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Concatenated γ-aminobutyric acid type A receptors revisited: Finding order in chaos

Concatenated γ-aminobutyric acid type A receptors revisited: Finding order in chaos

Title: Concatenated γ-aminobutyric acid type A receptors revisited: Finding order in chaos
Author: Liao, Vivian Wan Yu
Chua, Han Chow
Kowal, Natalia Magdalena
Chebib, Mary
Balle, Thomas
Ahring, Philip Kiær
Date: 2019-04-15
Language: English
Scope: 798-819
University/Institute: Háskóli Íslands
University of Iceland
School: Heilbrigðisvísindasvið (HÍ)
School of Health Sciences (UI)
Department: Lyfjafræðideild (HÍ)
Faculty of Pharmaceutical Sciences (UI)
Series: Journal of General Physiology;151(6)
ISSN: 0022-1295
1540-7748 (eISSN)
DOI: 10.1085/jgp.201812133
Subject: Nervous system; Heteromeric receptors; Neurotransmitter receptors; Taugakerfi; Lyfjafræði
URI: https://hdl.handle.net/20.500.11815/1697

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Vivian Wan Yu Liao, Han Chow Chua, Natalia Magdalena Kowal, Mary Chebib, Thomas Balle, Philip Kiær Ahring; Concatenated γ-aminobutyric acid type A receptors revisited: Finding order in chaos. The Journal of general physiology 3 June 2019; 151 (6): 798–819. doi: https://doi.org/10.1085/jgp.201812133


γ-aminobutyric acid type A receptors (GABAARs), the major inhibitory neurotransmitter receptors in the mammalian central nervous system, are arguably the most challenging member of the pentameric Cys-loop receptors to study due to their heteromeric structure. When two or more subunits are expressed together in heterologous systems, receptors of variable subunit type, ratio, and orientation can form, precluding accurate interpretation of data from functional studies. Subunit concatenation is a technique that involves the linking of individual subunits and in theory allows the precise control of the uniformity of expressed receptors. In reality, the resulting concatemers from widely used constructs are flexible in their orientation and may therefore assemble with themselves or free GABAAR subunits in unexpected ways. In this study, we examine functional responses of receptors from existing concatenated constructs and describe refinements necessary to allow expression of uniform receptor populations. We find that dimers from two commonly used concatenated constructs, β-23-α and α-10-β, assemble readily in both the clockwise and the counterclockwise orientations when coexpressed with free subunits. Furthermore, we show that concatemers formed from new tetrameric α-10-β-α-β and α-10-β-α-γ constructs also assemble in both orientations with free subunits to give canonical αβγ receptors. To restrict linker flexibility, we systematically shorten linker lengths of dimeric and pentameric constructs and find optimized constructs that direct the assembly of GABAARs only in one orientation, thus eliminating the ambiguity associated with previously described concatemers. Based on our data, we revisit some noncanonical GABAAR configurations proposed in recent years and explain how the use of some concatenated constructs may have led to wrong conclusions. Our results help clarify current contradictions in the literature regarding GABAAR subunit stoichiometry and arrangement. The lessons learned from this study may guide future efforts in understanding other related heteromeric receptors.


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