High Affinity Glutamate Transport in Rat Cortical Neurons in Culture

• Published in: Molecular pharmacology Vol. 53; no. 1; pp. 88 - 96
• Main Authors: Wang, G J, Chung, H J, Schnuer, J, Pratt, K, Zable, A C, Kavanaugh, M P, Rosenberg, P A
• Format: Journal Article
• Language: English
• Published: United States American Society for Pharmacology and Experimental Therapeutics 01.01.1998
• Subjects: 2-Aminoadipic Acid - pharmacology; Amino Acid Transport System X-AG; Animals; ATP-Binding Cassette Transporters - antagonists & inhibitors; ATP-Binding Cassette Transporters - metabolism; Biological Transport - drug effects; Cells, Cultured; Cerebral Cortex - cytology; Cerebral Cortex - drug effects; Cerebral Cortex - metabolism; Immunoblotting; Immunohistochemistry; Kainic Acid - analogs & derivatives; Kainic Acid - pharmacology; Kinetics; Neurons - drug effects; Neurons - metabolism; Oocytes - drug effects; Oocytes - metabolism; Rats; Rats, Sprague-Dawley; Serine - analogs & derivatives; Serine - pharmacology; Synaptosomes - drug effects; Synaptosomes - metabolism
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.53.1.88

We assayed glutamate transport activity in cultures of rat cortical neurons containing <0.2% astrocytes. Using [ 3 H] l -glutamate as the tracer, sodium-dependent high affinity glutamate transport was demonstrated [ K m = 17.2 ± 2.4 μ m ; V max = 3.3 ± 0.32 nmol/mg of protein/min ( n = 5)]. Dihydrokainate (1 m m ) inhibited uptake of radioactivity by 88 ± 3% and had a K i value of 65 ± 7 μ m . l -α-Aminoadipate (1 m m ) 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 μ m , 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 μ m ), and l -serine- O -sulfate produced a GLT1-mediated current with a K m value of 312 ± 33 μ 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.