Oligomerization and cell surface expression of recombinant GABA(A) receptors tagged in the delta subunit

Download

10.31083j.jin.2019.04.1207.pdf

Date

2019-12-01

Author

Oflaz, Furkan Enes
Son, Çağdaş Devrim
Arslan, Ayla

Metadata

Show full item record

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Item Usage Stats

311
views

205
downloads

The gamma-Aminobutyric acid type A receptors (GABA(A) Rs) are heteropentameric chloride channels responsible for primary inhibition in the mammalian brain. Studies have shown the expression of recombinant GABA(A) R 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 GABA(A) R subunits, the delta 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 delta, alpha 1, beta 2 subunits of GABA(A) Rs, and then developed two fusion proteins of delta 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 alpha 1 and/or beta 2 subunits in neuroblastoma 2a cells. Live cell confocal microscopy indicated that the cytoplasmically tagged delta subunits were targeted to the cell membrane when expressed in the presence of alpha 1 and beta 2 subunits in neuroblastoma 2a cells. However, this was not observed when they were expressed alone or only with alpha 1 or beta 2 subunits in the same cell line. These results confirm the general oligomerization and targeting pattern of GABA(A) R 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 delta subunits. To our knowledge, this is the first study showing the generation, expression and preliminary analysis of the delta-GABA(A) Rs tagged in the cytoplasmic domain of the delta subunit which can be further elaborated to probe intracellular protein interactions of GABA(A) Rs via the delta subunit.

Subject Keywords

General Neuroscience, General Medicine

URI

https://hdl.handle.net/11511/41973

Journal

JOURNAL OF INTEGRATIVE NEUROSCIENCE

DOI

https://doi.org/10.31083/j.jin.2019.04.1207

Collections

Department of Biology, Article

Suggestions

OpenMETU
Core

Electronic excited states and excitation transfer kinetics in the Fenna-Matthews-Olson protein of the photosynthetic bacterium Prosthecochloris aestuarii at low temperatures Iseri, EI; Gulen, D (Springer Science and Business Media LLC, 1999-01-01) The molecular structure-function relationship of the Fenna-Matthews-Olson light-harvesting complex of the photosynthetic green bacterium Prosthecochloris aestuarii has been investigated. It has been assumed that the electronic excited states responsible for the function (transfer of electronic excitation energy) result from the dipole-dipole interactions between the bacteriochlorophyll molecules bound to the polypeptide chain of the complex at a specific three-dimensional geometry. The molecular structure-e...
Ca2+ binding induced sequential allosteric activation of sortase A: An example for ion-triggered conformational selection Ugur, Iike; Schatte, Martin; Marıon, Antoıne; Glaser, Manuel; Boenitz-Dulat, Mara; Antes, Iris (Public Library of Science (PLoS), 2018-10-15) The allosteric activation of the intrinsically disordered enzyme Staphylococcus aureus sortase A is initiated via binding of a Ca2+ ion. Although Ca2+ binding was shown to initiate structural changes inducing disorder-to-order transitions, the details of the allosteric activation mechanism remain elusive. We performed long-term molecular dynamics simulations of sortase A without (3 simulations of 1.6 mu s) and with bound Ca2+ (simulations of 1.6 mu s, 1.8 mu s, and 2.5 mu s). Our results show that Ca2+ bind...
Biophysical and microbiological study of high hydrostatic pressure inactivation of Bovine Viral Diarrheavirus type 1 on serum CEYLAN, ÇAĞATAY; Severcan, Feride; ÖZKUL, AYKUT; Severcan, Mete; Bozoglu, Faruk; Taheri, Nusret (Elsevier BV, 2012-01-27) The effect of high hydrostatic pressure application on fetal bovine serum components and the model microorganism (Bovine Viral Diarrheavirus type 1 NADL strain) was studied at 132 and 220 MPa pressure for 5 min at 25 degrees C. Protein secondary structures were found to be unaffected by an artificial neural network application on the amide I region for both untreated and HHP treated samples. FTIR spectroscopy study of both the HHP-treated and control samples revealed changes in the intensity of some bands i...
Mitochondria-Targeting Selenophene-Modified BODIPY-Based Photosensitizers for the Treatment of Hypoxic Cancer Cells Karaman, Osman; Almammadov, Toghrul; Gedik, M. Emre; GÜNAYDIN, GÜRCAN; Kolemen, Safacan; Günbaş, Emrullah Görkem (Wiley, 2019-11-05) Two red-absorbing, water-soluble and mitochondria (MT)-targeting selenophene-substituted BODIPY-based photosensitizers (PSs) were realized (BOD-Se, BOD-Se-I), and their potential as photodynamic therapy (PDT) agents were evaluated. BOD-Se-I showed higher O-1(2) generation yield thanks to the enhanced heavy-atom effect, and this derivative was further tested in detail in cell culture studies under both normoxic and hypoxic conditions. BOD-Se-I not only effectively functioned under hypoxic conditions, but als...
Embryonic stem cell-derived motoneurons provide a highly sensitive cell culture model for botulinum neurotoxin studies, with implications for high-throughput drug discovery. Kiriş, Erkan; Burnett, JC; Kota, KP; Koh, DC; Wanner, LM; Torres-Melendez, E; Gussio, R; Tessarollo, L; Bavari, S (Elsevier BV, 2011-05-01) Botulinum neurotoxins (BoNTs) inhibit cholinergic synaptic transmission by specifically cleaving proteins that are crucial for neurotransmitter exocytosis. Due to the lethality of these toxins, there are elevated concerns regarding their possible use as bioterrorism agents. Moreover, their widespread use for cosmetic purposes, and as medical treatments, has increased the potential risk of accidental overdosing and environmental exposure. Hence, there is an urgent need to develop novel modalities to counter ...

Citation Formats

F. E. Oflaz, Ç. D. Son, and A. Arslan, “Oligomerization and cell surface expression of recombinant GABA(A) receptors tagged in the delta subunit,” JOURNAL OF INTEGRATIVE NEUROSCIENCE, pp. 341–350, 2019, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/41973.