The nicotinic acetylcholine receptor Dalpha7 is required for an escape behavior in Drosophila.
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ABSTRACT:
SUBMITTER: Fayyazuddin A
PROVIDER: S-EPMC1382016 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bulk RNAseq from whole Drosophila melanogaster guts expressing either mCherry-RNAi (control) or RNAi against nicotinic acetylcholine receptor subunits β1 and β3 (nAchRβ1 and nAchRβ3) under control of a enterocyte-specific driver to assess their role in barrier function.
2023-08-23 | GSE236069 | GEO
Project description:Description Drosophila melanogaster nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that present a target for insecticides. However, a better understanding of in vioreceptor subunit composition is needed for effective design of insecticides. Alpha-neurotxins are known as peptide toxins capable 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 nAChRs to a snake toxin by using styrene maleic acid lipid particles (SMALPs) extraction method. For the null alleles which are impaired in locomotor activities we examined the binding of nAChRs incorporated into SMALPs to alpha bungarotoxin by employing mass spectrometry. We show native ligand interactions to Dalpha5, Dalpha6 and Dalpha7 subunits and reveal glycosylation sites of Dalpha5 and Dalpha7 by a glycoproteomic approach. Finally, we report the localization of fluorescent tagged Dalpha6 across nervous system development
2021-10-11 | PXD028484 | Pride
Project description:Acetylcholine is an excitatory neurotransmitter in the central nervous system of insects and the nicotinic acetylcholine receptor (nAChR) is a target for neonicotinoid insecticides. Functional insect nAChRs are difficult to express in host cells, and hence difficult to study. In mammals, acetylcholine and nicotine evoke dopamine release, but the extent to which this mechanism is conserved in insects is unknown. In intact larval ventral nerve cords (VNCs), we studied dopamine evoked by acetylcholine, nicotine, or neonicotinoids. Using fast-scan cyclic voltammetry, we confirmed dopamine was measured by its cyclic voltammogram and also by feeding Drosophila the synthesis inhibitor, 3-iodotyrosine, which lowered the evoked dopamine response. Acetylcholine (1.8?pmol) evoked on average 0.43?±?0.04??M dopamine. Dopamine release significantly decreased after incubation with ?-bungarotoxin, demonstrating the release is mediated by nAChR, but atropine, a muscarinic AChR antagonist, had no effect. Nicotine (t1/2?=?71?s) and the neonicotinoids nitenpyram and imidacloprid (t1/2?=?86?s, 121?s respectively) also evoked dopamine release, which lasted longer than acetylcholine-stimulated release (t1/2?=?19?s). Nicotine-stimulated dopamine was significantly lower in the presence of sodium channel blocker, tetrodotoxin, showing that the release is exocytotic. Drosophila that have mutations in the nAChR subunit ?1 or ?2 have significantly lower neonicotinoid-stimulated release but no changes in nicotine-stimulated release. This work demonstrates that nAChR agonists mediate dopamine release in Drosophila larval VNC and that mutations in nAChR subunits affect how insecticides stimulate dopamine release.
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Project description:Alkaloids are a structurally complex group of natural products that have a diverse range of biological activities and significant therapeutic applications. In this study, we examined the acute, anxiolytic-like effects of nicotinic acetylcholine receptor (nAChR)-activating alkaloids with reported neuropharmacological effects but whose effects on anxiety are less well understood. Because α4β2 nAChRs can regulate anxiety, we first demonstrated the functional activities of alkaloids on these receptors in vitro. Their effects on anxiety-like behavior in zebrafish were then examined using the zebrafish novel tank test (NTT). The NTT is a relatively high-throughput behavioral paradigm that takes advantage of the natural tendency of fish to dive down when stressed or anxious. We report for the first time that cotinine, anatabine, and methylanatabine may suppress this anxiety-driven zebrafish behavior after a single 20-min treatment. Effective concentrations of these alkaloids were well above the concentrations naturally found in plants and the concentrations needed to induce anxiolytic-like effect by nicotine. These alkaloids showed good receptor interactions at the α4β2 nAChR agonist site as demonstrated by in vitro binding and in silico docking model, although somewhat weaker than that for nicotine. Minimal or no significant effect of other compounds may have been due to low bioavailability of these compounds in the brain, which is supported by the in silico prediction of blood-brain barrier permeability. Taken together, our findings indicate that nicotine, although not risk-free, is the most potent anxiolytic-like alkaloid tested in this study, and other natural alkaloids may regulate anxiety as well.
