High affinity glutamate transport in rat cortical neurons in culture

Guang Jian Wang, Hye Joo Chung, Jamie Schnuer, Kara Pratt, Anthony C. Zable, Michael P. Kavanaugh, Paul A. Rosenberg

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)88-96
Number of pages9
JournalMolecular Pharmacology
Volume53
Issue number1
DOIs
StatePublished - Jan 1998

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