**4.1. Hexokinases**

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 formed, glucose concentration, metabolic conditions, and the presence of tubulin-like effectors increasing VDAC voltage sensitivity [4]. Prevention of ATP production together with higher hexokinase activity favors glycolysis. Research has shown that cancer cells respire in the absence of glucose, suggesting that VDAC blocking is not absolute, and the respiration increases with the addition of glucose. Increase in respiration is attributed to the formation of ADP in the hexokinase-catalyzed reaction and subsequent stimulation of oxidative phosphorylation. Since not all VDACs are closed, preferential access to mitochondrial ATP may be allowed for the hexokinase reaction [8]. Another principle applies if ATP from cytosol is readily available for hexokinase, leading to so-called turbo effect of uncontrolled glycolysis activation and development of metabolic burst [4].

without mixing with bulk [59]. In IMS, MtCK renders a high affinity to cardiolipin and other anionic phospholipids forming a cross-link of two peripheral mitochondrial membranes [60] and to ANT, thus forming a complex of MtCK-VDAC-ANT and cardiolipin. The MtCK-VDAC association is enhanced at physiological calcium concentrations [61]. MtCK associates only with inner membrane and ANT in the cristae space. MtCK preferentially uses mitochondrial ATP that is exported via ANT to phosphorylate creatine, which has a higher diffusion rate in comparison to ATP, thus providing spatial energy shuttle. The locally produced ADP is immediately reimported into the mitochondrial matrix space via ANT, and phosphocreatine is then released into the cytosol via VDAC [62]. It keeps maintaining a relatively low [ATP]/[ADP] ratio in the mitochondrial matrix to stimulate oxidative phosphorylation. The degree of such metabolite channeling varies among different tissues, species, and developmental states [63]. Changed functionality of the CK essentially leads to changes in energy flows as well as calcium homeostasis, leading in particular to changes in muscle activity. Up to 40% of the cellular volume of the heart consists of mitochondria, with sMtCK activity being the highest among all tissues and representing up to 25% of CK activities [64]. Reducing its activity is the cause of congestive heart failure [65]. As is well reviewed in Schlattner et al. [62] with impairment of the CK system, dilated cardiomyopathy, hypertrophy, and heart failure were found in animal models. In addition, the protective effect of creatine supplementation and hence the enhancement of the CK system have been found to be beneficial in human Duchenne muscular dystrophy, mitochondrial cytopathies, and phosphorylase deficiency and in animal models of amyotrophic lateral sclerosis, Huntington's disease, Parkinsonism, and brain ischemia. However, as found in the study by Qian et al. [66], overexpression of uMtCK increased survival ability of cancer cells and downregulation of mitochondrial apoptotic pathway proteins.

Nuclear Encoded Mitochondrial Proteins in Metabolite Transport and Oxidation Pathway…

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

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**5. Some other factors affecting VDAC conductance**

kinase ε, NADH, Ca2+, ATP, and glutamate [68].

promotes VDAC opening [70].

homeostasis to ensure cell survival [72].

Besides the aforementioned intermediate filament proteins and mitochondrial kinases, the conductivity of VDAC as described by Lemasters et al. [67] is regulated by a number of other factors, for example, Bcl-2 family members, protein kinase A, glycogen synthase 3β, protein C

Protein kinase A phosphorylates VDAC and thus increases its sensitivity to tubulin and decreases VDAC conductance [69]. Glycogen synthase 3β–mediated VDAC phosphorylation

The Bcl-2 protein family belongs to the key factors in the regulation of apoptosis , modulation of Ca2+ and signal transduction pathway. The Bcl-2 protein as anti-apoptotic protein prevents the release of cytochrome c and the activity of caspase. Bax, the main regulator of Bcl-2 activity, can interact with VDAC to increase VDAC aperture and increases mitochondrial permeability, promoting apoptosis [71]. The proper proportion of Bax and Bcl-2 maintains the cell

Lemasters et al. [67] introduced a concept of aldehyde-dependent VDAC closure, mitochondrial uncoupling, and disruption of normal mitochondria functioning resulting from ethanol

The product hexokinase-catalyzed reaction, glucose-6-phosphate, was found to potentiate ATP release from mitochondria with the recovery of normal metabolism, and substrate replacement, even increased by induction of release of hexokinase and glucokinase from bound to VDAC [4, 41, 50]. Therefore, the effect of hexokinase dissociation from VDAC is the subject of many studies dealing with the development of potent chemotherapy of cancer diseases.

