Inhibition of the glutamate transporter EAAC1 expressed in Xenopus oocytes by phorbol esters.
Recent evidence indicates that second messengers and protein kinases regulate the activity and expression of glutamate transporters. The aim of the present study was to determine if direct activation of protein kinases C or A modulates the activity of the sodium-dependent glutamate transporter EAAC1. EAAC1 modulation was studied in cRNA-injected Xenopus oocytes by measuring [3H]L-glutamate uptake or glutamate-evoked uptake currents. We found that activation of PKA was ineffective, whereas treatment with the PKC agonist phorbol 12-myristate 13-acetate (PMA) caused a significant decrease in EAAC1 transport activity (IC(50)=44.7+/-12 nM). PMA-induced EAAC1 inhibition was PKC-mediated because the inhibition could be blocked by specific PKC inhibitors and incubation with the inactive 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) did not affect EAAC1. Saturation studies of glutamate-evoked uptake currents showed that PMA-mediated inhibition was due to a decrease in I(max) with no change in K(m). PMA simultaneously decreased membrane capacitance (C(m)) and transport-associated current and increased cytosolic accumulation of EAAC1 protein, compared to control. These results suggest that PKC activation inhibits EAAC1 by promoting its retrieval from the plasma membrane. PMA also significantly decreased glutamate uptake in a Madin-Darby canine kidney (MDCK) cell line stably transfected with EAAC1 but enhanced EAAC1-mediated glutamate uptake in the rat C6 glioma cells, consistent with previous observations. Because activation of PKC by phorbol esters leads to opposite effects on EAAC1 activity in different culture models, we conclude that the PKC-mediated regulation of EAAC1 is cell-type specific.
Trotti
D
D
Membrane Biology Program, Brigham & Women's Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. dtrotti@partners.org
Peng
J B
JB
Dunlop
J
J
Hediger
M A
MA
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D027322:Amino Acid Transport System X-AG; D000818:Animals; D002273:Carcinogens / Q000494:pharmacology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000378:metabolism* / Q000493:pharmacokinetics; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D010704:Phorbols / Q000494:pharmacology; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D018075:RNA, Complementary / Q000494:pharmacology; D027981:Symporters; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014316:Tritium / Q000493:pharmacokinetics; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D014982:Xenopus laevis
D027322:Amino Acid Transport System X-AG; D002273:Carcinogens; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D007211:Indoles; D008301:Maleimides; D010704:Phorbols; D018075:RNA, Complementary; C493400:SLC1A1 protein, human; D027981:Symporters; D014316:Tritium; D005576:Colforsin; D018698:Glutamic Acid; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C088060:bisindolylmaleimide; D013755:Tetradecanoylphorbol Acetate; D015056:1-Methyl-3-isobutylxanthine; C033085:phorbol