Role of synaptic vesicle proton gradient and protein phosphorylation on ATP-mediated activation of membrane-associated brain glutamate decarboxylase

J Biol Chem. 1999 Aug 20;274(34):24366-71. doi: 10.1074/jbc.274.34.24366.


Previously, we have shown that the soluble form of brain glutamic acid decarboxylase (GAD) is inhibited by ATP through protein phosphorylation and is activated by calcineurin-mediated protein dephosphorylation (Bao, J., Cheung, W. Y., and Wu, J. Y. (1995) J. Biol. Chem. 270, 6464-6467). Here we report that the membrane-associated form of GAD (MGAD) is greatly activated by ATP, whereas adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP), a non-hydrolyzable ATP analog, has no effect on MGAD activity. ATP activation of MGAD is abolished by conditions that disrupt the proton gradient of synaptic vesicles, e.g. the presence of vesicular proton pump inhibitor, bafilomycin A1, the protonophore carbonyl cyanide m-chorophenylhydrazone or the ionophore gramicidin, indicating that the synaptic vesicle proton gradient is essential in ATP activation of MGAD. Furthermore, direct incorporation of (32)P from [gamma-(32)P]ATP into MGAD has been demonstrated. In addition, MGAD (presumably GAD65, since it is recognized by specific monoclonal antibody, GAD6, as well as specific anti-GAD65) has been reported to be associated with synaptic vesicles. Based on these results, a model linking gamma-aminobutyric acid (GABA) synthesis by MGAD to GABA packaging into synaptic vesicles by proton gradient-mediated GABA transport is presented. Activation of MGAD by phosphorylation appears to be mediated by a vesicular protein kinase that is controlled by the vesicular proton gradient.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Triphosphate / physiology*
  • Animals
  • Biological Transport
  • Brain / enzymology*
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone / pharmacology
  • Cell Membrane / enzymology
  • Diabetes Mellitus, Type 1 / blood
  • Enzyme Activation
  • Glutamate Decarboxylase / metabolism*
  • Humans
  • Protons
  • Rabbits
  • Swine
  • Synaptic Vesicles / metabolism*
  • gamma-Aminobutyric Acid / metabolism


  • Protons
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone
  • gamma-Aminobutyric Acid
  • Adenosine Triphosphate
  • Glutamate Decarboxylase