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Project description:BackgroundImmunotherapy has proven to be an emerging treatment for non-small-cell lung cancer in recent years. Notably, smokers show higher programmed cell death ligand-1 (PD-L1) expression and better responses to PD-1/PD-L1 inhibitors than nonsmokers. Genome-wide association studies show that the CHRNΑ5 encoding α5-nicotinic acetylcholine receptor (α5-nAChR) is especially relevant to lung cancer and nicotine dependence. Jab1 is a key regulatory factor and promotes the stabilization of PD-L1. Our previous study reported that α5-nAChR mediates lung adenocarcinoma (LUAD) epithelial-mesenchymal transition (EMT) and metastasis via STAT3/Jab1. However, the link between α5-nAChR and PD-L1 is unclear in LUAD.MethodsWe used various bioinformatics databases to analyze the expression of related genes and their correlations. Expression and clinicopathologic significance of α5-nAChR and PD-L1 were detected by immunohistochemistry in a tissue microarray. α5-nAChR regulated LUAD cell immune escape by targeting the STAT3/Jab1-PD-L1 signalling by Western-blotting and ChIP in vitro. We used T cell coculture, flow cytometry, ELISA, CCK8 assay and crystal violet staining to detect the expression of regulatory T cell (Tregs), IFN-γ, IL-2 and the ability of T cell-mediated tumour cell killing respectively. IF assays were performed in both cancer cells and tumour xenograft paraffin sections to analyze the protein expression. The in vivo experiments in mouse model were performed to show the α5-nAChR-mediated immune escape via PD-L1 pathway.ResultsThe expression of α5-nAChR was correlated with PD-L1 expression, smoking status and lower survival of LUAD in vivo. In vitro, the expression of α5-nAChR mediated phosphorylated STAT3 (pSTAT3), Jab1 and PD-L1 expression. STAT3 bound to the Jab1 or PD-L1 promoter and mediated PD-L1 expression. Jab1 stabilized PD-L1 expression in LUAD cells. Furthermore, in primary T cell cocultured system, downregulation of α5-nAChR suppressed the function of CD4+CD25+FOXP3+ Tregs, enhanced IFN-γ secretion, and increased T cell-mediated killing of LUAD cells. In the Jurkat T cells and LUAD cells coculture assay, inhibition of α5-nAChR increased IL-2 secretion. In tumour xenograft tissues, α5-nAChR expression was related to PD-L1, Jab1, pSTAT3, CD4 and granzyme B expression (GB).ConclusionsOur results suggest that the novel α5-nAChR/STAT3-Jab1-PD-L1 axis is involved in LUAD immune escape, which could lead to potential therapeutic strategies for cancer immunotherapy. Video abstract.
| S-EPMC9377093 | biostudies-literature
Project description:Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of this mechanism is needed for effective insecticide design. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in a common genetic background. 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 Drosophila α5 (Dα5), Dα6, Dα7 subunits. Finally, we report the localisation of fluorescent tagged endogenous Dα6 during Drosophila CNS development. Taken together, this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.
