Role of Synaptic Vesicle Proton Gradient and Protein Phosphorylation on ATP-mediated Activation of Membrane-associated Brain Glutamate Decarboxylase

Che-Chang Hsu,Charles Thomas,Weiqing Chen,Kathleen M Davis,Todd Foos,Jeffrey L Chen,Elliott Wu,Erik Floor,John V Schloss,Jang-Yen Wu

doi:10.1074/jbc.274.34.24366

Abstract

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.

Highlights

␥-Aminobutyric acid (GABA),1 the major inhibitory neurotransmitter in brain, is synthesized by a single enzymatic reaction catalyzed by L-glutamate decarboxylase (EC 4.1.1.15; glutamic acid decarboxylase (GAD)) [1]
membraneassociated form of GAD (MGAD) is activated by ATP through phosphorylation modulated by the synaptic vesicle proton gradient and is inhibited by dephosphorylation
ATP Activation of MGAD—When SGAD and MGAD were assayed under conditions favoring protein phosphorylation, e.g. in the presence of ATP or protein phosphatase inhibitors, it was found that SGAD was inhibited whereas MGAD was activated (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Materials—Fresh porcine brains were obtained from a local abattoir. Benzethonium hydroxide (Hyamine base, 1 M solution in methanol), PLP, 2-aminoethylisothiuronium bromide, AMP-PNP, calf intestinal phosphatase (CIP), Triton X-100, bafilomycin A1, gramicidin, and carbonyl cyanide m-chorophenylhydrazone (CCCP) were purchased from Sigma. The pellet was washed six times in GAD buffer, which contains 1 mM 2-aminoethylisothiuronium bromide, 0.2 mM PLP in 50 mM Tris/citrate (KP) buffer at pH 7.2. Both the supernatant and pellet were assayed for GAD activity. The pellet obtained was sonicated and washed in GAD buffer solution This was the crude synaptosomal membrane, referred to as P2M. The mixture was further incubated with Triton X-100 (final concentration, 0.5%) for an additional 1 h to allow solubilization of MGAD and centrifuged at 100,000 ϫ g for 60 min. The immunoprecipitates were washed six times in GAD buffer, followed by GAD assay of the supernatant and the pellet. The Km values were obtained by a nonlinear least squares fit of the data to the MichaelisMenten rate equation by use of Prism by GraphPad Software, Inc

RESULTS

TABLE I Activation of ATP on synaptosomal MGAD under various conditions

SGAD Solubilized MGAD

DISCUSSION