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Physical and functional interaction of NCX1 and EAAC1 transporters leading to glutamate-enhanced ATP production in brain mitochondria.

Magi S, Lariccia V, Castaldo P, Arcangeli S, Nasti AA, Giordano A, Amoroso S - PLoS ONE (2012)

Bottom Line: Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides.The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart.The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Public Health, University Politecnica delle Marche, Ancona, Italy.

ABSTRACT
Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na(+)-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.

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Glutamate-induced ATP synthesis in rat liver mitochondria is independent on EAAT and NCX.(A) Mitochondria isolated from rat liver express EAAC1 and GLT1, but not GLAST. The panel shows three different western blot experiments performed using GLAST, GLT1 and EAAC1 antibodies on whole lysate or mitochondrial (mt) protein extracts from rat liver. Blots are representative of 3 different experiments. (B) Mitochondria isolated from rat liver do not express NCX1, NCX2 or NCX3. The panel shows 3 different western blot experiments, each representative of 2 sets of experiments performed with protein extracts from rat liver mitochondria blotted with NCX1, NCX2 and NCX3 antibodies. None of the NCX isoforms were expressed in rat liver mitochondria. In A and B, protein extracts from rat hippocampus or cortex were used as a positive control. In each IP lane, the lower band at around 50 KDa represents the immunoglobulin. (C, D) Glutamate-induced ATP synthesis in rat liver mitochondria is unaffected by EAAT or NCX pharmacological blockade with DL-TBOA and CGP-37157, respectively. The bar graphs show ATP production in the presence of 1 mM glutamate (black bars) or vehicle (white bars) for 1 h with or without DL-TBOA (C) or CGP-37157 (D). Each bar represents the mean ± SEM of data from 8 different experimental sessions. ATP data were normalized to the protein content of the different mitochondrial preparations. *p<0.05 vs control.
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pone-0034015-g015: Glutamate-induced ATP synthesis in rat liver mitochondria is independent on EAAT and NCX.(A) Mitochondria isolated from rat liver express EAAC1 and GLT1, but not GLAST. The panel shows three different western blot experiments performed using GLAST, GLT1 and EAAC1 antibodies on whole lysate or mitochondrial (mt) protein extracts from rat liver. Blots are representative of 3 different experiments. (B) Mitochondria isolated from rat liver do not express NCX1, NCX2 or NCX3. The panel shows 3 different western blot experiments, each representative of 2 sets of experiments performed with protein extracts from rat liver mitochondria blotted with NCX1, NCX2 and NCX3 antibodies. None of the NCX isoforms were expressed in rat liver mitochondria. In A and B, protein extracts from rat hippocampus or cortex were used as a positive control. In each IP lane, the lower band at around 50 KDa represents the immunoglobulin. (C, D) Glutamate-induced ATP synthesis in rat liver mitochondria is unaffected by EAAT or NCX pharmacological blockade with DL-TBOA and CGP-37157, respectively. The bar graphs show ATP production in the presence of 1 mM glutamate (black bars) or vehicle (white bars) for 1 h with or without DL-TBOA (C) or CGP-37157 (D). Each bar represents the mean ± SEM of data from 8 different experimental sessions. ATP data were normalized to the protein content of the different mitochondrial preparations. *p<0.05 vs control.

Mentions: Undifferentiated PC12 cells express EAAC1 (Figure 13A) but not NCX1 (Figure 13B). Conversely, NCX2 and NCX3 were detected (Figure 13C and D). When mitochondria obtained from PC12 cells were exposed to glutamate, an increase in ATP synthesis occurred (Figure 14A). However this phenomenon was independent from EAAC1 and NCX, since neither DL- TBOA nor CGP-37157 were able to inhibit glutamate-stimulated ATP synthesis (Figure 14A and B). Equivalent results were obtained in liver mitochondria, a system that does not express exchangers at all [54] (Fig. 15B), but does express EAAC1 protein (Figures 15A–D).


