Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors
Open Access
- 17 May 2004
- journal article
- Published by Rockefeller University Press in The Journal of general physiology
- Vol. 123 (6), 697-708
- https://doi.org/10.1085/jgp.200409042
Abstract
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.Keywords
This publication has 34 references indexed in Scilit:
- Monomeric and Dimeric Byproducts are the Principal Functional Elements of Higher Order P2X1ConcatamersPublished by American Society for Pharmacology & Experimental Therapeutics (ASPET) ,2003
- Forced Subunit Assembly in α1β2γ2 GABAAReceptors: INSIGHT INTO THE ABSOLUTE ARRANGEMENTJournal of Biological Chemistry, 2002
- Openings of the Rat Recombinant α1 Homomeric Glycine Receptor as a Function of the Number of Agonist Molecules BoundThe Journal of general physiology, 2002
- Formation of functional α3β4α5 human neuronal nicotinic receptors in Xenopus oocytes: a reporter mutation approachBritish Journal of Pharmacology, 2001
- Stoichiometry of human recombinant neuronal nicotinic receptors containing the β3 subunit expressed in Xenopus oocytesThe Journal of Physiology, 2000
- A Reporter Mutation Approach Shows Incorporation of the “Orphan” Subunit β3 into a Functional Nicotinic ReceptorPublished by Elsevier BV ,1998
- Cloning and sequence of full‐length cDNAs encoding the human neuronal nicotinic acetylcholine receptor (nAChR) subunits β3 and β4 and expression of seven nAChR subunits in the human neuroblastoma cell line SH‐SY5Y and/or IMR‐321FEBS Letters, 1997
- Functional contributions of α5 subunit to neuronal acetylcholine receptor channelsNature, 1996
- Chloride Channel Expression with the Tandem Construct of α6-β2 GABAA Receptor Subunit Requires a Monomeric Subunit of α6 or γ2Published by Elsevier BV ,1995
- Channel gating governed symmetrically by conserved leucine residues in the M2 domain of nicotinic receptorsNature, 1995