Unique luminal localization of VGAT-C terminus allows for selective labeling of active cortical GABAergic synapses.
Neurotransmitter uptake into synaptic vesicles is mediated by vesicular neurotransmitter transporters. Although these transporters belong to different families, they all are thought to share a common overall topology with an even number of transmembrane domains. Using epitope-specific antibodies and mass spectrometry we show that the vesicular GABA transporter (VGAT) possesses an uneven number of transmembrane domains, with the N terminus facing the cytoplasm and the C terminus residing in the synaptic vesicle lumen. Antibodies recognizing the C terminus of VGAT (anti-VGAT-C) selectively label GABAergic nerve terminals of live cultured hippocampal and striatal neurons as confirmed by immunocytochemistry and patch-clamp electrophysiology. Injection of fluorochromated anti-VGAT-C into the hippocampus of mice results in specific labeling of GABAergic synapses in vivo. Overall, our data open the possibility of studying novel GABA release sites, characterizing inhibitory vesicle trafficking, and establishing their contribution to inhibitory neurotransmission at identified GABAergic synapses.
Martens
Henrik
H
Synaptic Systems GmbH, Göttingen, Germany. lab@sysy.com
Weston
Matthew C
MC
Boulland
Jean-Luc
JL
Grønborg
Mads
M
Grosche
Jens
J
Kacza
Johannes
J
Hoffmann
Anke
A
Matteoli
Michela
M
Takamori
Shigeo
S
Harkany
Tibor
T
Chaudhry
Farrukh A
FA
Rosenmund
Christian
C
Erck
Christian
C
Jahn
Reinhard
R
Härtig
Wolfgang
W
D000818:Animals; D000918:Antibody Specificity; D003342:Corpus Striatum / Q000378:metabolism / Q000648:ultrastructure; D004705:Endocytosis / Q000502:physiology; D005089:Exocytosis / Q000502:physiology; D006624:Hippocampus / Q000378:metabolism / Q000648:ultrastructure; D007150:Immunohistochemistry / Q000379:methods*; D013058:Mass Spectrometry; D051379:Mice; D009433:Neural Inhibition / Q000502:physiology; D018408:Patch-Clamp Techniques; D016548:Prosencephalon / Q000378:metabolism* / Q000648:ultrastructure; D017434:Protein Structure, Tertiary / Q000502:physiology; D013194:Staining and Labeling / Q000379:methods*; D013569:Synapses / Q000378:metabolism* / Q000648:ultrastructure; D013570:Synaptic Membranes / Q000378:metabolism / Q000648:ultrastructure; D009435:Synaptic Transmission / Q000502:physiology; D013572:Synaptic Vesicles / Q000378:metabolism / Q000648:ultrastructure; D050495:Vesicular Inhibitory Amino Acid Transport Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D050495:Vesicular Inhibitory Amino Acid Transport Proteins; C493406:vesicular GABA transporter; D005680:gamma-Aminobutyric Acid