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Positive allosteric modulators of the α7 nicotinic acetylcholine (nACh) receptor (α7-PAMs) possess promnesic and procognitive properties and have potential in the treatment of cognitive and psychiatric disorders including Alzheimer's disease and schizophrenia. Behavioural studies in rodents have indicated that α7-PAMs can also produce antinociceptive and anxiolytic effects that may be associated with positive modulation of the GABAsubA/subreceptor. The overall goal of this study was to investigate the modulatory actions of selected α7-PAMs on the GABAsubA/subreceptor.We employed a combination of cell fluorescence imaging, electrophysiology, functional competition and site-directed mutagenesis to investigate the functional and structural mechanisms of modulation of the GABAsubA/subreceptor by three representative α7-PAMs.We show that the α7-PAMs at micromolar concentrations enhance the apparent affinity of the GABAsubA/subreceptor for the transmitter and potentiate current responses from the receptor. The compounds were equi-effective at binary αβ and ternary αβγ GABAsubA/subreceptors. Functional competition and site-directed mutagenesis indicate that the α7-PAMs bind to the classic anaesthetic binding sites in the transmembrane region in the intersubunit interfaces, which results in stabilization of the active state of the receptor.We conclude that the tested α7-PAMs are micromolar-affinity, intermediate- to low-efficacy allosteric potentiators of the mammalian αβγ GABAsubA/subreceptor. Given the similarities in the in vitro sensitivities of the α7 nACh and α1β2γ2L GABAsubA/subreceptors to α7-PAMs, we propose that doses used to produce nACh receptor-mediated behavioural effects in vivo are likely to modulate GABAsubA/subreceptor function.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast synaptic transmission and cell signaling, which contribute to learning, memory, and the execution of motor skills. Birdsong is a complex learned motor skill in songbirds. Although the existence of 15 nAChR subunits has been predicted in the avian genome, their expression patterns and potential contributions to song learning and production have not been comprehensively investigated. Here, we cloned all the 15 nAChR subunits (ChrnA1-10, B2-4, D, and G) from the zebra finch brain and investigated the mRNA expression patterns in the neural pathways responsible for the learning and production of birdsong during a critical period of song learning. Although there were no detectable hybridization signals for ChrnA1, A6, A9, and A10, the other 11 nAChR subunits were uniquely expressed in one or more major subdivisions in the song nuclei of the songbird brain. Of these 11 subunits, ChrnA3-5, A7, and B2 were differentially regulated in the song nuclei compared with the surrounding anatomically related regions. ChrnA5 was upregulated during the critical period of song learning in the lateral magnocellular nucleus of the anterior nidopallium. Furthermore, single-cell RNA sequencing revealed ChrnA7 and B2 to be the major subunits expressed in neurons of the vocal motor nuclei HVC and robust nucleus of the arcopallium, indicating the potential existence of ChrnA7-homomeric and ChrnB2-heteromeric nAChRs in limited cell populations. These results suggest that relatively limited types of nAChR subunits provide functional contributions to song learning and production in songbirds.

A subgroup of myasthenia gravis (MG) patients fail to respond adequately to recommended treatments, a condition referred to as refractory MG. During the refractory phase, patients experience persistent debilitating symptoms with potential life-threatening events or inability to reduce immunosuppressant dosages and minimize long-term toxicities.We conducted a retrospective, single-center study of 113 MG patients to investigate the autoantibody profile and clinical characteristics of refractory MG patients, compared with nonrefractory patients, based on predefined criteria.Fifteen patients (13.3%) were classified as refractory. Double-seronegative MG (DSNMG), without detectable nicotinic acetylcholine receptor (AChR) or muscle-specific tyrosine kinase (MuSK) antibodies, was identified in six refractory patients, significantly higher than those with nonrefractory MG (40% vs 16.3%; P = .031). None of the refractory patients had MuSK antibodies. Patients in the refractory group more frequently had an earlier disease onset, thymic pathology, and thymectomy (P ≤. 03 for all).In this study, patients with refractory MG were more likely than those with nonrefractory MG to be DSN; and refractory DSNMG patients had worse MGFA classes in their recent visit compared with anti-AChR positive refractory patients. Refractory DSNMG patients may represent a distinct group that requires more individualized and targeted treatment approaches.