Most interestingly, there is a view of influencing metabolism of the mitochondria by blocking VDAC after binding of glucokinase in pancreatic beta cells. The glucokinase acts as a glucose sensor to maintain glucose homeostasis also in neurons, pituitary, and endocrine K and L cells [51], as its *K*m of approximately 5 mM matches the set point of blood glucose [52]. An alternatively spliced glucokinase with an additional N-terminal sequence that promotes glucokinase binding to both mitochondria and secretory granules is expressed in pancreatic beta cells [53]. Glucose-6-phosphate formed after glucose enters beta cells acts to dissociate glucokinase from mitochondria and open VDAC permitting mitochondrial uptake of ADP, Pi and respiratory substrates, formation of ATP, and release of ATP into the cytosol. Increased cytosolic ATP/ADP then inhibits KATP channels, which causes plasmalemmal depolarization, activation of Ca2+ channels, and Ca2+-dependent exocytosis of insulin granules [8]. Then, respiratory substrates that require the passage through VDAC, such as pyruvate, lactate, and long-chain fatty acids, do not trigger insulin synthesis directly. But their permeable substrates without the need for VDAC transport such as short-chain fatty acids or methylpyruvate are capable of insulin synthesis induction [54]. Ahmed et al. [55] found that under glucotoxic conditions upregulation of VDAC1 expression occurs initiating a mitochondrial death cascade and beta cell dysfunction.

#### **4.2. Creatine kinases**

Two creatine kinase (CK) isoforms are expressed in vertebrate tissues, namely dimeric cytosolic and octameric mitochondrial located in the peripheral intermembrane space and the cristae MIM (MtCK) [56]. In addition, there are tissue-specific mitochondrial isoforms in the sarcomers of striated muscles (sMtCK), and in most other tissues, they are present as ubiquitous MtCK (uMtCK) [57]. Similar to hexokinase, creatine kinase acts as energy sensor and mediates antiapoptotic effect through VDAC-ANT complexes with preferential use of mitochondrial ATP [41, 58].

Isoenzymes are associated to subcellular structures, forming microcompartments that facilitate a functional coupling, e.g., a direct exchange of ADP and ATP between the association partners without mixing with bulk [59]. In IMS, MtCK renders a high affinity to cardiolipin and other anionic phospholipids forming a cross-link of two peripheral mitochondrial membranes [60] and to ANT, thus forming a complex of MtCK-VDAC-ANT and cardiolipin. The MtCK-VDAC association is enhanced at physiological calcium concentrations [61]. MtCK associates only with inner membrane and ANT in the cristae space. MtCK preferentially uses mitochondrial ATP that is exported via ANT to phosphorylate creatine, which has a higher diffusion rate in comparison to ATP, thus providing spatial energy shuttle. The locally produced ADP is immediately reimported into the mitochondrial matrix space via ANT, and phosphocreatine is then released into the cytosol via VDAC [62]. It keeps maintaining a relatively low [ATP]/[ADP] ratio in the mitochondrial matrix to stimulate oxidative phosphorylation. The degree of such metabolite channeling varies among different tissues, species, and developmental states [63].

Changed functionality of the CK essentially leads to changes in energy flows as well as calcium homeostasis, leading in particular to changes in muscle activity. Up to 40% of the cellular volume of the heart consists of mitochondria, with sMtCK activity being the highest among all tissues and representing up to 25% of CK activities [64]. Reducing its activity is the cause of congestive heart failure [65]. As is well reviewed in Schlattner et al. [62] with impairment of the CK system, dilated cardiomyopathy, hypertrophy, and heart failure were found in animal models. In addition, the protective effect of creatine supplementation and hence the enhancement of the CK system have been found to be beneficial in human Duchenne muscular dystrophy, mitochondrial cytopathies, and phosphorylase deficiency and in animal models of amyotrophic lateral sclerosis, Huntington's disease, Parkinsonism, and brain ischemia. However, as found in the study by Qian et al. [66], overexpression of uMtCK increased survival ability of cancer cells and downregulation of mitochondrial apoptotic pathway proteins.
