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Address correspondence to Lucia G. Sivilotti, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom. Fax: (44) 20-7679-7298; email: l.sivilotti{at}ucl.ac.uk
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 
(2–4, 6) and ß (ß2, ß4) subunits by a short linker of eight glutamine residues. Robust functional expression in oocytes was observed for several tandems (ß4_2, ß4_3, ß4_4, and ß2_4) when coexpressed with the corresponding ß monomer subunit. All tandems expressed when injected alone, except for ß4_3, which produced functional channels only together with ß4 monomer and was chosen for further characterization. These channels produced from ß4_3 tandem constructs plus ß4 monomer were identical with receptors expressed from monomer 3 and ß4 constructs in acetylcholine sensitivity and in the number of and ß subunits incorporated in the channel gate. However, separately mutating the ß 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 ß subunits from the tandem and one from the ß monomer construct, whereas the rest incorporated two or three ß 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.
Key Words: oocytes • two-electrode voltage-clamp • reporter mutation approach • acetylcholine • ion channels
Abbreviation used in this paper: nAChR, nicotinic acetylcholine receptor.
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