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This Article Full Text Full Text (PDF) All Versions of this Article: 297/4/C835    most recent 00212.2009v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Padovano, V. Articles by Pietrini, G. PubMed PubMed Citation Articles by Padovano, V. Articles by Pietrini, G.

MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

PKC induces internalization and retention of the EAAC1 glutamate transporter in recycling endosomes of MDCK cells

Department of Pharmacology, School of Medicine, Università degli Studi di Milano and Institute of Neuroscience-CNR, Milan, Italy

Submitted 13 May 2009 ; accepted in final form 10 July 2009

Here we show that stimulation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) treatment induces a time-dependent decrease in glutamate transport activity due to relocalization of the excitatory amino acid carrier 1 (EAAC1) glutamate transporter from the apical surface of polarized epithelial Madin-Darby canine kidney (MDCK) cells to intracellular compartments. The PKC-induced internalization of EAAC1 is negatively regulated by the calcineurin inhibitor cyclosporine A and by the expression of a dominant-negative mutant of the endocytic protein dynamin 1, a well-known target of the phosphatase activity of calcineurin. Using ³²P-metabolic labeling experiments, we found unchanged levels of phosphorylated EAAC1, indicating that EAAC1 relocalization does not depend on PKC and calcineurin modification of the transporter, while we found that a target of these modifications was the serine⁷⁷⁸ residue of dynamin, a calcineurin substrate that in its dephosphorylated form activates the endocytic functions of dynamin. These data suggest that PMA stimulates endogenous dynamin and that this activation is required to mediate internalization of EAAC1 in MDCK cells. By immunofluorescence experiments with endosomal markers we demonstrated that internalized EAAC1 accumulates in endosomes also containing the basolateral betaine-GABA transporter BGT1 and activated PKC. The sustained activation of PKC was required to maintain the transporters in the endosomal compartment, while a posttreatment with a PKC-specific inhibitor induced the recycling of the transporters to their appropriate surfaces. Taken together, our data indicate that PKC activity regulates EAAC1 surface density in MDCK cells by inducing its internalization and retention in PKC-labeled recycling endosomes common to apical and basolateral proteins.

common recycling endosomes; cyclosporine A; dynamin 1; pericentrion; protein kinase C; excitatory amino acid carrier