**9. Aspartate/glutamate carrier**

Glutamate can also enter mitochondria through aspartate/glutamate carrier (AGC1 and 2 isoforms, known as aralar and citrin) combining the input of glutamate to the release of aspartate [185]. The export of aspartate is favored in energized mitochondria. Moreover, in increased cytosolic calcium concentration, respiration is strongly increased associated with the reduction of mitochondrial membrane potential [185]. A decrease in ROS production could be expected given the opposite relationship between the mitochondrial membrane potential and ROS production [186]. Another attribute contributing to this effect is glutamate entry through AGC1 (SLC25A12) in cotransport with proton. The loss of membrane potential is compensated by the extrusion of four protons by the respiratory chain when one molecule of glutamate is processed through the citric acid cycle generating two molecules of NADH [187]. AGC together with the OGC plays a crucial role in the transport of NADH from cytosol to the mitochondria as a part of malate-aspartate shuttle [188]. Therefore, AGC1 and AGC2 (SLC25A13) are expressed in tissues differently according to their demands for maintenance of the redox balance between anaerobic and aerobic glycolysis. An interesting finding is that expression of AGC1 and AGC2 is almost completely restricted to neurons and photoreceptor cells [180, 189], in contrast to GC1 expressed in astrocytes. Cytosolic Ca2+ has a direct role in the regulation of *AGC1* gene expression via cAMP response element-binding protein in neuronal cells, underlining the key role of AGC1 in the central nervous system by upregulation in neuronal differentiation and downregulation in neuroinflammation [190]. AGC1 is also highly expressed in skeletal and heart muscle [191]. Upregulation of both isoforms was found in several cancers, which is also related to the change in glycolytic metabolism [187].

#### **9.1. Ornithine carriers**

Translocation of the ornithine and related substrates is mediated by mitochondrial ornithine carrier (ORC). The physiological importance of this carrier reclines on urea production, delivery-rate control of arginine, and interferential formation of NO, agmatine, creatine, glutamine, glutamate polyamines, and proline [192]. The human isoforms ORC1 (SLC25A15), ORC2 (SLC25A2), and ORC3 (SLC25A29) [193, 194] provide transport by exchange or by exchange for H+ but differ in substrate transport rates, substrate specificity, and tissue expression. They all facilitate passage of L-ornithine, L-lysine, and L-arginine. The ORC1 prefers transport of amino acid substrates with shorter and noncyclized side chains. It does not enable transport of L-homoarginine, D-ornithine, D-histidine, and D-arginine. The ORC2 transports all substrates with the same efficiency (L,D-forms of ornithine, lysine, histidine, arginine, and L-citrulline, L-homoarginine). The ORC3 enables transport of L-forms with longer side chains across MIM, e.g., lysine, arginine, and histidine [192]. The isoform expresses lower affinity to ornithine and does not transport citrulline [194].

Activity of ORC1 and 2 is enhanced by Pi , malate, and dicarboxylates and inhibited by pyridoxal 5′-phosphate (PLP), mercurials, spermine, and spermidine. The affinity of ORC2 to lysine and arginine is lower and to ornithine and citrulline is higher in comparison to ORC1. Moreover, ORC2 has been reported to be about three times less active than ORC1. The dispositions are also related to protein expression. The ORC1 is expressed in most tissues, with the highest levels in the liver, pancreas, lungs, kidney, and testis, unlike the ORC2 being more restricted to these organs [193]. ORC3 is expressed in heart, brain, liver, and kidney and is induced after partial hepatectomy or fasting [195, 196]. The import of arginine, lysine, and histidine allows for protein synthesis in mitochondria and that for ornithine enables degradation of arginine surplus. Transfer of ornithine out of the mitochondria allows for synthesis of polyamines reversibly inhibiting ORC activity. Ornithine is synthesized in mitochondria from glutamate in tissues with low arginase activity (except for the liver), from glutamine in intestinal mitochondria or when deficient in the diet [193]. Considering ornithine and citrulline transport efficiency and level of protein expression in the liver, the ORC1 isoform is of highest importance in urea cycle continuance [193].

Mutation in the gene encoding ORC1 isoform (localized on 13q14.1 chromosome) causes hyperornithinemia-hyperammonemia-homocitrullinuria (HHH syndrome), characterized by early-onset neurological deficits. Hyperammonemia results from impaired urea cycle due to ORC1 malfunction. Ornithine accumulates in the cytosol leading to hyperornithinemia and increases polyamine synthesis. Carbamoyl phosphate condensates with lysine in the absence of ornithine inside the mitochondria, leading to homocitrullinuria, or enters pyrimidine synthesis, thus increasing excretion of orotic acid and uracil [97, 197]. Overexpression of ORC2 might only partially compensate defective function of ORC1 due to lower affinity for ornithine and citrulline [196, 198]. ORC3 has not been found to compensate lack of ORC1 function but is probably responsible for lysine transport in patients with HHH syndrome [194].
