Regulation of the gamma-aminobutyric acid transporter activity by protein phosphatases in synaptic plasma membranes.
The influence of the phosphorylation dephosphorylation states on the gamma-aminobutyric acid (GABA) transporter activity of synaptic plasma membranes (SPM) was studied by using either specific phosphatase inhibitors or activators. Calyculin A and okadaic acid (phosphatase 1 and phosphatase 2A inhibitors) inhibited the GABA uptake by isolated SPM vesicles, whereas cyclosporin A (phosphatase 2B inhibitor) had a stimulatory effect (approximately 10%) which was higher (approximately 38%) when all these drugs were present in the reaction medium. On the other hand, intravesicular Ca2+, up to about 10 microM, inhibited the GABA uptake (approximately 50%) in a manner which appeared to be facilitated in the presence of PP1 and PP2A inhibitors and this inhibition was relieved by the calmodulin antagonist W-7. We also observed that isolated SPM vesicles contain both Ca(2+)-independent phosphatase activity that is significantly inhibited by PP1 and PP2A inhibitors, and Ca(2+)-dependent phosphatase activity that is abolished in the presence of the PP2B inhibitor, cyclosporin A. These results indicate that regulation of the SPM GABA transporter is determined by the internally localized Ca-calmodulin-dependent phosphatase activity (calcineurin), and that other phosphorylated sites, sensitive to PP1 and PP2A inhibitors, potentiate either the positive or negative effects exerted by those internal sites when they are in their phosphorylated or dephosphorylated states, respectively.
Gonçalves
P P
PP
Centro de Biologia Celular, Departamento de Biologia, Universidade de Aveiro, Portugal. pgoncalves@bio.ua.pt
Meireles
S M
SM
Vale
M G
MG
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D012756:Sheep; D013570:Synaptic Membranes / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D005680:gamma-Aminobutyric Acid; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2