Dual nicotinic acetylcholine receptor subunit gene knockouts reveal limits to functional redundancy

Abstract

The nicotinic acetylcholine receptor (nAChR) subunit gene family consists of ten members in Drosophila melanogaster. The mature nAChR is a pentamer assembled from these subunits. Despite recent advances in the in vitro expression of some receptor subunit combinations (nAChR subtypes), the in vivo combinations and stoichiometry of these subtypes remains poorly defined. In addition, there are many potential nAChR signalling roles for different subtypes in insect behaviour, development and physiology. Prior work has shown that nAChR subunit mutants can display altered sleep and mating behaviour, disrupted hormone signalling and reduced locomotion, climbing ability and longevity. Teasing out the specific receptor subunits that are involved in these different functions is potentially made more difficult given that the structural similarity between members of gene families often means that there is a degree of functional redundancy. In order to circumvent this, we created a dual knockout strain for the Dα1 and Dβ2 nAChR subunit genes and examined four traits including insecticide resistance. These subunits had been previously implicated in the response to a neonicotinoid insecticide, imidacloprid. The use of the dual knockout revealed that Dα1 and Dβ2 subunits are involved in signalling that leads to the inflation of wings following adult emergence from the pupal case. The Dβ1 subunit had previously been implicated as a contributor to this function. The lack of a phenotype or low penetrance of the phenotype in the Dα1 and Dβ2 single mutants compared to the dual knockout suggests that these subunits are, to some extent, functionally redundant. We also observed stronger reductions in climbing ability and longevity in the dual knockout. Our findings demonstrate that a dual knockout approach to examining members of the nAChR subunit gene family may increase the power of genetic approaches linking individual subunits and combinations thereof to particular biological functions. This approach will be valuable as the nAChRs are so widely expressed in the insect brain that they are likely to have many functions that hereto remain undetected.