The Drosophila nicotinic acetylcholine receptor subunits Dα5 and Dα7 form functional homomeric and heteromeric ion channels

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 (D alpha 1-D alpha 7 and D beta 1-D beta 3) and a similar number have been identified in other insect species. The focus of the present study is the D alpha 5, D alpha 6 and D alpha 7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate alpha 7 nAChR subunit. Results: A full-length cDNA clone encoding the Drosophila nAChR D alpha 5 subunit has been isolated and the properties of D alpha 5-, D alpha 6- and D alpha 7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the D alpha 5 and D alpha 7 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 (D alpha 5 + D alpha 6 + D alpha 7) with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [I-125]-alpha-bungarotoxin was detected in both Drosophila and mammalian cell lines when D alpha 5 was co-expressed with D alpha 6 and RIC-3. In contrast, co-expression of additional subunits (including D alpha 7) with D alpha 5 and D alpha 6 prevented specific binding of [I-125]-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: D alpha ta are presented demonstrating the ability of the Drosophila D alpha 5 and D alpha 7 subunits to generate functional homomeric and also heteromeric nAChRs.