Abstract
Synaptic vesicles from mammalian brain are among the best characterized trafficking organelles. However, so far it has not been possible to characterize vesicle subpopulations that are specific for a given neurotransmitter. Taking advantage of the recent molecular characterization of vesicular neurotransmitter transporters, we have used an antibody specific for the vesicular GABA transporter (VGAT) to isolate GABA-specific synaptic vesicles. The isolated vesicles are of exceptional purity as judged by electron microscopy. Immunoblotting revealed that isolated vesicles contain most of the major synaptic vesicle proteins in addition to VGAT and are devoid of vesicular monoamine and acetylcholine transporters. The vesicles are 10-fold enriched in GABA uptake activity when compared with the starting vesicle fraction. Furthermore, glutamate uptake activity and glutamate-induced but not chloride-induced acidification are selectively lost during immunoisolation. We conclude that the population of GABA-containing synaptic vesicles is separable and distinct from vesicle populations transporting other neurotransmitters.
MeSH terms
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Adenosine Triphosphate / pharmacology
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Amino Acid Sequence
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Animals
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Carrier Proteins / analysis
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Carrier Proteins / chemistry
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Carrier Proteins / genetics
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Carrier Proteins / physiology*
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Cell Line
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GABA Plasma Membrane Transport Proteins
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Humans
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Membrane Glycoproteins / analysis
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Membrane Glycoproteins / physiology
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Membrane Proteins / chemistry
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Membrane Proteins / genetics
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Membrane Proteins / physiology*
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Membrane Transport Proteins*
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Microscopy, Immunoelectron
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Molecular Sequence Data
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Neuropeptides*
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Organic Anion Transporters*
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Rats
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Synaptic Vesicles / physiology*
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Synaptic Vesicles / ultrastructure
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Synaptophysin / analysis
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Transfection
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Vesicular Acetylcholine Transport Proteins
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Vesicular Biogenic Amine Transport Proteins
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Vesicular Transport Proteins*
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gamma-Aminobutyric Acid / physiology*
Substances
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Carrier Proteins
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GABA Plasma Membrane Transport Proteins
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Membrane Glycoproteins
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Membrane Proteins
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Membrane Transport Proteins
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Neuropeptides
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Organic Anion Transporters
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SLC18A3 protein, human
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Slc18a3 protein, rat
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Synaptophysin
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Vesicular Acetylcholine Transport Proteins
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Vesicular Biogenic Amine Transport Proteins
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Vesicular Transport Proteins
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gamma-Aminobutyric Acid
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Adenosine Triphosphate