High Affinity Glutamate Transport in Rat Cortical Neurons in Culture
- Guang Jian Wang1,
- Hye Joo Chung1,
- Jamie Schnuer1,
- Kara Pratt1,
- Anthony C. Zable2,
- Michael P. Kavanaugh2 and
- Paul A. Rosenberg1
- 1Department of Neurology and Program in Neuroscience, Children’s Hospital and Harvard Medical School, Boston, Massachusetts 02115 (G.J.W., H.J.C., J.S., K.P., P.A.R.), and 2The Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201 (A.C.Z., M.P.K.)
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 [Km = 17.2 ± 2.4 μm; Vmax = 3.3 ± 0.32 nmol/mg of protein/min (n = 5)]. Dihydrokainate (1 mm) inhibited uptake of radioactivity by 88 ± 3% and had aKi 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 withKi 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 (Ki = 8 ± 1 μm), andl-serine-O-sulfate produced a GLT1-mediated current with a Kmvalue 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.
Footnotes
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Send reprint requests to: Dr. Paul A. Rosenberg, Enders Research Building, Department of Neurology, Children’s Hospital, 300 Longwood Avenue, Boston MA 02115. E-mail:rosenberg{at}a1.tch.harvard.edu
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This work was supported by National Institutes of Health Grants NS33270 (M.P.K.) and NS31353 and a Mental Retardation Core Grant to Children’s Hospital (P.A.R.) and by grants from the United Cerebral Palsy Foundation, Ron Shapiro Charitable Foundation, and Muscular Dystrophy Association (P.A.R.). G.J.W. and H.J.C. contributed equally to this work.
- Abbreviations:
- DHK
- dihydrokainate
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- SOS
- l-serine-O-sulfate
- l-α-AA
- l-α-aminoadipate
- NMDA
- N-methyl-d-aspartate
- PDC
- l-trans-2,4-pyrrolidine dicarboxylate
- GFAP
- glial fibrillary acidic protein
- BOAA
- b-N-oxalyl-l-a,b-diaminopropionate
- AMG
- α-methyl-dl-glutamate
- KA
- kainate
- SDS
- sodium dodecyl sulfate
- GLAST
- glutamate/aspartate transporter
- EAAC
- excitatory amino acid carrier
- EAAT
- excitatory amino acid transporter
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- Received May 30, 1997.
- Accepted September 26, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
