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

Previously, me have shown that the soluble form of brain glutamic acid deca rboxylase (GAD) is inhibited by ATP through protein phosphorylation and is activated by calcineurin-mediated protein dephosphorylation (Bao, J., Cheun g, W. Y., and Wu, J. Y. (1995) J. Biol. Chem. 270, 6464-6467); Here we repo rt that the membrane-associated form of GAD (MGAD) is greatly activated by ATP, whereas adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP), a non-h ydrolyzable ATP analog, has no effect on MGAD activity. ATP activation of M GAD is abolished by conditions that disrupt the proton gradient of synaptic vesicles, e.g. the presence of vesicular proton pump inhibitor, bafilomyci n Al, the protonophore carbonyl cyanide m-chorophenylhydrazone or the ionop hore gramicidin, indicating that the synaptic vesicle proton gradient is es sential in ATP activation of MGAD. Furthermore, direct incorporation of P-3 2 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 w ith synaptic vesicles. Based on these results, a model linking gamma-aminob utyric acid (GABA) synthesis by MGAD to GABA packaging into synaptic vesicl es by proton gradient-mediated GABA transport is presented. Activation of M GAD by phosphorylation appears to be mediated by a vesicular protein kinase that is controlled by the vesicular proton gradient.