Differential regulation by protein kinases of activity and cell surface expression of glutamate transporters in neuron-enriched cultures.
This study described the involvement of short-term PKA, PKC or PI3K phosphorylation-mediated processes in the regulation of activity and trafficking of the excitatory amino acid transporters EAAC1, GLAST and GLT-1 endogenously expressed in neuron-enriched cultures. Glutamate uptake was dose-dependently decreased by inhibitors of protein kinase A (PKA), [N-[2-(p-bromocinnamylamino)-ethyl]-5-(isoquinolinesulfonamide)] (H89) or phosphatidylinositol 3-kinase (PI3K) (wortmannin), but not altered after protein kinase C (PKC) inhibition (staurosporine) or activation phorbol-12-myristate-13-acetate (PMA). Biotinylation and immunoblotting results (% of controls) showed that EAAC1 membrane expression was significantly decreased by H89 (71.9+/-4.7%) and wortmannin (63.3+/-20.0%) and increased by PMA (137.7+/-15.5%). H89 and PMA induced a significant decrease of the cell surface fraction of GLAST (54.0+/-34.1% and 73.3+/-14.3%, respectively) whereas wortmannin significantly increased this fraction (119.8+/-9.3%). After treatment with H89, the GLT-1 membrane level showed a two-fold increase (179.4+/-19.7%). Conversely, PMA and wortmannin induced a significant decrease of the cell surface expression of GLT-1 (49.0+/-15.4% and 40.7+/-33.7%, respectively). Confocal microscopy revealed a wortmannin-induced clustering of EAAC1 in the intracellular compartment. These data suggest that trafficking of glutamate transporters can be differentially regulated by PKA-, PKC- and PI3K-dependent signaling pathways and could therefore control total glutamate uptake activity. These processes may represent rapid adaptive responses to changes in the cellular environment, which significantly contribute to regulation of EAA transmission and further prevent possible excitotoxic events.
Guillet
B A
BA
Laboratoire de Pharmacodynamie, Faculté de Pharmacie, Universite de la Mediterranee, UFR de Pharmacie, 27 bd Jean-Moulin, et IC2N (UMR 6181 CNRS), Marseille 13005, France.
Velly
L J
LJ
Canolle
B
B
Masmejean
F M
FM
Nieoullon
A L
AL
Pisano
P
P
D027322:Amino Acid Transport System X-AG / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D002451:Cell Compartmentation / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002490:Central Nervous System / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D027981:Symporters / Q000378:metabolism
D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C; D011494:Protein Kinases; D027981:Symporters; C493398:Slc1a1 protein, rat; C493392:Slc1a3 protein, rat