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The Journal of Neuroscience, December 3, 2008, 28(49):13125-13131; doi:10.1523/JNEUROSCI.3887-08.2008
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Brief Communications
Unique Luminal Localization of VGAT-C Terminus Allows for Selective Labeling of Active Cortical GABAergic Synapses
Henrik Martens,1 Matthew C. Weston,2 Jean-Luc Boulland,3 Mads Grønborg,4 Jens Grosche,5,6 Johannes Kacza,7 Anke Hoffmann,7 Michela Matteoli,8 Shigeo Takamori,9 Tibor Harkany,10,11 Farrukh A. Chaudhry,3 Christian Rosenmund,2 Christian Erck,1 Reinhard Jahn,4 andWolfgang Härtig5 1Synaptic Systems GmbH, D-37079 Göttingen, Germany, 2Department of Molecular and Human Genetics and Department of Neuroscience, Baylor College of Medicine, Houston, Texas 77030, 3Biotechnology Centre of Oslo and Centre for Molecular Biology and Neuroscience, University of Oslo, N-0317 Oslo, Norway, 4Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany, 5Paul Flechsig Institute for Brain Research, University of Leipzig, D-04109 Leipzig, Germany, 6Interdisciplinary Center of Clinical Research (IZKF), Faculty of Medicine and 7Department of Anatomy, Histology and Embryology, University of Leipzig, D-04103 Leipzig, Germany, 8Department of Medical Pharmacology, National Research Council Institute of Neuroscience, University of Milan and Italian Research Hospital Don Gnocchi, I-20129 Milan, Italy, 9Center for Brain Integration Research, Graduate School of Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113–8519, Japan, 10Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-17177 Stockholm, Sweden, and 11Institute of Medical Sciences, College of Life Sciences and Medicine, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom
Correspondence should be addressed to Dr. Henrik Martens, Synaptic Systems GmbH, Rudolf Wissell Strasse 28, D-37079 Göttingen, Germany. Email: lab{at}sysy.com
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.
Key words: endocytosis; fluorescence detection; synaptic plasticity; vesicular GABA transporter; live cell imaging; synaptic vesicle
Received Aug. 15, 2008; accepted Oct. 22, 2008.
Correspondence should be addressed to Dr. Henrik Martens, Synaptic Systems GmbH, Rudolf Wissell Strasse 28, D-37079 Göttingen, Germany. Email: lab{at}sysy.com
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