Structural selectivity and molecular nature of L-glutamate transport in cultured human fibroblasts

Benedict Cooper, Mary Chebib, Jie Shen, Nicholas J.C. King, Ivan G. Darvey, Philip W. Kuchel, Jeffrey D. Rothstein, Vladimir J. Balcar

Research output: Contribution to journalArticlepeer-review

Abstract

Uptake of L-[3H]glutamate by monolayers of fibroblasts cultured from human embryonic skin has been studied in the presence of several nonradioactive structural analogs of glutamate and aspartate. Results have suggested that the structural specificities of glutamate transporters in cultured human fibroblasts are similar to those of glutamate transporters in the mammalian brain. Only subtle differences have been detected: in the mammalian cerebral cortex, enantiomers of threo-3-hydroxyaspartate are almost equipotent as inhibitors of L-[3H]glutamate uptake while, in human fibroblasts, the D-isomer has been found to be an order of magnitude less potent than the corresponding L-isomer. Kinetic analysis of a model in which substrates are recognized by the glutamate transporter binding site(s) as both α- and β-amino acids indicated that such a mechanism cannot explain the apparent negative cooperativity characterizing the effects of D- and L- aspartate. Molecular modeling has been used to estimate the optimum conformation of L-glutamate as it interacts with the transporter(s). Flow cytometry has indicated that all fibroblasts in culture express at least moderate levels of four glutamate transporters cloned from human brain. Small subpopulations (<3%) of cells, however, were strongly labeled with antibodies against EAAT1 (GLAST) and EAAT2 (GLT-1) transporters. We conclude that these two transporters-known to be strongly expressed in brain tissue-can be principally responsible for the 'high affinity' transport of glutamate also in nonneural cells.

Original languageEnglish (US)
Pages (from-to)356-364
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume353
Issue number2
DOIs
StatePublished - May 15 1998

Keywords

  • EAAC1
  • EAAT4
  • Flow cytometry
  • GLAST
  • GLT- 1
  • Glutamate analogues
  • Molecular modeling
  • Skin fibroblasts
  • Transport of acidic amino acids

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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