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Journal of Integrative Neuroscience  2019, Vol. 18 Issue (4): 341-350    DOI: 10.31083/j.jin.2019.04.1207
Original Research | Next articles
Oligomerization and cell surface expression of recombinant GABAA receptors tagged in the δ subunit
Furkan Enes Oflaz1, 2, Çağdaş Devrim Son3, Ayla Arslan1, *()
Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, International University of Sarajevo, Hrasnička cesta 15, Ilidža 71210 Sarajevo, Bosnia and Herzegovina
Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Medical University of Graz, Neue Stiftingtalstraße 6/6 8010 Graz Austria
Department of Biological Sciences, Middle East Technical University, Üniversiteler Mahallesi, Dumlupınar Bulvarı No:1 06800 Çankaya, Ankara, Turkey
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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.

Key words:  GABAA receptor      Cys-loop receptorsion 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     
Submitted:  10 October 2019      Accepted:  19 December 2019      Published:  30 December 2019     
Fund: 
  • International University of Sarajevo
  • Middle East Technical University
*Corresponding Author(s):  Ayla Arslan     E-mail:  ayla.arslan@alumni.uni-heidelberg.de

Cite this article: 

Furkan Enes Oflaz, Çağdaş Devrim Son, Ayla Arslan. Oligomerization and cell surface expression of recombinant GABAA receptors tagged in the δ subunit. Journal of Integrative Neuroscience, 2019, 18(4): 341-350.

URL: 

https://jin.imrpress.com/EN/10.31083/j.jin.2019.04.1207     OR     https://jin.imrpress.com/EN/Y2019/V18/I4/341

Table 1  Optimized Nested PCR cycling conditions for δ subunit tagging
PCR Conditions Duration Temperature Cycle
Initial Denaturation 3 Minutes 95° C
Denaturation 30 seconds 95° C
18 cycles
Annealing 1 minute 51° C
Extension 15 Minutes 68° C
Final Extension 5 Minutes 68° C
Figure 1.  Schematic representation of the generated constructs of GABAARs. The cDNAs of α1, β2, δ, δ398EGFP399, and δ346EGFP347 are shown on the left column. The cDNAs were initially cloned into pTZ57R/T vector with T7 promoter and then cloned in to the mammalian expression vector under the control of CMV promoter. On the right, schematic representation of the α1, β2, δ, δ346EGFP347 and δ398EGFP399 subunits and the location of their extracellular N- and C- terminus as well as cytoplasmic domains in the absence or presence of EGFP tag are shown.

Figure 2.  Neuroblastoma 2a cells transfected with δ398EGFP399 construct in the absence or presence of-α-and/or β subunits. Yellow arrows show nucleous, red arrows show cell membrane, purple arrows show cytoplasmic/ER expression. (A): Transfection of δ398EGFP399 shows cytoplasmic expression as observed by the green signal (purple arrow) which is not observed in the nucleous (yellow arrow) (B): Co-transfection of δ398EGFP399 with α1 shows cytoplasmic expression as observed by the green signal (purple arrow) which is not observed in the nucleous (yellow arrow). (C): Co-transfection of δ398EGFP399 with β2 shows cytoplasmic expression as observed by the green signal (purple arrow) which is not observed in the nucleous (yellow arrow). (D): Co-transfection of δ398EGFP399 with α1and β2 in neuroblastoma 2a cells show membrane targeting (red arrows) as well as cytoplasmic expression excluding the nucleous (purple and yellow arrows, respectively). (E): Transfection of EGFP plasmid as control. The various EGFP intensity between the figures is due to the co-transfections which may lead to differential efficiency and thus variations in the signal intensity. Transfection of EGFP plasmid alone (E) showed a distribution of the EGFP signal not only in the cytoplasm but in the whole cell as EGFP can translocate to the nucleous.

Figure 3.  Neuroblastoma 2a cells transfected with δ346EGFP347 construct in the absence or presence of α and/or β subunits. Yellow arrows show nucleous, red arrows show cell membrane, purple arrows show cytoplasmic/ER expression. (A): Transfection of δ346EGFP347 shows the cytoplasmic expression as observed by the green signal (purple arrow) which is not observed in the nucleous (yellow arrow). (B): Co-transfection of δ346EGFP347 with α1 shows cytoplasmic expression as observed by the green signal (purple arrow) which is not observed in the nucleous (yellow arrow). (C): Co-transfection of δ346EGFP347 with β2 shows the cytoplasmic expression as observed by the green signal (purple arrow) which is not observed in the nucleous (yellow arrow). (D): Co-transfection of δ346EGFP347 with α1 and β2 which shows expression in the membrane (red arrows). Also some intracellular expression (purple) is seen. (E): Transfection of the EGFP plasmid as the control. The various EGFP intensity between the cells is due to the co-transfections which may lead to differential efficiency and thus variations in the signal intensity. Transfection of the EGFP plasmid alone (E) showed a strong EGFP signal in the whole cells as EGFP can translocate to the nucleous.

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