Oligomerization and cell surface expression of recombinant GABAA receptors tagged in the δ subunit

Abstract

The γ-Aminobutyric acid type A receptors (GABAARs) are heteropentameric chloride channels responsible for primary inhibition in the mammalian brain. Studies have shown the expression of recombinant GABAAR subunits tagged with the green fluorescent protein (GFP), a 26.9 kDa protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. This allows the formation of recombinant proteins essential for the development of relevant in-vitro and in-vivo methodologies. Among the GABAAR subunits, the δ subunit was never tagged in its cytoplasmic domain, an evolutionary conserved domain found in between the third and the fourth transmembrane domains. In this study, first, we have cloned the mouse cDNAs encoding for the δ, α1, β2 subunits of GABAARs, and then developed two fusion proteins of δ subunit each tagged with the GFP variant, EGFP (enhanced GFP) at unique sites in the cytoplasmic domain. The recombinant proteins were expressed alone or in combination with α1 and/or β2 subunits in neuroblastoma 2a cells. Live cell confocal microscopy indicated that the cytoplasmically tagged δ subunits were targeted to the cell membrane when expressed in the presence of α1 and β2 subunits in neuroblastoma 2a cells. However, this was not observed when they were expressed alone or only with α1 or β2 subunits in the same cell line. These results confirm the general oligomerization and targeting pattern of GABAAR subtypes described in the other in–vitro studies in the literature. Thus, our results suggest that the EGFP tagging in the ctoplasmic domain did not interfere with the oligomerization and cell surface expression of recombinant δ subunits. To our knowledge, this is the first study showing the generation, expression and preliminary analysis of the δ-GABAARs tagged in the cytoplasmic domain of the δ subunit which can be further elaborated to probe intracellular protein interactions of GABAARs via the δ subunit.

Keywords

• gabaa receptor
• cys-loop receptors
• ion channel
• delta subunit
• extrasynaptic
• recombinant protein expression
• egfp
• oligomerization
• protein tagging
• fusion protein
• cdna cloning
• ta cloning
• confocal microscopy
• live cell imaging
• fluorescence imaging
• neuroblastoma