Analysis of GABAA receptor assembly in mammalian cell lines and hippocampal neurons using γ2 subunit green fluorescent protein chimeras

Josef T. Kittler, Jianfeng Wang, Christopher N. Connolly, Stefano Vicini, Trevor G. Smart, Stephen J. Moss

    Research output: Contribution to journalArticle

    65 Citations (Scopus)

    Abstract

    Type A ?-aminobutyric acid receptors (GABA(A)), the major sites of fast
    synaptic inhibition in the brain, are believed to be predominantly
    composed of a, ß, and ? subunits. To examine the membrane trafficking of
    GABA(A) receptors we have produced ?2L subunit chimeras with green
    fluorescent protein (GFP). Addition of GFP to the N-terminus of the ?2
    subunit (?2L-GFPN) was functionally silent for a1ß2?2L-GFPN receptors
    expressed in A293 cells. Furthermore, this chimera allowed the
    visualization of receptor membrane targeting and endocytosis in live
    cells. In contrast, incorporation of GFP at the C-terminus reduced
    subunit stability, impairing assembly with receptor a and ß subunits.
    Using ?2L-GFPN we were able to demonstrate that targeting of the ?2
    subunit to GABAergic synapses in hippocampal neurons was dependent upon
    coassembly with receptor a and subunits. Together our results
    demonstrate that the assembly and membrane targeting of GABA(A)
    receptors composed of a1ß2?2L-GFPN subunits follow similar itineraries
    in heterologous systems and neurons.

    Original languageEnglish
    Pages (from-to)440-452
    Number of pages13
    JournalMolecular and Cellular Neuroscience
    Volume16
    Issue number4
    DOIs
    Publication statusPublished - 2000

    Cite this

    Kittler, Josef T. ; Wang, Jianfeng ; Connolly, Christopher N. ; Vicini, Stefano ; Smart, Trevor G. ; Moss, Stephen J. / Analysis of GABAA receptor assembly in mammalian cell lines and hippocampal neurons using γ2 subunit green fluorescent protein chimeras. In: Molecular and Cellular Neuroscience. 2000 ; Vol. 16, No. 4. pp. 440-452.
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    title = "Analysis of GABAA receptor assembly in mammalian cell lines and hippocampal neurons using γ2 subunit green fluorescent protein chimeras",
    abstract = "Type A ?-aminobutyric acid receptors (GABA(A)), the major sites of fast synaptic inhibition in the brain, are believed to be predominantly composed of a, {\ss}, and ? subunits. To examine the membrane trafficking of GABA(A) receptors we have produced ?2L subunit chimeras with green fluorescent protein (GFP). Addition of GFP to the N-terminus of the ?2 subunit (?2L-GFPN) was functionally silent for a1{\ss}2?2L-GFPN receptors expressed in A293 cells. Furthermore, this chimera allowed the visualization of receptor membrane targeting and endocytosis in live cells. In contrast, incorporation of GFP at the C-terminus reduced subunit stability, impairing assembly with receptor a and {\ss} subunits. Using ?2L-GFPN we were able to demonstrate that targeting of the ?2 subunit to GABAergic synapses in hippocampal neurons was dependent upon coassembly with receptor a and subunits. Together our results demonstrate that the assembly and membrane targeting of GABA(A) receptors composed of a1{\ss}2?2L-GFPN subunits follow similar itineraries in heterologous systems and neurons.",
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    Analysis of GABAA receptor assembly in mammalian cell lines and hippocampal neurons using γ2 subunit green fluorescent protein chimeras. / Kittler, Josef T.; Wang, Jianfeng; Connolly, Christopher N.; Vicini, Stefano; Smart, Trevor G.; Moss, Stephen J.

    In: Molecular and Cellular Neuroscience, Vol. 16, No. 4, 2000, p. 440-452.

    Research output: Contribution to journalArticle

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    AU - Kittler, Josef T.

    AU - Wang, Jianfeng

    AU - Connolly, Christopher N.

    AU - Vicini, Stefano

    AU - Smart, Trevor G.

    AU - Moss, Stephen J.

    PY - 2000

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    AB - Type A ?-aminobutyric acid receptors (GABA(A)), the major sites of fast synaptic inhibition in the brain, are believed to be predominantly composed of a, ß, and ? subunits. To examine the membrane trafficking of GABA(A) receptors we have produced ?2L subunit chimeras with green fluorescent protein (GFP). Addition of GFP to the N-terminus of the ?2 subunit (?2L-GFPN) was functionally silent for a1ß2?2L-GFPN receptors expressed in A293 cells. Furthermore, this chimera allowed the visualization of receptor membrane targeting and endocytosis in live cells. In contrast, incorporation of GFP at the C-terminus reduced subunit stability, impairing assembly with receptor a and ß subunits. Using ?2L-GFPN we were able to demonstrate that targeting of the ?2 subunit to GABAergic synapses in hippocampal neurons was dependent upon coassembly with receptor a and subunits. Together our results demonstrate that the assembly and membrane targeting of GABA(A) receptors composed of a1ß2?2L-GFPN subunits follow similar itineraries in heterologous systems and neurons.

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