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Project description:Polymodal nociceptors sense and integrate information on injurious mechanical, thermal, and chemical stimuli. Chemical signals either activate nociceptors or modulate their responses to other stimuli. One chemical known to activate or modulate responses of nociceptors is acetylcholine (ACh). Across evolution nociceptors express subunits of the nicotinic acetylcholine receptor (nAChR) family, a family of ACh-gated ion channels. The roles of ACh and nAChRs in nociceptor function are, however, poorly understood. Caenorhabditis elegans polymodal nociceptors, PVD, express nAChR subunits on their sensory arbor. Here we show that mutations reducing ACh synthesis and mutations in nAChR subunits lead to defects in PVD function and morphology. A likely cause for these defects is a reduction in cytosolic calcium measured in ACh and nAChR mutants. Indeed, overexpression of a calcium pump in PVD mimics defects in PVD function and morphology found in nAChR mutants. Our results demonstrate, for the first time, a central role for nAChRs and ACh in nociceptor function and suggest that calcium permeating via nAChRs facilitates activity of several signaling pathways within this neuron.
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Project description:How does an agonist activate a receptor? In this article I consider the activation process in muscle nicotinic acetylcholine receptors (AChRs), a prototype for understanding the energetics of binding and gating in other ligand-gated ion channels. Just as movements that generate gating currents activate voltage-gated ion channels, movements at binding sites that generate an increase in affinity for the agonist activate ligand-gated ion channels. The main topics are: i) the schemes and intermediate states of AChR activation, ii) the energy changes of each of the steps, iii) the sources of the energies, iv) the three kinds of AChR agonist binding site and v) the correlations between binding and gating energies. The binding process is summarized as sketches of different conformations of an agonist site. The results suggest that agonists lower the free energy of the active conformation of the protein in stages by establishing favorable, local interactions at each binding site, independently. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.
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Project description:The nicotinic acetylcholine receptor (nAChR) is a cation-selective channel central to both neuronal and muscular processes and is considered the prototype for ligand-gated ion channels, motivating a structural determination effort that spanned several decades [Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4 A resolution. J Mol Biol 346:967-989]. Purified nAChR must be reconstituted in a mixture containing cholesterol to function. Proposed modes of interaction between cholesterol and the protein range from specific binding to indirect membrane-mediated mechanisms. However, the underlying cause of nAChR sensitivity to cholesterol remains controversial, in part because the vast majority of functional studies were conducted before a medium resolution structure was reported. We show that the nAChR contains internal sites capable of containing cholesterol, whose occupation stabilizes the protein structure. We detect sites at the protein-lipid interface as conventionally predicted from functional data, as well as deeply buried sites that are not usually considered. Molecular dynamics simulations reveal that occupation of both superficial and deeply buried sites most effectively preserves the experimental structure; the structure collapses in the absence of bound cholesterol. In particular, we find that bound cholesterol directly supports contacts between the agonist-binding domain and the pore that are thought to be essential for activation of the receptor. These results likely apply to those other ion channels within the Cys-loop superfamily that depend on cholesterol, such as the GABA receptor.
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Project description:RIC-3 is a transmembrane protein which acts as a molecular chaperone of nicotinic acetylcholine receptors (nAChRs). For some nAChR subtypes (such as homomeric alpha7 neuronal nAChRs), RIC-3 is required for efficient receptor folding, assembly and functional expression. In contrast, for other nAChR subtypes (such as heteromeric alpha4beta2 neuronal nAChRs) there have been reports that RIC-3 can both enhance and reduce levels of functional expression. There is also evidence that RIC-3 can modulate maturation of the closely related 5-hydroxytryptamine (5-HT) receptor (5-HT(3)R). As with heteromeric nAChRs, apparently contradictory results have been reported for the influence of RIC-3 on 5-HT(3)R maturation in different expression systems. Recent evidence indicates that these differences in RIC-3 chaperone activity may be influenced by the host cell, suggesting that other proteins may play an important role in modulating the effects of RIC-3 as a chaperone. RIC-3 was originally identified in the nematode Caenorhabditis elegans as the protein encoded by the gene ric-3 (resistance to inhibitors of cholinesterase) and has subsequently been cloned and characterized from mammalian and insect species. This review provides a brief history of RIC-3; from the identification of the ric-3 gene in C. elegans in 1995 to the more recent demonstration of its activity as a nAChR chaperone.
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