Regular Article
Analysis of GABAA Receptor Assembly in Mammalian Cell Lines and Hippocampal Neurons Using γ2 Subunit Green Fluorescent Protein Chimeras

https://doi.org/10.1006/mcne.2000.0882Get rights and content

Abstract

Type A γ-aminobutyric acid receptors (GABAA), the major sites of fast synaptic inhibition in the brain, are believed to be predominantly composed of α, β, and γ subunits. To examine the membrane trafficking of GABAA receptors we have produced γ2L subunit chimeras with green fluorescent protein (GFP). Addition of GFP to the N-terminus of the γ2 subunit (γ2L-GFPN) was functionally silent for α1β2γ2L-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 α and β subunits. Using γ2L-GFPN we were able to demonstrate that targeting of the γ2 subunit to GABAergic synapses in hippocampal neurons was dependent upon coassembly with receptor α and β subunits. Together our results demonstrate that the assembly and membrane targeting of GABAA receptors composed of α1β2γ2L-GFPN subunits follow similar itineraries in heterologous systems and neurons.

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