Limits...
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.

Show MeSH

Related in: MedlinePlus

Real-time membrane potential analysis in permeabilized cells.Experiments performed in digitonin-permeabilized SH-SY5Y (A,B) and C6 (C,D) cells using TMRE at non-quencing concentration (10 nM). Glutamate perfusion induced a mitochondrial depolarization (blue line). Both DL-TBOA (300 µM) and CGP-37157 (3 µM) prevented the glutamate-stimulated mitochondrial depolarization (pink and yellow lines, respectively). FCCP (5 µM) was added at the end as an internal control in each experiment. Mitochondria response was analyzed as the ratio of fluorescence during maximal glutamate depolarization versus pre stimulus levels. Each bar represents the mean ± SEM of 50–100 cells recorded in 5 different sessions (B,D). *** p<0.001 vs control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3316532&req=5

pone-0034015-g007: Real-time membrane potential analysis in permeabilized cells.Experiments performed in digitonin-permeabilized SH-SY5Y (A,B) and C6 (C,D) cells using TMRE at non-quencing concentration (10 nM). Glutamate perfusion induced a mitochondrial depolarization (blue line). Both DL-TBOA (300 µM) and CGP-37157 (3 µM) prevented the glutamate-stimulated mitochondrial depolarization (pink and yellow lines, respectively). FCCP (5 µM) was added at the end as an internal control in each experiment. Mitochondria response was analyzed as the ratio of fluorescence during maximal glutamate depolarization versus pre stimulus levels. Each bar represents the mean ± SEM of 50–100 cells recorded in 5 different sessions (B,D). *** p<0.001 vs control.

Mentions: In order to completely exclude any contamination of the plasma membrane potential to the TMRE signals, we performed a set of experiments with digitonin-permeabilized SH-SY5Y and C6 cells. As shown in Figures 7A–D, when permeabilized cells were exposed to glutamate, a rapid decrease in TMRE fluorescence was observed (reflecting in these conditions mitochondrial depolarization) that reverted to baseline levels after washout. Exposure to FCCP (5 µM) at the end of the experiments abolished TMRE fluorescence, as expected. When the cells were treated with DL-TBOA (300 µM), the glutamate-dependent drop in ΔΨmit was significantly prevented (Figures 7A–D) in agreement with the TMRE data previously obtained in non permeabilized cells.


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)

Real-time membrane potential analysis in permeabilized cells.Experiments performed in digitonin-permeabilized SH-SY5Y (A,B) and C6 (C,D) cells using TMRE at non-quencing concentration (10 nM). Glutamate perfusion induced a mitochondrial depolarization (blue line). Both DL-TBOA (300 µM) and CGP-37157 (3 µM) prevented the glutamate-stimulated mitochondrial depolarization (pink and yellow lines, respectively). FCCP (5 µM) was added at the end as an internal control in each experiment. Mitochondria response was analyzed as the ratio of fluorescence during maximal glutamate depolarization versus pre stimulus levels. Each bar represents the mean ± SEM of 50–100 cells recorded in 5 different sessions (B,D). *** p<0.001 vs control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316532&req=5

pone-0034015-g007: Real-time membrane potential analysis in permeabilized cells.Experiments performed in digitonin-permeabilized SH-SY5Y (A,B) and C6 (C,D) cells using TMRE at non-quencing concentration (10 nM). Glutamate perfusion induced a mitochondrial depolarization (blue line). Both DL-TBOA (300 µM) and CGP-37157 (3 µM) prevented the glutamate-stimulated mitochondrial depolarization (pink and yellow lines, respectively). FCCP (5 µM) was added at the end as an internal control in each experiment. Mitochondria response was analyzed as the ratio of fluorescence during maximal glutamate depolarization versus pre stimulus levels. Each bar represents the mean ± SEM of 50–100 cells recorded in 5 different sessions (B,D). *** p<0.001 vs control.
Mentions: In order to completely exclude any contamination of the plasma membrane potential to the TMRE signals, we performed a set of experiments with digitonin-permeabilized SH-SY5Y and C6 cells. As shown in Figures 7A–D, when permeabilized cells were exposed to glutamate, a rapid decrease in TMRE fluorescence was observed (reflecting in these conditions mitochondrial depolarization) that reverted to baseline levels after washout. Exposure to FCCP (5 µM) at the end of the experiments abolished TMRE fluorescence, as expected. When the cells were treated with DL-TBOA (300 µM), the glutamate-dependent drop in ΔΨmit was significantly prevented (Figures 7A–D) in agreement with the TMRE data previously obtained in non permeabilized cells.

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