**2.1. Voltage-dependent anion channel**

phosphorylation, and the basis of the aerobic oxidation is the citric acid cycle interconnection representing the final metabolic pathway of oxidation of all major nutrients to the respiratory chain where oxidation of reduced coenzymes results in ATP formation. The nutrient to be oxidized must transfer the mitochondria by means of the transporters, as pyruvate produced in glycolysis in the cytosol. After fatty acids released by hydrolysis of lipoproteins or triacylglycerols transport across the mitochondrial membranes, acetyl-CoA arising from β-oxidation enters the citric acid cycle. Similarly, amino acids or their catalytic products enter the citric acid cycle at different sites. The production of energy in mitochondria from various nutrients is controlled by the availability of the individual nutrients that a given organ or tissue can use. For example, in excess of carbohydrates, the energy is obtained from glycolysis rather than from fatty acids and amino acids. The needs for ATP produced in oxidative phosphorylation vary in different cell compartments, and therefore it is efficiently transported out of mitochondria. Through the activity of uncoupler proteins, mitochondria also regulate energy production in the form of heat. In addition to providing different forms of energy, mitochondria are involved in other important metabolic processes. In the excess of saccharides, the acetyl-CoA resulting from pyruvate is not used in the citric acid cycle but is transported to the cytosol for the synthesis of more energy-efficient reserve, triacylglycerols. When there is a lack of glucose, mitochondria provide the intermediates for gluconeogenesis, but also participate in the synthesis of various substances, such as urea, heme, and polyamines. Reactive oxygen and nitrogen species production and triggering the intrinsic apoptotic pathway are other significant functions. They synthesize proteins from their own DNA, but most mitochondrial proteins are encoded by nuclear genes. Mitochondria are the sole site for Fe-S cluster biogenesis, which is also the only fully conservative function. The diversity and importance of biochemical pathways taking place in the mitochondria require the access of substrates and transport products generated outside the mitochondria. In terms of ensuring the normal physiological functions of the mitochondria, it is therefore crucial to ensure the transfer of the substances through the mitochondrial membranes separating the organelle from the cytoplasm, thus allowing the course of these specialized metabolic processes. Therefore, the chapter is focused on the mitochondrial transport proteins, transporters of citric acid cycle intermediates, localized in mitochondrial outer and inner membrane, since their activities significantly affect the functions of mitochondria and subsequently functions of the given organ, tissues, and the whole organism.

252 Mitochondrial Diseases

**2. Mitochondrial outer membrane transport proteins**

The mitochondrial outer membrane (MOM) is characterized by higher lipid content than inner membrane and is permeable to small molecules such as sucrose, salts, adenine nucleotides, coenzyme A, and tRNA. It is not permeable to larger molecules such as inulin, polyglucose, cytochrome c, or albumin [1]. The outer mitochondrial membrane contains three integral membrane protein families. The entire translocation and insertion of nearly all newly synthesized proteins destined to the mitochondrial organelle is mediated through channels as part of larger protein complexes, translocase of the outer membrane (TOM complex), the sorting and assembly machinery (SAM) complex (followed by translocase of the inner membrane of mitochondria (TIM)) [2]. Channels, generally, are used to conduct ions and cycle between open and closed states, with The functions of VDAC related to four main aspects are controlling of transport of metabolites and ATP transport between mitochondria and cytoplasm, forming part of mitochondrial permeability transition pore; modulation of inner mitochondrial Ca2+ level through connection to endoplasmic reticulum calcium release channel IP3R with glucose-related protein 75 and through phosphorylation cluster sorting protein 2 (PACS2), regulating Bid of Bcl2 proapoptotic factor family-mediated apoptotic pathways; and regulation of intracellular redox substances [9]. VDAC is a way of transiting reactive oxygen species (ROS) from mitochondria to cytoplasm, though it reacts directly with the NO leading to decrease in permeability and inhibition of mitochondrial transition pore [10].

VDAC, mitochondrial porin, forms a barrel comprised of a transmembrane alpha helix and 13- and more transmembrane beta strands. Beta barrel encloses a channel large in diameter (~3 nm), which is permeable to molecules up to ~5 kDa in the open configuration [11]. *In vitro* studies have shown a conserved property of eukaryotic VDAC channels to adopt multiple conductance states [12]. In humans, three isoforms of VDAC (VDAC 1–3) located on chromosome 5, each of 30 kDa, are known [13]. VDAC1 and 2 have prototypic voltage gating, but VDAC2 also has a second discrete lower conductance and ion selective state. VDAC3 is not fully voltage-dependent [14], and unlike the previous two types, VDAC3 is evenly distributed [15].

