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 (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.


RESULTS
A 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.


CONCLUSIONS
Data are presented demonstrating the ability of the Drosophila Dα5 and Dα7 subunits to generate functional homomeric and also heteromeric nAChRs.
Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK.
Stuart J
SJ
Lansdell
Toby
T
Collins
Jim
J
Goodchild
Neil S
NS
Millar
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000493:pharmacokinetics; D002038:Bungarotoxins / Q000493:pharmacokinetics; D002460:Cell Line; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D018722:Nicotinic Agonists / Q000493:pharmacokinetics; D018408:Patch-Clamp Techniques; D011485:Protein Binding / Q000187:drug effects; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000493:pharmacokinetics; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D044406:Receptors, Serotonin, 5-HT3 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014982:Xenopus laevis; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
D019086:Bridged Bicyclo Compounds, Heterocyclic; D002038:Bungarotoxins; D029721:Drosophila Proteins; D007473:Ion Channels; D018722:Nicotinic Agonists; D021122:Protein Subunits; D011725:Pyridines; C102569:RIC protein, Drosophila; D019275:Radiopharmaceuticals; D011978:Receptors, Nicotinic; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins; C113681:nicotinic acetylcholine receptor alpha-subunits, Drosophila; D018631:ras Proteins; C082748:epibatidine; D000109:Acetylcholine