title: Modulation of human glutamate transporter activity by phorbol ester.
abstract: Termination of synaptic glutamate transmission depends on rapid removal of glutamate by neuronal and glial high-affinity transporters. Molecular biological and pharmacological studies have demonstrated that at least five subtypes of Na+-dependent mammalian glutamate transporters exist. Our study demonstrates that Y-79 human retinoblastoma cells express a single Na+-dependent glutamate uptake system with a Km of 1.7 +/- 0.42 microM that is inhibited by dihydrokainate and DL-threo-beta-hydroxyaspartate (IC50 = 0.29 +/- 0.17 microM and 2.0 +/- 0.43 microM, respectively). The protein kinase C activator phorbol 12-myristate 13-acetate caused a concentration-dependent inhibition of glutamate uptake (IC50 = 0.56 +/- 0.05 nM), but did not affect Na+-dependent glycine uptake significantly. This inhibition of glutamate uptake resulted from a fivefold decrease in the transporter's affinity for glutamate, without significantly altering the Vmax. 4Alpha-phorbol 12,13-didecanoate, a phorbol ester that does not activate protein kinase C, did not alter glutamate uptake significantly. The phorbol 12-myristate 13-acetate-induced inhibition of glutamate uptake was reversed by preincubation with staurosporine. The biophysical and pharmacological profile of the human glutamate transporter expressed by the Y-79 cell line indicates that it belongs to the dihydrokainate-sensitive EAAT2/GLT-1 subtype. This conclusion was confirmed by western blot analysis. Protein kinase C modulation of glutamate transporter activity may represent a mechanism to modulate extracellular glutamate and shape postsynaptic responses.
authors.affiliation: Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock 79430-0001, USA.
authors.forename: R
authors.initials: R
authors.lastname: Ganel
authors.forename: C E
authors.initials: CE
authors.lastname: Crosson
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D027322:Amino Acid Transport System X-AG; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002273:Carcinogens / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005971:Glutamates / Q000494:pharmacology; D018698:Glutamic Acid / Q000493:pharmacokinetics; D006801:Humans; D007608:Kainic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D010703:Phorbol Esters / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism; D012175:Retinoblastoma; D019311:Staurosporine / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology
chemical_list: D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D000900:Anti-Bacterial Agents; D002273:Carcinogens; D004791:Enzyme Inhibitors; D005971:Glutamates; D010703:Phorbol Esters; C011274:3-hydroxyaspartic acid; C008876:phorbol-12,13-didecanoate; D001224:Aspartic Acid; D018698:Glutamic Acid; C025648:2-methylglutamic acid; C020889:dihydrokainic acid; D011493:Protein Kinase C; D019311:Staurosporine; D007608:Kainic Acid