High affinity glutamate transport in rat cortical neurons in culture.
We assayed glutamate transport activity in cultures of rat cortical neurons containing < 0.2% astrocytes. Using [3H]L-glutamate as the tracer, sodium-dependent high affinity glutamate transport was demonstrated [K(m) = 17.2 +/- 2.4 microM; Vmax = 3.3 +/- 0.32 nmol/mg of protein/min (n = 5)]. Dihydrokainate (1 mM) inhibited uptake of radioactivity by 88 +/- 3% and had a Ki value of 65 +/- 7 microM. L-alpha-Aminoadipate (1 mM) inhibited uptake by only 25 +/- 4%. L-trans-2,4-Pyrrolidine dicarboxylate, L-serine-O-sulfate, and kainate potently inhibited transport activity with Ki values of 5.1 +/- 0.3, 56 +/- 6, and 103 +/- 9 microM, respectively (n = 3). Voltage-clamp studies of GLT1-expressing oocytes showed that, as in cortical neurons, glutamate transport was not inhibited by L-alpha-aminoadipate. Dihydrokainate was a potent inhibitor (Ki = 8 +/- 1 microM), and L-serine-O-sulfate produced a GLT1-mediated current with a K(m) value of 312 +/- 33 microM. Immunoblot analysis showed that neuronal cultures express excitatory amino acid carrier 1 (EAAC1), shown previously to be relatively insensitive to dihydrokainate, plus a trace amount of GLT1, but no GLAST. These studies establish that a major component of the glutamate transport activity of cortical neurons is dihydrokainate sensitive and distinct from the previously recognized neuronal transporter excitatory amino acid carrier 1.
Wang
G J
GJ
Department of Neurology, Children's Hospital, Boston, Massachusetts, USA.
Chung
H J
HJ
Schnuer
J
J
Pratt
K
K
Zable
A C
AC
Kavanaugh
M P
MP
Rosenberg
P A
PA
D015074:2-Aminoadipic Acid / Q000494:pharmacology; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000378:metabolism*; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015151:Immunoblotting; D007150:Immunohistochemistry; D007608:Kainic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000031:analogs & derivatives / Q000494:pharmacology; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D015074:2-Aminoadipic Acid; D012694:Serine; C020889:dihydrokainic acid; C036040:serine O-sulfate; D007608:Kainic Acid