The insect nicotinic acetylcholine receptor (nAChR) is a pentameric channel protein and also a target for neonicotinoids. There are few reported studies on the molecular interactions of Leptinotarsa decemlineata nAChRs with neonicotinoids. In this study, we analyzed the response of acetylcholine and neonicotinoids (thiamethoxam [TMX], imidacloprid [IMI], and clothianidin [CLO]) on hybrid receptors constructed by nAChR α1 and α8 subunits of L. decemlineata (Ldα1 and Ldα8) co-expressed with rat β2 subunit (rβ2) at different capped RNA (cRNA) ratios in Xenopus oocytes. In addition, we evaluated the expression changes of Ldα1 and Ldα8 after median lethal dose of TMX treatment for 72 h by quantitative polymerase chain reaction (qPCR). The resulting functional nAChRs Ldα1/rβ2 and Ldα1/Ldα8/rβ2 showed different pharmacological characteristics. The neonicotinoids tested showed lower agonist affinity on Ldα1/Ldα8/rβ2 compared to Ldα1/rβ2 at same ratios of subunit cRNAs. The sensitivities of neonicotinoids tested for Ldα1/rβ2 and Ldα1/Ldα8/rβ2 at cRNA ratios of 5:1, 1:1 and 5:5:1, 1:1:1, respectively, were lower than those for nAChRs at ratios of 1:5 and 1:1:5, respectively, whereas the values of maximum response (I

The peach-potato aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae), is a major pest worldwide. The intensive use of insecticides has led to the development of resistance against neonicotinoid insecticides. The R81T mutation in the nicotinic acetylcholine receptor (nAChR) beta1 subunit is considered a crucial mechanism adaptation to neonicotinoid resistance in M. persicae and Aphis gossypii.Resistance-related mutations (R81T and V101I) were detected in the imidacloprid-resistant M. persicae AH19 population. The V101I mutation is reported for the first time. The V101I and R81T mutations existed separately, indicating that the two mutations evolved independently. Imidacloprid resistance in the AH19 population was stable without insecticide exposure. Four mutant strains were selected from the population with stable resistance. The resistance of the AH19-T, AH19-I, and AH19-TI strains to imidacloprid, thiamethoxam, and dinotefuran was significantly increased compared with the AH19-W strain. Synergism bioassays showed that the inhibition of three detoxification enzymes did not affect imidacloprid resistance in the AH19-T and AH19-I strains. Expression of nAChR β1 subunits in the AH19-W, AH19-T, and AH19-I strains remained unchanged.The V101I mutation is associated with neonicotinoid resistance in M. persicae. The resistance of the AH19-T and AH19-I strains to neonicotinoids appears to be mainly due to the R81T and V101I mutations, whereas these mutations, together with changes in the cytochrome P450 monooxygenases and nAChR expression may be responsible for the development of neonicotinoid resistance in the AH19-TI strain. © 2021 Society of Chemical Industry.

The identification of proteins that are altered following nicotine/tobacco exposure can facilitate and positively impact the investigation of related diseases. In this report, we investigated the effects of chronic (–)-menthol exposure in fourteen murine brain regions for changes in total β2 subunit protein levels and changes in epibatidine binding levels using immunoblotting and radioligand binding assays. We identified the habenula as a region of interest due to the region’s marked decreases in β2 subunit and nAChR levels in response to chronic (–)-menthol alone. Thus, we further examined the habenula, a brain region associated with both the reward and withdrawal components of addiction, for additional protein level alterations using mass spectrometry. A total of 552 proteins with altered levels were identified after chronic (–)-menthol exposure. Enriched in the proteins with altered levels after (–)-menthol exposure were proteins associated with signaling, immune systems, RNA regulation, and protein transport. The continuation and expansion of the brain region-specific protein profiling in response to (–)-menthol will provide a better understanding of how this common flavorant in tobacco and e-liquid products may affect addiction and general health.

The use of areca nuts (areca) in the form of betel quids constitutes the fourth most common addiction in the world, associated with high risk for oral disease and cancer. Areca is a complex natural product, making it difficult to identify specific components associated with the addictive and carcinogenic properties. It is commonly believed that the muscarinic agonist arecoline is at the core of the addiction. However, muscarinic receptor activation is not generally believed to support drug-taking behaviour. Subjective accounts of areca use include descriptions of both sedative and stimulatory effects, consistent with the presence of multiple psychoactive agents. We have previously reported partial agonism of α4-containing nicotinic acetylcholine receptors by arecoline and subsequent inhibition of those receptors by whole areca broth. In the present study, we report the inhibition of nicotinic acetylcholine receptors and other types of neurotransmitter receptors with compounds of high molecular weight in areca and the ability of low molecular weight areca extract to activate GABA and glutamate receptors. We confirm the presence of a high concentration of GABA and glutamate in areca. Additionally, data also indicate the presence of a dopamine and serotonin transporter blocking activity in areca that could account for the reported stimulant and antidepressant activity. Our data suggest that toxic elements of high molecular weight may contribute to the oral health liability of betel quid use, while two distinct low molecular weight components may provide elements of reward, and the nicotinic activity of arecoline contributes to the physical dependence of addiction.

