Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins

Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that present a target for insecticides. However, a better understanding of receptor subunit composition is needed for effective design of insecticides. Peptide neurotoxins are known to block nAChRs by binding to its target subunits. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles we determined the effects of α-Bungarotoxin (α-Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm α-Btx interactions with the Dα5, Dα6, Dα7 subunits. Finally, we report the localization of fluorescent tagged endogenous Dα6 during nervous system development. Taken together this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins.

Postsynaptic plasticity of cholinergic synapses underlies the induction and expression of appetitive memories in Drosophila

In vertebrates, memory-relevant synaptic plasticity involves postsynaptic rearrangements of glutamate receptors. In contrast, previous work indicates that Drosophila and other invertebrates store memories using presynaptic plasticity of cholinergic synapses. Here, we provide evidence for postsynaptic plasticity at cholinergic output synapses from the Drosophila mushroom bodies (MBs). We find that the nicotinic acetylcholine receptor (nAChR) subunit α5 is required within specific MB output neurons (MBONs) for appetitive memory induction, but is dispensable for aversive memories. In addition, nAChR α2 subunits mediate memory expression downstream of α5 and the postsynaptic scaffold protein Dlg. We show that postsynaptic plasticity traces can be induced independently of the presynapse, and that in vivo dynamics of α2 nAChR subunits are changed both in the context of associative and non-associative memory formation, underlying different plasticity rules. Therefore, regardless of neurotransmitter identity, key principles of postsynaptic plasticity support memory storage across phyla.

The α5 Nicotinic Acetylcholine Receptor Subunit Differentially Modulates α4β2* and α3β4* Receptors

Nicotine, the principal reinforcing compound in tobacco, acts in the brain by activating neuronal nicotinic acetylcholine receptors (nAChRs). This review summarizes our current knowledge regarding how the α5 accessory nAChR subunit, encoded by the CHRNA5 gene, differentially modulates α4β2* and α3β4* receptors at the cellular level. Genome-wide association studies have linked a gene cluster in chromosomal region 15q25 to increased susceptibility to nicotine addiction, lung cancer, chronic obstructive pulmonary disease, and peripheral arterial disease. Interestingly, this gene cluster contains a non-synonymous single-nucleotide polymorphism (SNP) in the human CHRNA5 gene, causing an aspartic acid (D) to asparagine (N) substitution at amino acid position 398 in the α5 nAChR subunit. Although other SNPs have been associated with tobacco smoking behavior, efforts have focused predominantly on the D398 and N398 variants in the α5 subunit. In recent years, significant progress has been made toward understanding the role that the α5 nAChR subunit—and the role of the D398 and N398 variants—plays on nAChR function at the cellular level. These insights stem primarily from a wide range of experimental models, including receptors expressed heterologously in Xenopus oocytes, various cell lines, and neurons derived from human induced pluripotent stem cells (iPSCs), as well as endogenous receptors in genetically engineered mice and—more recently—rats. Despite providing a wealth of available data, however, these studies have yielded conflicting results, and our understanding of the modulatory role that the α5 subunit plays remains incomplete. Here, we review these reports and the various techniques used for expression and analysis in order to examine how the α5 subunit modulates key functions in α4β2* and α3β4* receptors, including receptor trafficking, sensitivity, efficacy, and desensitization. In addition, we highlight the strikingly different role that the α5 subunit plays in Ca2+ signaling between α4β2* and α3β4* receptors, and we discuss whether the N398 α5 subunit variant can partially replace the D398 variant.