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Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors

Tandem constructs are increasingly being used to restrict the composition of recombinant multimeric channels. It is therefore important to assess not only whether such approaches give functional channels, but also whether such channels completely incorporate the subunit tandems. We have addressed th...

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Autores principales: Groot-Kormelink, Paul J., Broadbent, Steven D., Boorman, James P., Sivilotti, Lucia G.
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 2004
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2234567/
https://www.ncbi.nlm.nih.gov/pubmed/15148328
http://dx.doi.org/10.1085/jgp.200409042
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author Groot-Kormelink, Paul J.
Broadbent, Steven D.
Boorman, James P.
Sivilotti, Lucia G.
author_facet Groot-Kormelink, Paul J.
Broadbent, Steven D.
Boorman, James P.
Sivilotti, Lucia G.
author_sort Groot-Kormelink, Paul J.
collection PubMed
description Tandem constructs are increasingly being used to restrict the composition of recombinant multimeric channels. It is therefore important to assess not only whether such approaches give functional channels, but also whether such channels completely incorporate the subunit tandems. We have addressed this question for neuronal nicotinic acetylcholine receptors, using a channel mutation as a reporter for subunit incorporation. We prepared tandem constructs of nicotinic receptors by linking α (α2–α4, α6) and β (β2, β4) subunits by a short linker of eight glutamine residues. Robust functional expression in oocytes was observed for several tandems (β4_α2, β4_α3, β4_α4, and β2_α4) when coexpressed with the corresponding β monomer subunit. All tandems expressed when injected alone, except for β4_α3, which produced functional channels only together with β4 monomer and was chosen for further characterization. These channels produced from β4_α3 tandem constructs plus β4 monomer were identical with receptors expressed from monomer α3 and β4 constructs in acetylcholine sensitivity and in the number of α and β subunits incorporated in the channel gate. However, separately mutating the β subunit in either the monomer or the tandem revealed that tandem-expressed channels are heterogeneous. Only a proportion of these channels contained as expected two copies of β subunits from the tandem and one from the β monomer construct, whereas the rest incorporated two or three β monomers. Such inaccuracies in concatameric receptor assembly would not have been apparent with a standard functional characterization of the receptor. Extensive validation is needed for tandem-expressed receptors in the nicotinic superfamily.
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spelling pubmed-22345672008-03-21 Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors Groot-Kormelink, Paul J. Broadbent, Steven D. Boorman, James P. Sivilotti, Lucia G. J Gen Physiol Article Tandem constructs are increasingly being used to restrict the composition of recombinant multimeric channels. It is therefore important to assess not only whether such approaches give functional channels, but also whether such channels completely incorporate the subunit tandems. We have addressed this question for neuronal nicotinic acetylcholine receptors, using a channel mutation as a reporter for subunit incorporation. We prepared tandem constructs of nicotinic receptors by linking α (α2–α4, α6) and β (β2, β4) subunits by a short linker of eight glutamine residues. Robust functional expression in oocytes was observed for several tandems (β4_α2, β4_α3, β4_α4, and β2_α4) when coexpressed with the corresponding β monomer subunit. All tandems expressed when injected alone, except for β4_α3, which produced functional channels only together with β4 monomer and was chosen for further characterization. These channels produced from β4_α3 tandem constructs plus β4 monomer were identical with receptors expressed from monomer α3 and β4 constructs in acetylcholine sensitivity and in the number of α and β subunits incorporated in the channel gate. However, separately mutating the β subunit in either the monomer or the tandem revealed that tandem-expressed channels are heterogeneous. Only a proportion of these channels contained as expected two copies of β subunits from the tandem and one from the β monomer construct, whereas the rest incorporated two or three β monomers. Such inaccuracies in concatameric receptor assembly would not have been apparent with a standard functional characterization of the receptor. Extensive validation is needed for tandem-expressed receptors in the nicotinic superfamily. The Rockefeller University Press 2004-06 /pmc/articles/PMC2234567/ /pubmed/15148328 http://dx.doi.org/10.1085/jgp.200409042 Text en Copyright © 2004, The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Article
Groot-Kormelink, Paul J.
Broadbent, Steven D.
Boorman, James P.
Sivilotti, Lucia G.
Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors
title Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors
title_full Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors
title_fullStr Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors
title_full_unstemmed Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors
title_short Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors
title_sort incomplete incorporation of tandem subunits in recombinant neuronal nicotinic receptors
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2234567/
https://www.ncbi.nlm.nih.gov/pubmed/15148328
http://dx.doi.org/10.1085/jgp.200409042
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