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 μM; V(max) = 3.3 ± 0.32 nmol/mg of protein/min (n = 5)]. Dihydrokainate (1 mM) inhibited uptake of radioactivity by 88 ± 3% and had a K(i) value of 65 ± 7 μM. L-α-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 μM, respectively (n = 3). Voltage-clamp studies of GLT1-expressing oocytes showed that, as in cortical neurons, glutamate transport was not inhibited by L-α-aminoadipate. Dihydrokainate was a potent inhibitor (K(i) = 8 ± 1 μM), and L-serine-O-sulfate produced a GLT1 - mediated current with a K(m) value of 312 ± 33 μM. 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.