The role of membrane potentials (Ψ) in the physiological regulation of VDAC conductance is considered with regard to appearance of Donnan potential across the outer membrane [16]. However, ambiguity is not confirmed due to the presence of charged macromolecules on both sides of outer membrane, and high ionic strength of intracellular environment decreasing Donnan potentials and causing closure of channels. Differences in pH across the outer membrane indicate the presence of Donnan potential of ~−40 mV, close to a gating potential for VDAC [17]. Positive and negative ΔΨ close VDAC symmetrically with half maximal closure at ±50 mV. In the open state, anions are favored over cations, but the selectivity is weak. In the closed state, VDAC becomes a cation selective pore of 1.8 nm in diameter that still conducts small cations, such as K+ , Na+ , and Ca2+, as well as Cl<sup>−</sup> , whose movement through VDAC collapses electrical potentials [16]. In most conductive, open state, VDAC shows significant preference especially for metabolic anions. The states of lower conductance reduce permeability to metabolic anions, thus greatly diminishing metabolite flux across the outer mitochondrial membrane [18]. The flux of charged metabolites does not significantly contribute to the membrane potential because it is confined by the internal mitochondrial membrane transport, which is even 2 orders of magnitude less than the flux of small ions through VDAC in the closed state. However, when VDAC closes, the transition of major anionic metabolites (as creatine phosphate, ATP, ADP, Pi, and respiratory substrates) is prevented [19]. A number of papers have been published that confirm the VDAC conductance regulation by several factors contributing to the explanation of mitochondria dysfunction and affection by the energy metabolism of cells.

Mitochondrial abnormalities cause cardiomyocyte death and myocardial degeneration, accompanied by inflammation and fibrosis, resulting in dilated cardiomyopathy and heart failure [35–38]. The cardiac-specific small heat-shock protein, αB-crystallin, was proven to rescue desmin-deficient heart failure and maintain mitochondrial functions through inhibition of mtPTP. In addition, similarly as tubulin, desmin affects mitochondrial bioenergetics

Nuclear Encoded Mitochondrial Proteins in Metabolite Transport and Oxidation Pathway…

http://dx.doi.org/10.5772/intechopen.72937

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Association of vimentin with mitochondria increases mitochondrial membrane potential and has an important function in controlling the production of ATP to various sites in the cytoplasm [40]. As a possible mechanism of action, the similarity of the mitochondrial binding site on vimentin with the domains targeting outer mitochondrial membrane is reported. The domains interact with the VDAC, increasing its permeability for several negatively charged compounds (such as pyruvate, succinate, ADP, etc.), thus compensating decreasing membrane potential effects of hexokinase [41], or tubulin. A study by Winter et al. [42] suggests that plectin 1b also plays an important role in regulating mitochondrial outer membrane per-

Mitochondrial localized kinases, hexokinase, and creatine kinase have been shown to regulate outer VDAC conductance [43–45]. There are three isoforms of hexokinases (fourth is glucokinase) known to mammals, whose role is to retain glucose in the cell by phosphorylation at position 6, thereby subsequently metabolizing in glycolysis and pentose phosphate pathways. Isoenzymes are of different subcellular localization: I and II are localized in the outer mitochondrial membrane reflecting controlling glycolysis, and III and IV in the nuclei and cytoplasm more reflecting the synthesis of glycogen and the pentose-phosphate pathway. Along with highly elevated levels of aerobic glycolysis (and suppression of mitochondrial respiration, Warburg effect) when compared to healthy tissue, high expression of hexokinase and more than doubled activity concurrently with VDAC closure were found in cancer cells [8, 46–48].

Both glucose phosphorylation reaction and hexokinase binding to VDAC have been found to pronounce protective effects against cell death [49]. A study by Azouylay-Zohar et al. [41] shows hexokinase-I acting through its N-terminal mitochondrial binding domain block conductance of rat liver mitochondrial VDAC and block opening of mtPTPs. An outer membrane potential generation (OMP) by hexokinase bound to VDAC allows electrical suppression of mitochondria and calcium extrusion from mitochondrial intermembrane space (IMS). Interestingly, a significant limitation on the permeability of the MOM is the reaction velocity of hexokinase binding to VDAC that is only mild per se but the OMP becomes high enough to prevent release of ADP from mitochondrial inner membrane (MIM), which should lead to inhibition of the hexokinase reaction. Yet, OMP values directly depend on percentage bounds

through interaction with VDAC and ATP synthase [39].

meability to ADP and ATP through VDAC.

**4. VDAC regulation by mitochondrial kinases**

**3.3. Vimentin and plectin**

**4.1. Hexokinases**
