Lansdell, Stuart John; (1998) The assembly and folding of neuronal nicotinic acetylcholine receptors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Nicotinic acetylcholine receptors (nAChRs) are pentameric hetero-oligomeric ion channels. To date five vertebrate muscle subunits (α,β,γ,δ and ε) and eleven vertebrate neuronal nAChR subunits (α2-α9 and β2-β4) have been cloned. In Drosophila only four nAChR subunits have been reported to date. These include two subunits (ALS and SAD) which show homology to the vertebrate α subunits and are assumed to be agonist binding subunits. The remaining two subunits (ARD and SBD) resemble the vertebrate non-α subunits in that they lack the two adjacent cysteine residues characteristic of the α subunits and are thought to have structural roles. A central aim of this research project was to investigate the folding, subunit assembly and functional properties of cloned nAChRs. Invertebrate (Drosophila and C. elegans) and vertebrate (rat) nAChR subunits were expressed in a variety of mammalian and invertebrate cell lines. In contrast to vertebrate nAChRs, no functional expression has been reported for any combination of the four Drosophila subunits expressed in Xenopus oocytes or cultured mammalian cells. Whereas the Drosophila SBD cDNA, reported previously, lacked part of the SBD coding sequence, here the construction and expression of a full-length SBD cDNA is reported. It seems plausible that the problems in expressing functional Drosophila nAChRs in Xenopus oocytes or in mammalian cell lines might be due to an inability of these expression systems to correctly assemble Drosophila nAChRs. Despite expression in what might be considered a more native cellular environment, functional nAChRs were not detected in a Drosophila cell line unless the Drosophila nAChR subunit cDNAs were co-expressed with vertebrate nAChR subunits. The results also indicate that the folding of Drosophila nAChR subunits is temperature-sensitive and strongly suggest that the inability of these subunits to generate functional channels in the absence of vertebrate subunits is due to a requirement for co-assembly with as yet unidentified Drosophila nAChR subunits. In the latter stages of this project a new (fifth) Drosophila nAChR subunit, Dα3 (isolated by another group) was obtained and examined by heterologous expression. Drosophila cDNA libraries have been screened for novel nAChR subunits and a partial cDNA clone of a novel α subunit has been isolated and sequenced.

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