α-Conotoxins are small disulfide-rich peptides found in the venom of marine cone snails and are potent antagonists of nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential therapeutic applications for the treatment of chronic pain or neurological diseases and disorders. In the present study, we synthesized and functionally characterized a novel α-conotoxin Bt1.8, which was cloned from Conus betulinus. Bt1.8 selectively inhibited ACh-evoked currents in Xenopus oocytes expressing rat(r) α6/α3β2β3 and rα3β2 nAChRs with an IC50 of 2.1 nM and 9.4 nM, respectively, and similar potency for human (h) α6/α3β2β3 and hα3β2 nAChRs. Additionally, Bt1.8 had higher binding affinity with a slower dissociation rate for the rα6/α3β2β3 subtype compared to rα3β2. The amino acid sequence of Bt1.8 is significantly different from other reported α-conotoxins targeting the two nAChR subtypes. Further Alanine scanning analyses demonstrated that residues Ile9, Leu10, Asn11, Asn12 and Asn14 are critical for its inhibitory activity at the α6/α3β2β3 and α3β2 subtypes. Moreover, the NMR structure of Bt1.8 indicated the presence of a relatively larger hydrophobic zone than other α4/7-conotoxins which may explain its potent inhibition at α6/α3β2β3 nAChRs.

Background and purpose α4β2 nicotinic acetylcholine receptor (nAChR), a subtype of the ligand-gated ion channel, is abundantly expressed in the brain and implicated in several neurological disorders. The endocytosis of nAChR plays important roles in the pathogenesis of neurological diseases, but its molecular mechanisms remain poorly understood. Experimental approach Loss-of-function approaches and mutants of α4β2 nAChR that display different endocytic properties were used to identify the cellular components and processes responsible for endocytosis. The signaling cascade that leads to endocytosis was deduced via protein interactions in predicted cellular components. The endocytosis of α4β2 nAChR was determined and crosschecked using an enzyme-linked immunosorbent assay and radioligand assay. Key results Endocytosis of α4β2 nAChR occurred through clathrin-mediated endocytosis in a dynamin-dependent manner. 14-3-3η-dependent Src-mediated phosphorylation of the nAChR α4 subunit at Y575 was required for nAChR endocytosis, and this occurred with the assistance of β-arrestin1 and G protein-coupled receptor (GPCR) kinase 2 (GRK2) without the need for kinase activity. Endocytosis triggered the mouse double minute 2 homolog-mediated ubiquitination and subsequent downregulation of α4β2 nAChR. Conclusions and implications α4β2 nAChR, an ionophore receptor, employs the metabotropic signaling pathway required for endocytosis, which leads to ubiquitination and downregulation. Further, GRK2 and β-arrestin1, representative players of GPCRs, are involved in the endocytosis of α4β2 nAChR via different mechanisms. Considering the functional and pathological implications of nAChR endocytosis, results obtained in this study are crucial for the progression of basic research and clinical investigations.

Nicotine is a highly addictive compound present in tobacco, which causes the release of dopamine in different regions of the brain. Recent studies have shown that astrocytes express nicotinic acetylcholine receptors (nAChRs) and mediate calcium signaling. In this study, we examine the morphological and functional adaptations of astrocytes due to nicotine exposure. Utilizing a combination of fluorescence and atomic force microscopy, we show that nicotine-treated astrocytes exhibit time-dependent remodeling in the number and length of both proximal and fine processes. Blocking nAChR activity with an antagonist completely abolishes nicotine's influence on astrocyte morphology indicating that nicotine's action is mediated by these receptors. Functional studies show that 24-hr nicotine treatment induces higher levels of calcium activity in both the cell soma and the processes with a more substantial change observed in the processes. Nicotine does not induce reactive astrocytosis even at high concentrations (10 μM) as determined by cytokine release and glial fibrillary acidic protein expression. We designed tissue clearing experiments to test whether morphological changes occur in vivo using astrocyte specific Aldh1l1-tdTomato knock in mice. We find that nicotine induces a change in the volume of astrocytes in the prefrontal cortex, CA1 of the hippocampus, and the substantia nigra. These results indicate that nicotine directly alters the functional and morphological properties of astrocytes potentially contributing to the underlying mechanism of nicotine abuse.

