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Demonstration of coupling between GABA synthesis and vesicular GABA transport in synaptic vesicles



Demonstration of coupling between GABA synthesis and vesicular GABA transport in synaptic vesicles



Society for Neuroscience Abstracts 27(1): 25



Previously, we have shown that the activity of GAD65 is activated by conditions favoring protein phosphorylation and the integrity of the electrochemical proton gradient of synaptic vesicles (Hsu et al., J. Biol. Chem. 274:24366-24371 (1999)). Furthermore, We have reported that GAD65 is anchored to synaptic vesicles through first forming a protein complex with heat shock cognate 70, followed by subsequent protein interaction with cysteine string proteins (Hsu et al., J. Biol. Chem. 275:20822-20828 (2000)). Here, we report that highly purified GABAergic synaptic vesicles could be obtained using anti-GAD immunoaffinity procedure. The synaptic vesicle preparations thus obtained are highly enriched in GABAergic vesicles as indicated in a 8-fold increase of GAD activity. This is further confirmed from immunoblotting results using specific antibodies against synaptic vesicle specific protein, SV2. A direct demonstration of (3H)GABA uptake into synaptic vesicles was obtained by incubation of (3H)L-glutamate with purified GABAergic synaptic vesicles. Under similar conditions, no specific vesicular uptake of pre-existing GABA was observed. These results suggest that the newly synthesized GABA from glutamate by synaptic vesicle-associated GAD is preferably taken up by synaptic vesicles and support our hypothesis that GABA synthesis is functionally coupled to GABA packaging into synaptic vesicles.

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