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 [H-3]L-glutamate as the tracer, sodium-dependent high affinity glutamate transport was demonstrated [K-m, = 17.2 +/- 2.4 mu M; V-max,, = 3.3 +/-: 0.32 nmol/mg of protein/min (n = 5)]. Dojudrplaomate (1 mM) inhibited uptake of radioactivity by 88 +/- 3% and had a K-i value of 65 +/- 7 mu M 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 K-i, values of 5.1 +/- 0.3, 56 +/- 6, and 103 +/- 9 mu M, 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 (K-i,= +/- 1 mu M), and L-serine-O-sulfate produced a GLT1-mediated current with a K-m, value of 312 +/- 33 mu M. 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.