Aspartate/glutamate Carrier (glutamate + carrier)

Distribution by Scientific Domains


Selected Abstracts


Mass spectrometrical analysis of the mitochondrial carrier Aralar1 from mouse hippocampus

ELECTROPHORESIS, Issue 11 2010
Seok Heo
Abstract Aralar1 is a mitochondrial aspartate/glutamate carrier and a key component of the malate,aspartate NADH shuttle system. An analytical approach to obtain high sequence coverage is important to predict conformation, identify splice variants and binding partners or generate specific antibodies. Moreover, a method allowing determination of Aralar1 from brain samples is a prerequisite for evaluating a biological role. Sucrose gradient ultracentrifugation was applied to enrich native membrane protein fractions and these were run on blue-native PAGE, followed by multidimensional gel electrophoresis. Spots from the third-dimensional gel electrophoresis were in-gel digested with trypsin, chymotrypsin and subtilisin. Subsequently, peptides were analyzed by nano-ESI-LC-MS/MS using collision-induced dissociation and electron transfer dissociation modes. ModiroÔ v1.1 along with Mascot v2.2 software was used for data handling. Aralar1 could be clearly separated, unambiguously identified and characterized from protein extracts of mouse hippocampus by the use of the multidimensional gel electrophoretic steps. The combined sequence coverage of Aralar1 from trypsin, chymotrypsin and subtilisin digestions was 99.85%. The results provide the basis for future studies of Aralar1 at the protein chemical rather than at the immunochemical level in the brain and thus challenge and enable determination of Aralar1 levels required for understanding biological functions in health and disease. [source]


Expression of the aspartate/glutamate mitochondrial carriers aralar1 and citrin during development and in adult rat tissues

FEBS JOURNAL, Issue 13 2002
Araceli Del Arco
Aralar1 and citrin are members of the subfamily of calcium-binding mitochondrial carriers and correspond to two isoforms of the mitochondrial aspartate/glutamate carrier (AGC). These proteins are activated by Ca2+ acting on the external side of the inner mitochondrial membrane. Although it is known that aralar1 is expressed mainly in skeletal muscle, heart and brain, whereas citrin is present in liver, kidney and heart, the precise tissue distribution of the two proteins in embryonic and adult tissues is largely unknown. We investigated the pattern of expression of aralar1 and citrin in murine embryonic and adult tissues at the mRNA and protein levels. In situ hybridization analysis indicates that both isoforms are expressed strongly in the branchial arches, dermomyotome, limb and tail buds at early embryonic stages. However, citrin was more abundant in the ectodermal components of these structures whereas aralarl had a predominantly mesenchymal localization. The strong expression of citrin in the liver was acquired postnatally, whereas the characteristic expression of aralar1 in skeletal muscle was detected at E18 and that in the heart began early in development (E11) and was preferentially localized to auricular myocardium in late embryonic stages. Aralar1 was also expressed in bone marrow, T-lymphocytes and macrophages, including Kupffer cells in the liver, indicating that this is the major AGC isoform present in the hematopoietic system. Both aralar1 and citrin were expressed in fetal gut and adult stomach, ovary, testis, and pancreas, but only aralar1 is enriched in lung and insulin-secreting ,,cells. These results show that aralar1 is expressed in many more tissues than originally believed and is absent from hepatocytes, where citrin is the only AGC isoform present. This explains why citrin deficiency in humans (type II citrullinemia) only affects the liver and suggests that aralar1 may compensate for the lack of citrin in other tissues. [source]


Energy sources for glutamate neurotransmission in the retina: absence of the aspartate/glutamate carrier produces reliance on glycolysis in glia