The critical role of acetylcholine (ACh) in the basal ganglia is evident from the effect of cholinergic agents in patients suffering from several related neurological disorders, such as Parkinson's disease, Tourette syndrome, or dystonia. The striatum possesses the highest density of ACh markers in the basal ganglia underlying the importance of ACh in this structure. Striatal cholinergic interneurons (CINs) are responsible for the bulk of striatal ACh, although extrinsic cholinergic afferents from brainstem structures may also play a role. CINs are tonically active, and synchronized pause in their activity occurs following the presentation of salient stimuli during behavioral conditioning. However, the synaptic mechanisms involved are not fully understood in this physiological response. ACh modulates striatal circuits by acting on muscarinic and nicotinic receptors existing in several combinations both presynaptically and postsynaptically. While the effects of ACh in the striatum through muscarinic receptors have received particular attention, nicotinic receptors function has been less studied. Here, after briefly reviewing relevant results regarding muscarinic receptors expression and function, I will focus on striatal nicotinic receptor expressed presynaptically on glutamatergic and dopaminergic afferents and postsynaptically on diverse striatal interneurons populations. I will also review recent evidence suggesting the involvement of different GABAergic sources in two distinct nicotinic-receptor-mediated striatal circuits: the disynaptic inhibition of striatal projection neurons and the recurrent inhibition among CINs. A better understanding of striatal nicotinic receptors expression and function may help to develop targeted pharmacological interventions to treat brain disorders such as Parkinson's disease, Tourette syndrome, dystonia, or nicotine addiction.

Background and PurposeThe α7 and α4β2* (“*” denotes possibly assembly with another subunit) nicotinic acetylcholine receptors (nAChRs) are the most abundant nAChRs in the mammalian brain. These receptors are the most targeted nAChRs in drug discovery programmes for brain disorders. However, the development of subtype-specific agonists remains challenging due to the high degree of sequence homology and conservation of function in nAChRs. We have developed C(10) variants of cytisine, a partial agonist of α4β2 nAChR that has been used for smoking cessation. The C(10) methyl analogue used in this study displays negligible affinity for α7 nAChR, while retaining high affinity for α4β2 nAChR.Experimental ApproachThe structural underpinning of the selectivity of 10-methylcytisine for α7 and α4β2 nAChRs was investigated using molecular dynamic simulations, mutagenesis and whole-cell and single-channel current recordings.Key ResultsWe identified a conserved arginine in the β3 strand that exhibits a non-conserved function in nAChRs. In α4β2 nAChR, the arginine forms a salt bridge with an aspartate residue in loop B that is necessary for receptor expression, whereas in α7 nAChR, this residue is not stabilised by electrostatic interactions, making its side chain highly mobile. This lack of constrain produces steric clashes with agonists and affects the dynamics of residues involved in agonist binding and the coupling network.Conclusion and ImplicationsWe conclude that the high mobility of the β3-strand arginine in the α7 nAChR influences agonist binding and possibly gating network and desensitisation. The findings have implications for rational design of subtype-selective nAChR agents.

Background and purpose Mucociliary clearance is an innate immune process of the airways, essential for removal of respiratory pathogens. It depends on ciliary beat and ion and fluid homeostasis of the epithelium. We have shown that nicotinic ACh receptors (nAChRs) activate ion transport in mouse tracheal epithelium. Yet the receptor subtypes and signalling pathways involved remained unknown. Experimental approach Transepithelial short circuit currents (ISC ) of freshly isolated mouse tracheae were recorded using the Ussing chamber technique. Changes in [Ca2+ ]i were studied on freshly dissociated mouse tracheal epithelial cells. Key results Apical application of the nAChR agonist nicotine transiently increased ISC . The nicotine effect was abolished by the nAChR antagonist mecamylamine. α-Bungarotoxin (α7 antagonist) had no effect. The agonists epibatidine (α3β2, α4β2, α4β4 and α3β4) and A-85380 (α4β2 and α3β4) increased ISC . The antagonists dihydro-β-erythroidine (α4β2, α3β2, α4β4 and α3β4), α-conotoxin MII (α3β2) and α-conotoxin PnIA (α3β2) reduced the nicotine effect. Nicotine- and epibatidine-induced currents were unaltered in β2-/- mice, but in β4-/- mice no increase was observed. In the presence of thapsigargin (endoplasmatic reticulum Ca2+ -ATPase inhibitor) or the ryanodine receptor antagonists JTV-519 and dantrolene there was a reduction in the nicotine-effect, indicating involvement of Ca2+ release from intracellular stores. Additionally, the PKA inhibitor H-89 and the TMEM16A (Ca2+ -activated chloride channel) inhibitor T16Ainh-A01 significantly reduced the nicotine-effect. Conclusion and implications α3β4 nAChRs are responsible for the nicotine-induced current changes via Ca2+ release from intracellular stores, PKA and ryanodine receptor activation. These nAChRs might be possible targets to stimulate chloride transport via TMEM16A.

