Inhibition of the glutamate transporter EAAC1 expressed in Xenopus oocytesby phorbol esters

Recent evidence indicates that second messengers and protein kinases regula te the activity and expression of glutamate transporters. The aim of the pr esent 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 measurin g [ H]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 EAAC 1 transport activity (IC50=44.7 +/- 12 nM). PMA-induced EAAC1 inhibition wa s PKC-mediated because the inhibition could be blocked by specific PKC inhi bitors and incubation with the inactive 4 alpha -phorbol-12,13-didecanoate (4 alpha -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 capac itance (C.) and transport-associated current and increased cytosolic accumu lation of EAAC1 protein, compared to control. These results suggest that PK C activation inhibits EAAC1 by promoting its retrieval from the plasma memb rane. PMA also significantly decreased glutamate uptake in a Madin-Darby ca nine kidney (MDCK) cell line stably transfected with EAAC1 but enhanced EAA C1-mediated glutamate uptake in the rat C6 glioma cells, consistent with pr evious observations. Because activation of PKC by phorbol esters leads to o pposite effects on EAAC1 activity in different culture models, we conclude that the PKC-mediated regulation of EAAC1 is cell-type specific. (C) 2001 E lsevier Science BY. All rights reserved.