Constitutive phosphorylation of the vesicular inhibitory amino acid transporter in rat central nervous system.
gamma-Aminobutyric acid (GABA) and glycine are stored into synaptic vesicles by a recently identified vesicular inhibitory amino acid transporter [VIAAT, also called vesicular GABA transporter (VGAT)]. Immunoblotting analysis revealed that rat brain VIAAT migrated as a doublet during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a predominant slower band in all areas examined except olfactory bulb and retina. The slower band corresponded to a phosphorylated form of VIAAT as it was converted to the faster one by treating brain homogenates with alkaline phosphatase or with an endogenous phosphatase identified as type 2A protein-serine/threonine phosphatase using okadaic acid. In contrast, the recombinant protein expressed in COS-7 or PC12 cells co-migrated with the faster band of the brain doublet and was insensitive to alkaline phosphatase. To investigate the influence of VIAAT phosphorylation on vesicular neurotransmitter loading, purified synaptic vesicles were treated with alkaline phosphatase and assayed for amino acid uptake. However, neither GABA nor glycine uptake was affected by VIAAT phosphorylation. These results indicate that VIAAT is constitutively phosphorylated on cytosolic serine or threonine residues in most, but not all, regions of the rat brain. This phosphorylation does not regulate the vesicular loading of GABA or glycine, suggesting that it is involved at other stages of the synaptic vesicle life cycle.
Bedet
C
C
Centre National de la Recherche Scientifique UPR 1929, Institut de Biologie Physico-Chimique, Paris, France.
Isambert
M F
MF
Henry
J P
JP
Gasnier
B
B
D000469:Alkaline Phosphatase / Q000378:metabolism; D000581:Amidohydrolases / Q000378:metabolism; D026905:Amino Acid Transport Systems; D000818:Animals; D001921:Brain / Q000378:metabolism*; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005998:Glycine / Q000493:pharmacokinetics; D015151:Immunoblotting; D008297:Male; D009830:Olfactory Bulb / Q000378:metabolism; D016716:PC12 Cells; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011479:Protamines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013116:Spinal Cord / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000378:metabolism; D016708:Synaptophysin / Q000187:drug effects; D050495:Vesicular Inhibitory Amino Acid Transport Proteins; D033921:Vesicular Transport Proteins; D005680:gamma-Aminobutyric Acid / Q000493:pharmacokinetics
D026905:Amino Acid Transport Systems; D002352:Carrier Proteins; D011479:Protamines; C118623:Slc32a1 protein, rat; D016708:Synaptophysin; D050495:Vesicular Inhibitory Amino Acid Transport Proteins; D033921:Vesicular Transport Proteins; D012967:Sodium Dodecyl Sulfate; D005680:gamma-Aminobutyric Acid; D000469:Alkaline Phosphatase; D010749:Phosphoprotein Phosphatases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D005998:Glycine