As a cis‐acting non‐depolarizing neuromuscular blocker through a nicotinic acetylcholine receptor (nAChR), cisatracurium (CAC) is widely used in anaesthesia and intensive care units. nAChR may be present on Leydig cells to mediate the action of CAC. Here, by Western blotting, immunohistochemistry and immunofluorescence, we identified that CHRNA4 (a subunit of nAChR) exists only on rat adult Leydig cells. We studied the effect of CAC on the synthesis of testosterone in rat adult Leydig cells and mouse MLTC‐1 tumour cells. Rat Leydig cells and MLTC‐1 cells were treated with CAC (5, 10 and 50 μmol/L) or nAChR agonists (50 μmol/L nicotine or 50 μmol/L lobeline) for 12 hours, respectively. We found that CAC significantly increased testosterone output in rat Leydig cells and mouse MLTC‐1 cells at 5 μmol/L and higher concentrations. However, nicotine and lobeline inhibited testosterone synthesis. CAC increased intracellular cAMP levels, and nicotine and lobeline reversed this change in rat Leydig cells. CAC may increase testosterone synthesis in rat Leydig cells and mouse MLTC‐1 cells by up‐regulating the expression of Lhcgr and Star. Up‐regulation of Scarb1 and Hsd3b1 expression by CAC was also observed in rat Leydig cells. In addition to cAMP signal transduction, CAC can induce ERK1/2 phosphorylation in rat Leydig cells. In conclusion, CAC binds to nAChR on Leydig cells, and activates cAMP and ERK1/2 phosphorylation, thereby up‐regulating the expression of key genes and proteins in the steroidogenic cascade, resulting in increased testosterone synthesis in Leydig cells.

There is an urgent need to address the devastating pandemic, COVID‐19, caused by SARS‐CoV‐2. The efforts to understand the details of this disease in hope of providing effective treatments is commendable. It is clear now that the virus can cause far more damage in patients with co‐morbid conditions ‐ particularly in those with respiratory, cardiovascular or immune‐compromised system ‐ than in patients without such co‐morbidities. Drug use can further exacerbate the condition. In this regard, the ill effects of smoking are amply documented, and no doubt can be a confounding factor in COVID‐19 progression. Although conflicting hypotheses on the potential role of nicotine in COVID‐19 pathology have recently been offered, we believe that nicotine itself, through its interaction with the nicotinic cholinergic system, as well as ACE2, may not only be of use in a variety of neuropsychiatric and neurodegenerative diseases, but may also be of potential use in COVID‐19. Thus, on one hand, while we strongly support smoking cessation as a means of harm reduction associated with COVID‐19, on the other hand, we support a potential therapeutic role for nicotine, nicotinic agonists or positive allosteric modulators of nicotinic cholinergic receptors in COVID‐19, owing to their varied effects including mood regulation, anti‐inflammatory as well as purported interference with SARS‐CoV‐2 entry and/or replication.

