Lansdell_2012_BMC.Neurosci_13_73

BACKGROUND: Nicotinic acetylcholine receptors (nAChRs) play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect Drosophila melanogaster (Dalpha1-Dalpha7 and Dbeta1-Dbeta3) and a similar number have been identified in other insect species. The focus of the present study is the Dalpha5, Dalpha6 and Dalpha7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate alpha7 nAChR subunit.
RESULTS: A full-length cDNA clone encoding the Drosophila nAChR Dalpha5 subunit has been isolated and the properties of Dalpha5-, Dalpha6- and Dalpha7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dalpha5 and Dalpha7 subunits in Xenopus oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, we have demonstrated the functional expression of a heteromeric 'triplet' nAChR (Dalpha5 + Dalpha6 + Dalpha7) with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [125I]-alpha-bungarotoxin was detected in both Drosophila and mammalian cell lines when Dalpha5 was co-expressed with Dalpha6 and RIC-3. In contrast, co-expression of additional subunits (including Dalpha7) with Dalpha5 and Dalpha6 prevented specific binding of [125I]-alpha-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in some cellular environments. CONCLUSION: Data are presented demonstrating the ability of the Drosophila Dalpha5 and Dalpha7 subunits to generate functional homomeric and also heteromeric nAChRs.