Abstract
BackgroundNicotinic 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 (Dα1-Dα7 and Dβ1-Dβ3) and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit.ResultsA full-length cDNA clone encoding the Drosophila nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 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 (Dα5 + Dα6 + Dα7) with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [125I]-α-bungarotoxin was detected in both Drosophila and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7) with Dα5 and Dα6 prevented specific binding of [125I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in some cellular environments.ConclusionData are presented demonstrating the ability of the Drosophila Dα5 and Dα7 subunits to generate functional homomeric and also heteromeric nAChRs.
Highlights
Nicotinic acetylcholine receptors play an important role as excitatory neurotransmitters in vertebrate and invertebrate species
Molecular cloning of Dα5 A full-length cDNA encoding the Drosophila nicotinic acetylcholine receptor (nAChR) Dα5 subunit was isolated from a preparation of Drosophila embryo mRNA
In agreement with previous studies [41], the Dα5 cDNA isolated in this study contains an open reading frame encoding an unusually large N-terminal domain, extending some 300 amino acids upstream of the start methionine in most nAChR subunits
Summary
Nicotinic acetylcholine receptors (nAChRs) play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. Ten nAChR subunits have been identified in the model insect Drosophila melanogaster (Dα1-Dα7 and Dβ1-Dβ3) and a similar number have been identified in other insect species. Ten nAChR subunits (Dα1-Dα7 and Dβ1-Dβ3) have been identified in the model insect Drosophila melanogaster and a similar number of subunits have been identified in other insect species [1,2]. Experimental approaches that have had some success in overcoming problems associated with expression of insect nAChRs include the expression of subunit chimeras containing domains from other neurotransmitter receptors [15], co-expression of insect nAChRs with vertebrate subunits [16,17] or a combination of these approaches [18]. This includes nAChRs cloned from the honeybee Apis mellifera [2325], diamondback moth Plutella xylostella [26,27], house fly Musca domestica [28,29,30], locust Locusta migratoria [31], mosquito Anopheles gambiae [25], red flour beetle Tribolium castaneum [25,32], silkworm Bombyx mori [25,33] and tobacco hornworm Manduca sexta [34]
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