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.

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Biochemical and functional properties of distinct nicotinic acetylcholine receptors in the superior cervical ganglion of mice with targeted deletions of nAChR subunit genes

European Journal of Neuroscience ◽

10.1111/j.1460-9568.2010.07133.x ◽

2010 ◽

Vol 31 (6) ◽

pp. 978-993 ◽

Cited By ~ 39

Author(s):

Reinhard David ◽

Anna Ciuraszkiewicz ◽

Xenia Simeone ◽

Avi Orr-Urtreger ◽

Roger L. Papke ◽

...

Keyword(s):

Functional Properties ◽

Nicotinic Acetylcholine Receptors ◽

Superior Cervical Ganglion ◽

Acetylcholine Receptors ◽

Nachr Subunit ◽

Nicotinic Acetylcholine ◽

Cervical Ganglion

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Nicotinic acetylcholine receptors in rat cochlear nucleus: [125I]-?-bungarotoxin receptor autoradiography and in situ hybridization of ?7 nAChR subunit mRNA

The Journal of Comparative Neurology ◽

10.1002/(sici)1096-9861(19980727)397:2<163::aid-cne2>3.0.co;2-z ◽

1998 ◽

Vol 397 (2) ◽

pp. 163-180 ◽

Cited By ~ 24

Author(s):

H. Kevin Happe ◽

Barbara J. Morley

Keyword(s):

In Situ Hybridization ◽

Nicotinic Acetylcholine Receptors ◽

Cochlear Nucleus ◽

Acetylcholine Receptors ◽

Receptor Autoradiography ◽

Nachr Subunit ◽

Nicotinic Acetylcholine ◽

Subunit Mrna

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Polypeptide and peptide toxins, magnifying lenses for binding sites in nicotinic acetylcholine receptors

Biochemical Pharmacology ◽

10.1016/j.bcp.2009.05.032 ◽

2009 ◽

Vol 78 (7) ◽

pp. 720-731 ◽

Cited By ~ 54

Author(s):

Victor Tsetlin ◽

Yuri Utkin ◽

Igor Kasheverov

Keyword(s):

Nicotinic Acetylcholine Receptors ◽

Binding Sites ◽

Acetylcholine Receptors ◽

Nicotinic Acetylcholine ◽

Peptide Toxins

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Assembly and subunit diversity of nicotinic acetylcholine receptors

Biochemical Society Transactions ◽

10.1042/bst0310869 ◽

2003 ◽

Vol 31 (4) ◽

pp. 869-874 ◽

Cited By ~ 152

Author(s):

N.S. Millar

Keyword(s):

Ion Channels ◽

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Nicotinic Receptors ◽

Subunit Composition ◽

Receptor Subtypes ◽

Receptor Subunit ◽

Vertebrate Species ◽

Nachr Subunit ◽

Nicotinic Acetylcholine

Nicotinic acetylcholine receptors (nAChRs) are a diverse family of neurotransmitter-gated ion channels which contain five transmembrane subunits arranged around a central pore. Distinct receptor subtypes are expressed at the vertebrate skeletal neuromuscular junction, in mechanosensory cells and within the central and peripheral nervous systems. A total of 17 nAChR subunits (α1–α10, β1–β4, γ, δ and ∊) have been identified in vertebrate species, which can co-assemble to generate a wide variety of nAChRs. Nicotinic receptors also constitute an abundant and diverse family of receptors in invertebrates. As a consequence of studies which have been conducted with both native and recombinant nAChRs, the subunit composition of nAChRs and the rules governing subunit co-assembly are becoming clearer. In this paper the extent of nAChR subunit diversity and evidence for receptor subunit composition is reviewed.

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ShAR2β, a divergent nicotinic acetylcholine receptor subunit from the blood fluke Schistosoma

Parasitology ◽

10.1017/s0031182006002162 ◽

2007 ◽

Vol 134 (6) ◽

pp. 833-840 ◽

Cited By ~ 9

Author(s):

G. N. BENTLEY ◽

A. K. JONES ◽

A. AGNEW

Keyword(s):

Amino Acid ◽

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Transmembrane Domain ◽

Structural Features ◽

Amino Acid Residues ◽

Nachr Subunit ◽

Nicotinic Acetylcholine ◽

Blood Fluke ◽

Nematode Parasites

SUMMARYNicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate the fast actions of the neurotransmitter, acetylcholine. Invertebrate nAChRs are of interest as they are targets of widely-selling insecticides and drugs that control nematode parasites. Here, we report the cloning of ShAR2β, a candidate nAChR subunit from the blood fluke, Schistosoma haematobium, which is the third trematode nAChR subunit to be characterized. While ShAR2β possesses key structural features common to all nAChRs, its amino acid sequence shares considerably low identity with those of insect, nematode and vertebrate nAChR subunits. In particular, the second transmembrane domain of ShAR2β, which lines the ion channel, bears unusual amino acid residues which will likely give rise to a receptor with distinct functional properties. Phylogenetic analysis shows that ShAR2β is a divergent nAChR subunit that may define a clade of trematode-specific subunits. We discuss our findings in the context of potentially exploiting this receptor as a target for controlling schistosome parasites.

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