Inhibition of the high-affinity brain glutamate transporter GLAST-1 via direct phosphorylation

J Neurochem. 1997 Mar;68(3):1244-51. doi: 10.1046/j.1471-4159.1997.68031244.x.


Neurotransmission at excitatory glutamatergic synapses is terminated by the reuptake of the neurotransmitter by high-affinity transporters, which keep the extracellular glutamate concentration below excitotoxic levels. The amino acid sequence of the recently isolated and cloned brain-specific glutamate/aspartate transporter (GLAST-1) of the rat reveals three consensus sequences of putative phosphorylation sites for protein kinase C (PKC). The PKC activator phorbol 12-myristate 13-acetate (PMA) decreased glutamate transport activity in Xenopus oocytes and human embryonic kidney cells (HEK293) expressing the cloned GLAST-1 cDNA, within 20 min, to 25% of the initial transport activity. This downregulation was blocked by the PKC inhibitor staurosporine. GLAST-1 transport activity remains unimpaired by phorbol 12-monomyristate. Removal of all putative PKC sites of wild-type GLAST-1 by site-directed mutagenesis did not abolish inhibition of glutamate transport. [32P]Phosphate-labeled wild-type and mutant transport proteins devoid of all predicted PKC sites were detected by immunoprecipitation after stimulation with PMA. Immunoprecipitation of [35S]methionine-labeled transporter molecules indicates a similar stability of phosphorylated and nonphosphorylated GLAST-1 protein. Immunofluorescence staining did not differentiate surface staining of HEK293 cells expressing GLAST-1 with and without PMA treatment. These data suggest that the neurotransmitter transporter activity of GLAST-1 is inhibited by phosphorylation at a non-PKC consensus site.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Amino Acid Transport System X-AG
  • Animals
  • Binding Sites
  • Brain / metabolism*
  • Carrier Proteins / antagonists & inhibitors*
  • Carrier Proteins / drug effects
  • Carrier Proteins / metabolism
  • Cell Membrane / metabolism
  • Female
  • Glycoproteins / antagonists & inhibitors*
  • Glycoproteins / drug effects
  • Glycoproteins / metabolism
  • Humans
  • Oocytes / metabolism
  • Phosphates / metabolism
  • Phosphorylation
  • Protein Kinase C / metabolism
  • Rats
  • Tetradecanoylphorbol Acetate / pharmacology
  • Xenopus laevis


  • Amino Acid Transport System X-AG
  • Carrier Proteins
  • Glycoproteins
  • Phosphates
  • Adenosine Triphosphate
  • Protein Kinase C
  • Tetradecanoylphorbol Acetate