JOURNAL OF NEUROCHEMISTRY, Issue 1 2007
Y. Xu
Abstract The mitochondrial transporter, the aspartate/glutamate carrier (AGC), is a necessary component of the malate/aspartate cycle, which promotes the transfer into mitochondria of reducing equivalents generated in the cytosol during glycolysis. Without transfer of cytosolic reducing equivalents into mitochondria, neither glucose nor lactate can be completely oxidized. In the present study, immunohistochemistry was used to demonstrate the absence of AGC from retinal glia (Müller cells), but its presence in neurons and photoreceptor cells. To determine the influence of the absence of AGC on sources of ATP for glutamate neurotransmission, neurotransmission was estimated in both light- and dark-adapted retinas by measuring flux through the glutamate/glutamine cycle and the effect of light on ATP-generating reactions. Neurotransmission was 80% faster in the dark as expected, because photoreceptors become depolarized in the dark and this depolarization induces release of excitatory glutamate neurotransmitter. Oxidation of [U- 14C]glucose, [1- 14C]lactate, and [1- 14C]pyruvate in light- and dark-adapted excised retinas was estimated by collecting 14CO2. Neither glucose nor lactate oxidation that require participation of the malate/aspartate shuttle increased in the dark, but pyruvate oxidation that does not require the malate/aspartate shuttle increased to 36% in the dark. Aerobic glycolysis was estimated by measuring the rate of lactate appearance. Glycolysis was 37% faster in the dark. It appears that in the retina, ATP consumed during glutamatergic neurotransmission is replenished by ATP generated glycolytically within the retinal Müller cells and that oxidation of glucose within the Müller cells does not occur or occurs only slowly. [source]


Mitochondrial transport proteins of the brain

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 15 2007
D.A. Berkich
Abstract In this study, cellular distribution and activity of glutamate and ,-aminobutyric acid (GABA) transport as well as oxoglutarate transport across brain mitochondrial membranes were investigated. A goal was to establish cell-type-specific expression of key transporters and enzymes involved in neurotransmitter metabolism in order to estimate neurotransmitter and metabolite traffic between neurons and astrocytes. Two methods were used to isolate brain mitochondria. One method excludes synaptosomes and the organelles may therefore be enriched in astrocytic mitochondria. The other method isolates mitochondria derived from all regions of the brain. Immunological and enzymatic methods were used to measure enzymes and carriers in the different preparations, in addition to studying transport kinetics. Immunohistochemistry was also employed using brain slices to confirm cell type specificity of enzymes and carriers. The data suggest that the aspartate/glutamate carriers (AGC) are expressed predominantly in neurons, not astrocytes, and that one of two glutamate/hydroxyl carriers is expressed predominantly in astrocytes. The GABA carrier and the oxoglutarate carrier appear to be equally distributed in astrocytes and neurons. As expected, pyruvate carboxylase and branched-chain aminotransferase were predominantly astrocytic. Insofar as the aspartate/glutamate exchange carriers are required for the malate/aspartate shuttle and for reoxidation of cytosolic NADH, the data suggest a compartmentation of glucose metabolism in which astrocytes catalyze glycolytic conversion of glucose to lactate, whereas neurons are capable of oxidizing both lactate and glucose to CO2 + H2O. © 2007 Wiley-Liss, Inc. [source]


Post-translational regulation of EAAT2 function by co-expressed ubiquitin ligase Nedd4-2 is impacted by SGK kinases

JOURNAL OF NEUROCHEMISTRY, Issue 4 2006
Christoph Boehmer
Abstract The human excitatory amino acid transporter (EAAT)2 is the major glutamate carrier in the mammalian CNS. Defective expression of the transporter results in neuroexcitotoxicity that may contribute to neuronal disorders such as amyotrophic lateral sclerosis (ALS). The serum and glucocorticoid inducible kinase (SGK) 1 is expressed in the brain and is known to interact with the ubiquitin ligase Nedd4-2 to modulate membrane transporters and ion channels. The present study aimed to investigate whether SGK isoforms and the related kinase, protein kinase B (PKB), regulate EAAT2. Expression studies in Xenopus oocytes demonstrated that glutamate-induced inward current (IGLU) was stimulated by co-expression of SGK1, SGK2, SGK3 or PKB. IGLU is virtually abolished by Nedd4-2, an effect abrogated by additional co-expression of either kinase. The kinases diminish the effect through Nedd4-2 phosphorylation without altering Nedd4-2 protein abundance. SGKs increase the transporter maximal velocity without significantly affecting substrate affinity. Similar to glutamate-induced currents, [3H] glutamate uptake and cell surface abundance of the transporter were increased by the SGK isoforms and down-regulated by the ubiquitin ligase Nedd4-2. In conclusion, all three SGK isoforms and PKB increase EAAT2 activity and plasma membrane expression and thus, may participate in the regulation of neuroexcitability. [source]