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The Subcellular Localization of GABA Transporters and Its Implication for Seizure Management

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Abstract

The ability to modulate the synaptic GABA levels has been demonstrated by using the clinically effective and selective GAT1 inhibitor tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid]. N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (EF1502) which not only inhibits GAT1 like tiagabine but also BGT1 has been shown to modulate extrasynaptic GABA levels. The simultaneous inhibition of synaptic and extrasynaptic GABA transporters using tiagabine and EF1502, respectively has been demonstrated to exert a synergistic anticonvulsant effect in several seizure models in mice. The pharmacological profile of these and similar compounds has been thoroughly investigated in in vitro systems, comparing the GAT subtype selectivity with the ability to inhibit GABA uptake in primary cultures of neurons and astrocytes. However, an exact explanation has not yet been found. In the present study, the ability of GATs to form homo and/or heterodimers was investigated as well as to which membrane micro environment the GATs reside. To investigate dimerization of GATs, fusion proteins of GATs tagged with either yellow fluorescent protein or cerulean fluorescent protein were made and fluorescence resonance energy transfer (FRET) was measured. It was found that GATs form both homo- and hetero-dimers in N2A and HEK-293 cells. Microdomain localization of GATs as investigated by detergent resistant membrane fractions after treatment of tissue with Brij-98 or Triton X-100 revealed that BGT1 and GAT1 mostly localize to non-membrane rafts independent of the detergent used. However, GAT3 localizes to membrane rafts when using Brij-98. Taken together, these results suggest that the observed hetero dimerization of GATs in the FRET study is unlikely to have functional implications since the GATs are located to very different cellular compartments and cell types.

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Acknowledgments

We would like to thank Ph.D. Steven S. Vogel, National Institute of Alcohol Abuse and Alcoholism, Bethesda, Maryland (20892), for providing cDNA of Cerulean and Venus and Professor Kathryn M. Partin, Colorado State University, Fort Collins (80523-1617), for providing the positive FRET constructs of AMPA receptors tagged with CFP and YFP (R1i15CFP and R1i46YFP). The work has been financially supported by the Carlsberg Foundation (2009_01_0501).

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Authors and Affiliations

  1. Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen Ø, Denmark

    Karsten K. Madsen & Arne Schousboe

  2. Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen Ø, Denmark

    Gert H. Hansen & E. Michael Danielsen

Authors
  1. Karsten K. Madsen
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  2. Gert H. Hansen
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  3. E. Michael Danielsen
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  4. Arne Schousboe
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Correspondence to Karsten K. Madsen.

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Special Issue: In Honor of Michael Norenberg.

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Madsen, K.K., Hansen, G.H., Danielsen, E.M. et al. The Subcellular Localization of GABA Transporters and Its Implication for Seizure Management. Neurochem Res 40, 410–419 (2015). https://doi.org/10.1007/s11064-014-1494-9

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  • DOI: https://doi.org/10.1007/s11064-014-1494-9

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