Physical and functional interaction of NCX1 and EAAC1 transporters leading to glutamate-enhanced ATP production in brain mitochondria.

Magi S, Lariccia V, Castaldo P, Arcangeli S, Nasti AA, Giordano A, Amoroso S - PLoS ONE (2012)

Glutamate-induced ATP synthesis in rat liver mitochondria is independent on EAAT and NCX.(A) Mitochondria isolated from rat liver express EAAC1 and GLT1, but not GLAST. The panel shows three different western blot experiments performed using GLAST, GLT1 and EAAC1 antibodies on whole lysate or mitochondrial (mt) protein extracts from rat liver. Blots are representative of 3 different experiments. (B) Mitochondria isolated from rat liver do not express NCX1, NCX2 or NCX3. The panel shows 3 different western blot experiments, each representative of 2 sets of experiments performed with protein extracts from rat liver mitochondria blotted with NCX1, NCX2 and NCX3 antibodies. None of the NCX isoforms were expressed in rat liver mitochondria. In A and B, protein extracts from rat hippocampus or cortex were used as a positive control. In each IP lane, the lower band at around 50 KDa represents the immunoglobulin. (C, D) Glutamate-induced ATP synthesis in rat liver mitochondria is unaffected by EAAT or NCX pharmacological blockade with DL-TBOA and CGP-37157, respectively. The bar graphs show ATP production in the presence of 1 mM glutamate (black bars) or vehicle (white bars) for 1 h with or without DL-TBOA (C) or CGP-37157 (D). Each bar represents the mean ± SEM of data from 8 different experimental sessions. ATP data were normalized to the protein content of the different mitochondrial preparations. *p<0.05 vs control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316532&req=5

pone-0034015-g015: Glutamate-induced ATP synthesis in rat liver mitochondria is independent on EAAT and NCX.(A) Mitochondria isolated from rat liver express EAAC1 and GLT1, but not GLAST. The panel shows three different western blot experiments performed using GLAST, GLT1 and EAAC1 antibodies on whole lysate or mitochondrial (mt) protein extracts from rat liver. Blots are representative of 3 different experiments. (B) Mitochondria isolated from rat liver do not express NCX1, NCX2 or NCX3. The panel shows 3 different western blot experiments, each representative of 2 sets of experiments performed with protein extracts from rat liver mitochondria blotted with NCX1, NCX2 and NCX3 antibodies. None of the NCX isoforms were expressed in rat liver mitochondria. In A and B, protein extracts from rat hippocampus or cortex were used as a positive control. In each IP lane, the lower band at around 50 KDa represents the immunoglobulin. (C, D) Glutamate-induced ATP synthesis in rat liver mitochondria is unaffected by EAAT or NCX pharmacological blockade with DL-TBOA and CGP-37157, respectively. The bar graphs show ATP production in the presence of 1 mM glutamate (black bars) or vehicle (white bars) for 1 h with or without DL-TBOA (C) or CGP-37157 (D). Each bar represents the mean ± SEM of data from 8 different experimental sessions. ATP data were normalized to the protein content of the different mitochondrial preparations. *p<0.05 vs control.
Mentions: Undifferentiated PC12 cells express EAAC1 (Figure 13A) but not NCX1 (Figure 13B). Conversely, NCX2 and NCX3 were detected (Figure 13C and D). When mitochondria obtained from PC12 cells were exposed to glutamate, an increase in ATP synthesis occurred (Figure 14A). However this phenomenon was independent from EAAC1 and NCX, since neither DL- TBOA nor CGP-37157 were able to inhibit glutamate-stimulated ATP synthesis (Figure 14A and B). Equivalent results were obtained in liver mitochondria, a system that does not express exchangers at all [54] (Fig. 15B), but does express EAAC1 protein (Figures 15A–D).

Bottom Line: Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides.The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart.The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Public Health, University Politecnica delle Marche, Ancona, Italy.

ABSTRACT
Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na(+)-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.

Show MeSH
Related in: MedlinePlus