Abstract
Glutamate uptake into nerve cells and astrocytes via high-affinity transporters controls the extracellular glutamate concentration in the brain, with major implications for physiological excitatory neurotransmission and the prevention of excitotoxicity. We report here that three recently cloned rat glutamate transporter subtypes, viz. EAAC1 (neuronal), GLT1 and GLAST (glial), possess a redox-sensing property, undergoing opposite functional changes in response to oxidation or reduction of reactive sulphydryls present in their structure. In particular, thiol oxidation with 5,5'-dithio-bis(2-nitrobenzoic) acid (DTNB) and disulphide reduction with dithiothreitol (DTT) result, respectively, in reduced and increased uptake capacity by a preparation of partially purified brain transporters as well as by the three recombinant proteins reconstituted into liposomes. In this model system, EAAC1, GLT1 and GLAST react similarly to DTT/DTNB exposures despite their different contents of cysteines, suggesting that only the conserved residues might be involved in redox modulation. Redox sensitivity is a property of the glutamate transporters also when present in their native cell environment. Thus, by using cultured cortical astrocytes and the whole-cell patch-clamp technique we were able to observe dynamic increase and decrease of the glutamate uptake current in response to application of DTT and DTNB in sequence. Moreover, in the same paradigm, DDT-reversible current inhibition was observed with hydrogen peroxide instead of DTNB, indicating that the SH-based redox modulatory site is targeted by endogenous oxidants and might constitute an important physiological or pathophysiological regulatory mechanism of glutamate uptake in vivo.
| Original language | English |
|---|---|
| Pages (from-to) | 1236-1243 |
| Number of pages | 8 |
| Journal | European Journal of Neuroscience |
| Volume | 9 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - 1997 |
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Keywords
- Endogenous oxidants and reductants
- Excitatory neurotransmission
- Excitotoxicity
- GLT1 and GLAST transporters
- Oxidative stress
- Rat EAAC1
ASJC Scopus subject areas
- Neuroscience(all)
Cite this
Neuronal and glial glutamate transporters possess an SH-based redox regulatory mechanism. / Trotti, Davide; Rizzini, Barbara Lodi; Rossi, Daniela; Haugeto, Oyvind; Racagni, Giorgio; Danbolt, Niels C.; Volterra, Andrea.
In: European Journal of Neuroscience, Vol. 9, No. 6, 1997, p. 1236-1243.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Neuronal and glial glutamate transporters possess an SH-based redox regulatory mechanism
AU - Trotti, Davide
AU - Rizzini, Barbara Lodi
AU - Rossi, Daniela
AU - Haugeto, Oyvind
AU - Racagni, Giorgio
AU - Danbolt, Niels C.
AU - Volterra, Andrea
PY - 1997
Y1 - 1997
N2 - Glutamate uptake into nerve cells and astrocytes via high-affinity transporters controls the extracellular glutamate concentration in the brain, with major implications for physiological excitatory neurotransmission and the prevention of excitotoxicity. We report here that three recently cloned rat glutamate transporter subtypes, viz. EAAC1 (neuronal), GLT1 and GLAST (glial), possess a redox-sensing property, undergoing opposite functional changes in response to oxidation or reduction of reactive sulphydryls present in their structure. In particular, thiol oxidation with 5,5'-dithio-bis(2-nitrobenzoic) acid (DTNB) and disulphide reduction with dithiothreitol (DTT) result, respectively, in reduced and increased uptake capacity by a preparation of partially purified brain transporters as well as by the three recombinant proteins reconstituted into liposomes. In this model system, EAAC1, GLT1 and GLAST react similarly to DTT/DTNB exposures despite their different contents of cysteines, suggesting that only the conserved residues might be involved in redox modulation. Redox sensitivity is a property of the glutamate transporters also when present in their native cell environment. Thus, by using cultured cortical astrocytes and the whole-cell patch-clamp technique we were able to observe dynamic increase and decrease of the glutamate uptake current in response to application of DTT and DTNB in sequence. Moreover, in the same paradigm, DDT-reversible current inhibition was observed with hydrogen peroxide instead of DTNB, indicating that the SH-based redox modulatory site is targeted by endogenous oxidants and might constitute an important physiological or pathophysiological regulatory mechanism of glutamate uptake in vivo.
AB - Glutamate uptake into nerve cells and astrocytes via high-affinity transporters controls the extracellular glutamate concentration in the brain, with major implications for physiological excitatory neurotransmission and the prevention of excitotoxicity. We report here that three recently cloned rat glutamate transporter subtypes, viz. EAAC1 (neuronal), GLT1 and GLAST (glial), possess a redox-sensing property, undergoing opposite functional changes in response to oxidation or reduction of reactive sulphydryls present in their structure. In particular, thiol oxidation with 5,5'-dithio-bis(2-nitrobenzoic) acid (DTNB) and disulphide reduction with dithiothreitol (DTT) result, respectively, in reduced and increased uptake capacity by a preparation of partially purified brain transporters as well as by the three recombinant proteins reconstituted into liposomes. In this model system, EAAC1, GLT1 and GLAST react similarly to DTT/DTNB exposures despite their different contents of cysteines, suggesting that only the conserved residues might be involved in redox modulation. Redox sensitivity is a property of the glutamate transporters also when present in their native cell environment. Thus, by using cultured cortical astrocytes and the whole-cell patch-clamp technique we were able to observe dynamic increase and decrease of the glutamate uptake current in response to application of DTT and DTNB in sequence. Moreover, in the same paradigm, DDT-reversible current inhibition was observed with hydrogen peroxide instead of DTNB, indicating that the SH-based redox modulatory site is targeted by endogenous oxidants and might constitute an important physiological or pathophysiological regulatory mechanism of glutamate uptake in vivo.
KW - Endogenous oxidants and reductants
KW - Excitatory neurotransmission
KW - Excitotoxicity
KW - GLT1 and GLAST transporters
KW - Oxidative stress
KW - Rat EAAC1
UR - http://www.scopus.com/inward/record.url?scp=0031001036&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0031001036&partnerID=8YFLogxK
U2 - 10.1111/j.1460-9568.1997.tb01478.x
DO - 10.1111/j.1460-9568.1997.tb01478.x
M3 - Article
C2 - 9215707
AN - SCOPUS:0031001036
VL - 9
SP - 1236
EP - 1243
JO - European Journal of Neuroscience
JF - European Journal of Neuroscience
SN - 0953-816X
IS - 6
ER -