintain high cytosolic ATP concentrations for reactions that require energy [27]. Wherever the cell needs it, energy is released from ATP by stripping it of a phosphate. This leaves behind ADP and phosphate that within the mitochondrion can be recharged to ATP again. What is exquisitely elaborate is how mitochondria put this plan into action.
It was after 1982, i.e., when the first amino acid sequence, that of the ADP/ATP carrier [28], was identified, that it became clear that all carriers belong to the same “family” by virtue of common characteristics of their molecules.
After the first communications on the discovery of the ADP/ATP exchange [22], a full characterization of the ADP/ATP transport in mitochondria was given in two detailed key papers [28,29] by the Klingenberg group. All these studies were performed on rat liver mitochondria, which were the classical mitochondria for studying OXPHOS.
The main features to be briefly mentioned [22] are:(1)ADP is exchanged against intramitochondrial ATP with a one-to-one stoichiometry when mitochondria are actively respiring;(2)The only physiological substrates are ADP and ATP; surprisingly in their free forms, i.e., Mg-ADP and Mg-ATP, they are not recognized by the carrier;(3)Very high selectivity for the substrates: It does not transport AMP;(4)The highly charged and very hydrophilic nature of the large substrates;(5)The strong regulation by high electrical forces of the mitochondrial membrane potential of the highly charged substrates ADP3− and ATP4−;(6)The kinetic parameters of the carrier are consistent with the mitochondrial ATP production and the cell nucleotide concentrations under physiological conditions;(7)The high levels of the underlying carrier in mitochondria to cope with the high demand for ATP in most aerobic cells;(8)The unique existence of highly specific inhibitors, as represented first by atractyloside (ATR) and then by other atractylogenin glucosides, in particular by carboxyatractyloside (CAT), greatly facilitating the characterization of the transport sites;(9)Another type inhibitor was represented by bongkrekic acid (BKA), which became a key element to elucidating the transport mechanism of transport of the ADP/ATP carrier at the molecular level;(10)Stabilized by these inhibitors toward detergents, in particular by CAT and thanks to its high levels in heart mitochondria, the ANT protein became the first carrier protein to be isolated in the native state;(11)The translocation mechanism of ANT was further elucidated and the involved conformation changes were demonstrated;(12)The first rational reconstitution of a carrier into vesicles was achieved, with the isolated ANT, and the transport parameters were determined in detail, in particular the differences between ADP and ATP and the control by
the membrane potential and lastly;(13)The complete primary structure was determined by amino acid sequencing with large amounts of purified ANT protein.,. The elucidation of the complete primary structures of ANT in 1982 [28] and UCP1 in 1985 [29] by Klingenberg’s group, together with the sequence of the phosphate carrier, led to the initial definition of the homologous family of mitochondrial solute transporters.
The concept of a mitochondrial carrier for adenine nucleotides arose in the 1950s–1960s.
In a pioneering study from 1953, Siekevitz and Potter [30] established that two distinct pools of adenine nucleotides operate cooperatively in the OXPHOS pathway, one located in the mitochondrial matrix and the other in the cytosol. Soon after, Pressman [31] reported that “an extremely dynamic interchange of nucleotides” occurs between the two pools.
In 1962, Bruni et al. [32] and Vignais et al. [33] reported that ATR, a poisonous natural drug, specifically inhibits the OXPHOS of extramitochondrial, but not intramitochondrial, ADP, results that were also confirmed by several others groups [34]. The obvious conclusion was that ATR inhibits the entry of ADP into mitochondria, and consequently, the existence of a transporter for ADP was hypothesized.
In 1970, Henderson and Lardy [35] introduced BKA, a complex fatty acid derivative, as an ADP/ATP carrier inhibitor.
From that time to 1982, i.e., when the protein sequence of the ADP/ATP carrier was determined (see above), a large quantity of results dealing with the properties/characteristics of the carrier were collected.
A paper by the mitochondrial carrier research group at the MRC MBU in Cambridge, UK, titled “The molecular mechanism of transport by the mitochondrial ADP/ATP carrier” and published two years ago by the journal Cell [36], represents a major step forward in understanding how the mitochondrial ADP/ATP carrier works at the molecular level. The carrier plays a vitally important role of transporting ADP into mitochondria and newly synthesized ATP out, replenishing the cell with energy. This process is vital to keep us alive, every second of our lives, for all of our lives, and makes us understand how mutations as well as effectors of various types can affect the function of this protein, resulting in a range of neuromuscular, metabolic, and developmental diseases.
Since we have only a small amount of ATP in our body, we need to remake it from the spent product ADP and phosphate using an enzyme complex, called ATP synthase, which is located in mitochondria. In this way, every molecule of ATP is recycled roughly 1300 times a day. For ADP to reach the enzyme, and for the product ATP to refuel the cell, each molecule has to cross an impermeable lipid membrane that surrounds the mitochondria.
The mitochondrial ADP/ATP carrier is involved in the transport of ADP into the mitochondria and of ATP out.
The ADP/ATP exchange follows Michaelis–Menten kinetics, with a Km of 1–10 μM for ADP and a Km of 1–150 μM for ATP for rat liver ANT [22]. The activity of this electrogenic transport is moderate, catalyzing the transport of 1500–2000 molecules per min. This relatively low specific activity is compensated by the high density of ANT, which represents about 10% of the total IM proteins.
ANT proteins have a characteristic structure with a tripartite repeat of ~100 amino acids and a total of six transmembrane domains that cluster in a generally cylindrical shape [37]. From a topological point of view, ANT proteins have carboxy- and amino-terminals that face the intramembrane space, with two exposed cytosolic loops (C1–C2), whereas the matrix side of ANT is composed of three hydrophilic loops (M1–M3) that are available for protein interaction and modification [37].
The concept of “induced transition fit” for carrier catalysis, by analogy with enzyme kinetics, was introduced by Klingenberg [38]. Briefly, he proposed a transition state between c- and m-states, according to which substrates, initially weakly binding to the c- or m-state, induce conformational changes that lead to tighter binding and the formation of the transition state. The carrier-substrate binding energy lowers the activation barrier for the formation of the transition state, then catalyzing transport.