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Transfection of GABAA Receptor with GFP-Tagged Subunits in Neurons and HEK 293 Cells

  • Stefano Vicini
  • Jin Hong Li
  • Wei Jian Zhu
  • Karl Krueger
  • Jian Feng Wang

Abstract

We describe an approach that may allow to study changes in the γ-aminobutyric acid (GABAA)receptor distribution with development and pharmacological treatments in living neurons. We produced expression vectors containing chimeras of the green fluorescent protein (GFP)linked to the C terminus of GABAA receptors α1, γ2, or the δ subunits. Human embryonic kidney (HEK) 293 cells were successfully transfected with α1-GFP cDNAs together with β3 subunit as indicated by the formation of green fluorescent clusters of receptor subunits that colocalized with immunospecific staining for the α1 subunits and by whole-cell recordings of GABA-activated Cl currents. Although the current density was lower in these cells, GABA, bicuculline, and ZnCl2 actions were unaltered. Similarly, transfection with cDNAs encoding for the γ-GFP chimera together with α1 β3 subunit cDNAs produced clusters of subunits and GABA-activated chloride currents that were insensitive to blockade by ZnCl2 and that were potentiated by zolpidem. Lastly, δ-GFP tion by the neurosteroid THDOC. We then successfully transfected primary cultures of neocortical and cerebellar neurons with these GABAA receptor subunits—GFP chimeras. We obtained evidence of elongated cluster formation in both cell types that matched well, although not completely, endogenous receptor clusters as indicated by β2∖3 staining, and also partially corresponded to synaptophysin positive punctae indicating synaptic localization of transfected subunits. Electrophysiological recordings from transfected neurons indicated that functional GABAergic synapses were still maintained. This approach will allow to follow targeting and distribution of GABAA receptor clusters in living neurons during development in culture and in different experimental conditions.

Keywords

Green Fluorescent Protein GABAA Receptor Receptor Subunit Receptor Cluster Living Neuron 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. l.
    Ango, E., S. Albani-Torregrossa, C. Joly, D. Robbe, J. M. Michel, J.-P. Pin, J. Bockaert, and L. Fagni. 1999. A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu5 receptor in cerebellar granule cells Neuropharmacology 38:793–803.PubMedCrossRefGoogle Scholar
  2. 2.
    Awaji, T., A. Hirasawa, M. Kataoka, H. Shinoura, Y. Nakayama, T. Sugawara, S. Izumi, and G. Tsujimoto. 1998. Real-time optical monitoring of ligand-mediated internalization of (αlb-adrenoceptor with green fluorescent protein Mol. Endocrinol. 12:1099–1111.PubMedCrossRefGoogle Scholar
  3. 3.
    Barak, L.S., S.S. Ferguson, J. Zhang, C. Martenson, T. Meyer, and M.G. Caron. 1997. Internal trafficking and surface mobility of a functionally intact β2-adrenergic receptor-green fluorescent protein conjugate. Mol. Pharmacol. 51:177–184.PubMedGoogle Scholar
  4. 4.
    Chen, C. and H. Okayama. 1987. High efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell Biol. 7:2745–2752.PubMedGoogle Scholar
  5. 5.
    Connor, J.X., A.J. Boileau, and C. Czajkowski. 1998. A GABAA receptor αl subunit tagged with green fluorescent protein requires a beta subunit for functional surface expression. J. Biol. Chem. 273:28906–28911.PubMedCrossRefGoogle Scholar
  6. 6.
    Cubitt, A.B., R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, and R.Y. Tsien. 1995. Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20:448–455.PubMedCrossRefGoogle Scholar
  7. 7.
    Cubitt, A.B., L.A. Woollenweber, and R. Heim. 1999. Understanding structure-function relationships in the Aequorea victoria green fluorescent protein. Methods Cell Biol. 58:19–30.PubMedCrossRefGoogle Scholar
  8. 8.
    David-Watine, B., S.L. Shorte, S. Fucile, D. de SaintJan, H. Korn, and P. Bregestovski. 1999. Functional integrity of green fluorescent protein conjugated glycine receptor channels. Neuropharmacology 38:785–792.PubMedCrossRefGoogle Scholar
  9. 9.
    Grabner, M., R.T. Dirksen, and K.G. Beam. 1998. Tagging with green fluorescent protein reveals a distinct subcellular distribution of L-type and non-L-type Ca2+ channels expressed in dysgenic myotubes. Proc. Nad. Acad. Sei. USA 17:1903–1908.CrossRefGoogle Scholar
  10. l0.
    Hirasawa, A., T. Sugawara, T. Awaji, K. Tsumaya, H. Ito, and G. Tsujimoto. 1997. Subtype-specific differences in subcellular localization of αl-adrenoceptors: chlorethylclonidine preferentially alkylates the accessible cell surface αl-adrenoceptors irrespective of the subtype. Mol. Pharmacol. 52:764–770.PubMedGoogle Scholar
  11. ll.
    MacDonald, R.L. and R.W. Olsen. 1994. GABAA receptor channels. Annu. Rev. Neurosci. 17:569–602.PubMedGoogle Scholar
  12. 12.
    Marshall, J., R. Molloy, G.W. Moss, J.R. Howe, and T.E. Hughes. 1995. The jellyfish green fluorescent protein: a new tool for studying ion channel expression and function. Neuron 14:211–215.PubMedCrossRefGoogle Scholar
  13. 13.
    Rao, A. and A.M. Craig. 1997. Activity regulates the synaptic localization of the NMDA receptor in hippocampal Neurons. Neuron 19:801–812.PubMedCrossRefGoogle Scholar
  14. 14.
    Rongo, C., C.W. Whitfield, A. Rodai, S.K. Kim, and J.M. Kaplan. 1998. LIN-10 is a shared component of the polarized protein localization pathways in neurons and epithelia. Cell 94:751–759.PubMedCrossRefGoogle Scholar
  15. 15.
    Shi, S.H., Y. Hayashi, R.S. Petralia, S.H. Zaman, R.J. Wenthold, K. Svoboda, and R. Malinow. 1999. Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Science 284:1811–1816.PubMedCrossRefGoogle Scholar
  16. l6.
    Van den Pol, A.N. and P.K. Ghosh. 1998. Selective neuronal expression of green fluorescent protein with cytomegalovirus promoter reveals entire neuronal arbor in transgenic mice. J. Neurosci. 18:10640–10651.PubMedGoogle Scholar
  17. 17.
    Wan, Q., Z.G. Xiong, H.Y. Man, C.A. Ackerley, J. Braunton, W.Y. Lu, L.E. Becker, J.E MacDonald, and Y.T. Wang. 1997. Recruitment of functional GABA(A) receptors to postsynaptic domains by insulin. Nature 388:686–690.PubMedCrossRefGoogle Scholar
  18. 18.
    Zhu, W.J., J.E Wang, K.E. Krueger, and S. Vicini. 1996. Subunit inhibits neurosteroid modulation of GABAA receptors. J. Neurosci. 16:6648–6656.PubMedGoogle Scholar

Copyright information

© Eaton Publishing 2001

Authors and Affiliations

  • Stefano Vicini
    • 1
  • Jin Hong Li
    • 1
  • Wei Jian Zhu
    • 1
  • Karl Krueger
    • 1
  • Jian Feng Wang
    • 1
  1. 1.Department of Physiology and BiophysicsGeorgetown University Medical SchoolWashington, DCUSA

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