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L-Glutamic acid decarboxylase (GAD) exists as both membrane-associated and soluble forms in the mammalian brain. Here, we propose that there is a functional and structural coupling between the synthesis of [gamma]-aminobutyric acid (GABA) by membrane-associated GAD and its packaging into synaptic vesicles (SVs) by vesicular GABA transporter (VGAT). This notion is supported by the following observations. First, newly synthesized [[sup.3]H]GABA from [3H]L-glutamate by membrane-associated GAD is taken up preferentially over preexisting GABA by using immunoaffinity-purified GABAergic SVs. Second, the activity of SV-associated GAD and VGAT seems to be coupled because inhibition of GAD also decreases VGAT activity. Third, VGAT and SV-associated [Ca.sup.2+]/ calmodulin-dependent kinase II have been found to form a protein complex with GAD. A model is also proposed to link the neuronal stimulation to enhanced synthesis and packaging of GABA into SVs.

Presents a demonstration of functional coupling between gamma-aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles. Isolation of GABAergic SVs by anti-GAF[sub 65]-conjugated beads; Comparison of vesicular uptake of newly synthesized GABA and the preexisting GABA in cystosolic pools.

Abstract: In GAD65-knockout mice, lack of GAD65 expression was confirmed. The expression level of vesicular GABA transporter (VGAT) was upregulated, and no change in the synaptic vesicles (SV)-associated GAD67 was found. GAD65(−/−) SV transported cytosolic GABA much more efficiently than that of the wild type, further supporting our model that there is a structural and functional coupling between GABA synthesis and packaging into SV. Both full-length and truncated forms of GAD65 could bind to GABAergic SV, indicating the N-terminus is not required for the anchoring of GAD65 to SV. Although both GAD65(−/−) SV reconstituted with either GAD65 or GAD67 could synthesize GABA from [3H] glutamate and transport this newly synthesized GABA into SV, the combined evidence suggests that GAD65 plays a major role in GABA transmission in normal physiological condition. However, GAD67 could serve this role under some pathological conditions. [Copyright &y& Elsevier]