Constitutive phosphorylation of the vesicular inhibitory amino acid transporter in rat central nervous system

gamma-Aminobutyric acid (GABA) and glycine are stored into synaptic vesicle s by a recently identified vesicular inhibitory amino acid transporter [VIA AT, 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 cor responded to a phosphorylated form of VIAAT as it was converted to the fast er one by treating brain homogenates with alkaline phosphatase or with an e ndogenous phosphatase identified as type 2A protein-serine/threonine phosph atase 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 o f VIAAT phosphorylation on vesicular neurotransmitter loading, purified syn aptic vesicles were treated with alkaline phosphatase and assayed for amino acid uptake, However, neither GABA nor glycine uptake was affected by VIAA T phosphorylation, These results indicate that VIAAT is constitutively phos phorylated on cytosolic serine or threonine residues in most, but not all, regions of the rat brain. This phosphorylation does not regulate the vesicu lar loading of GABA or glycine, suggesting that it is involved at other sta ges of the synaptic vesicle life cycle.