Molecular characterization of Dα6 and Dα7 nicotinic acetylcholine receptor subunits fromDrosophila: formation of a high‐affinity α‐bungarotoxin binding site revealed by expression of subunit chimeras

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Abstract
Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in the insect brain and are target sites for neonicotinoid insecticides. Seven nAChR subunits (four α-type and three β-type) have been cloned previously from Drosophila melanogaster, the model insect system and characterized by heterologous expression. Recently, three further putative nAChR α subunits (Dα5, Dα6 and Dα7) with sequence similarity to the vertebrate α7 subunit have been identified from Drosophila genome sequence data but there have been no reports, as yet, of their characterization by heterologous expression. In the present study, we report the first isolation of a full-length Dα7 cDNA and the independent molecular cloning of Dα6. Binding of nicotinic radioligands was not detected to full-length Dα6 or Dα7 subunits when expressed alone or when or co-expressed with other nAChR subunits in Drosophila or mammalian cell lines, but specific cell-surface binding of [125I]α-bungarotoxin (Kd = 0.68 ± 0.22 nm) and [3H]methyllycaconitine (Kd = 0.27 ± 0.06 nm) was detected after expression of a subunit chimera containing the ligand-binding domains of Dα6 fused to the C-terminal domain of the 5-hydroxytryptamine receptor 5HT3A. Although cell-surface binding was not detected with a Dα7/5HT3Α chimera expressed alone, co-expression of the two subunit chimeras resulted in significantly enhanced levels of nicotinic radioligand binding (with no change in affinity). This is the first evidence for the formation of a nAChR binding site by heterologously expressed Drosophila nAChR subunits in the absence of a co-expressed vertebrate nAChR subunit. In addition to the formation of homomeric nAChR complexes, evidence has been obtained from both radioligand binding and co-immunoprecipitation studies for the co-assembly of Dα6 and Dα7 into heteromeric cell surface complexes.

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