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.
Highlights
The γ-Aminobutyric acid type A receptors (GABAARs) are GABA gated heteropentameric chloride channels and responsible for major inhibition in the brain
In order to show that Enhanced Green Fluorescent Protein (EGFP) tagging in the cytoplasmic domain does not interfere with any possible interaction involved in the subunit assembly and localization, we tested the pattern of oligomerization and cell surface expression of recombinant receptors to see if they are oligomerized and localized in the cell membrane, and they were
Our data provide the first study focusing on the EGFP tagging of the cytoplasmic domain of the δ subunit, in which the tagging was performed in two distinct locations
Summary
The γ-Aminobutyric acid type A receptors (GABAARs) are GABA gated heteropentameric chloride channels and responsible for major inhibition in the brain. They belong to the superfamily of ligand-gated ion channels ("Cys-loop receptors") including the nicotinic acetylcholine receptors (nAChR), the 5hydroxytryptamine type 3 (5-HT3) receptors, and the glycine receptors (Sigel and Steinmann, 2012). The topological organization is confirmed by structural studies of Cys-loop receptors, including the subunits of acetyl choline receptors and the recently published β3 subunit of the GABAAR (Miller and Aricescu, 2014; Unwin, 2013). Structures of assembled heteropentameric GABAAR had been hard to pin down until recently, when several cryo-electron microscopy studies (Laverty et al, 2019; Masiulis et al, 2019; Phulera et al, 2018; Zhu et al, 2018), have led to the structural resolution of heteropentameric receptors
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