The first toxin to give rise to the three-finger protein (TFP) family was α-bungarotoxin (α-Bgt) from Bungarus multicinctus krait venom. α-Bgt was crucial for research on nicotinic acetylcholine receptors (nAChRs), and in this Review article we focus on present data for snake venom TFPs and those of the Ly6/uPAR family from mammalians (membrane-bound Lynx1 and secreted SLURP-1) interacting with nAChRs. Recently isolated from Bungarus candidus venom, αδ-bungarotoxins differ from α-Bgt: they bind more reversibly and distinguish two binding sites in Torpedo californica nAChR. Naja kaouthia α-cobratoxin, classical blocker of nAChRs, was shown to inhibit certain GABA-A receptor subtypes, whereas α-cobratoxin dimer with 2 intermolecular disulfides has a novel type of 3D structure. Non-conventional toxin WTX has additional 5th disulfide not in the central loop, as α-Bgt, but in the N-terminal loop, like all Ly6/uPAR proteins, and inhibits α7 and Torpedo nAChRs. A water-soluble form of Lynx1, ws-Lynx1, was expressed in E. coli, its 1 H-NMR structure and binding to several nAChRs determined. For SLURP-1, similar information was obtained with its recombinant analogue rSLURP-1. A common feature of ws-Lynx1, rSLURP-1, and WTX is their activity against nAChRs and muscarinic acetylcholine receptors. Synthetic SLURP-1, identical to the natural protein, demonstrated some differences from rSLURP-1 in distinguishing nAChR subtypes. The loop II fragment of the Lynx1 was synthesized having the same µM affinity for the Torpedo nAChR as ws-Lynx1. This review illustrates the productivity of parallel research of nAChR interactions with the two TFP groups.

Aims/Introduction Under irremediable endoplasmic reticulum (ER) stress, hyperactivated inositol‐requiring enzyme 1α (IRE1α) triggers the terminal unfolded protein response (T‐UPR), causing crucial cell dysfunction and apoptosis. We hypothesized that nicotinic acetylcholine receptor (nAChR) signaling regulates IRE1α activation to protect β‐cells from the T‐UPR under ER stress. Materials and Methods The effects of nicotine on IRE1α activation and key T‐UPR markers, thioredoxin‐interacting protein and insulin/proinsulin, were analyzed by real‐time polymerase chain reaction and western blotting in rat INS‐1 and human EndoC‐βH1 β‐cell lines. Doxycycline‐inducible IRE1α overexpression or ER stress agents were used to induce IRE1α activation. An α7 subunit‐specific nAChR agonist (PNU‐282987) and small interfering ribonucleic acid for α7 subunit‐specific nAChR were used to modulate nAChR signaling. Results Nicotine inhibits the increase in thioredoxin‐interacting protein and the decrease in insulin 1/proinsulin expression levels induced by either forced IRE1α hyperactivation or ER stress agents. Nicotine attenuated X‐box‐binding protein‐1 messenger ribonucleic acid site‐specific splicing and IRE1α autophosphorylation induced by ER stress. Furthermore, PNU‐282987 attenuated T‐UPR induction by either forced IRE1α activation or ER stress agents. The effects of nicotine on attenuating thioredoxin‐interacting protein and preserving insulin 1 expression levels were attenuated by pharmacological and genetic inhibition of α7 nAChR. Finally, nicotine suppressed apoptosis induced by either forced IRE1α activation or ER stress agents. Conclusions Our findings suggest that nAChR signaling regulates IRE1α activation to protect β‐cells from the T‐UPR and apoptosis under ER stress partly through α7 nAChR. Targeting nAChR signaling to inhibit the T‐UPR cascade may therefore hold therapeutic promise by thwarting β‐cell death in diabetes., Nicotinic acetylcholine receptor signaling attenuated inositol‐requiring enzyme 1α signaling and Terminal Unfolded Protein Response (T‐UPR) under irremediable endoplasmic reticulum stress in rat INS‐1 and human EndoC‐βH1 β‐cell lines. The effects of nicotine on terminal unfolded protein response were reduced by pharmacological and genetic inhibition of nicotinic acetylcholine receptor α7 subunit. Finally, nicotine suppressed apoptosis induced by either forced inositol requiring enzyme 1α activation or endoplasmic reticulum stress.

The serious coronavirus disease‐2019 (COVID‐19) was first reported in December 2019 in Wuhan, China. COVID‐19 is an infectious disease caused by severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2). Angiotensin converting enzyme 2(ACE2) is the cellular receptor for SARS‐CoV‐2. Considering the critical roles of testicular cells for the transmission of genetic information between generations, we analyzed single‐cell RNA‐sequencing (scRNA‐seq) data of adult human testis. The mRNA expression of ACE2 was expressed in both germ cells and somatic cells. Moreover, the positive rate of ACE2 in testes of infertile men was higher than normal, which indicates that SARS‐CoV‐2 may cause reproductive disorders through pathway activated by ACE2 and the men with reproductive disorder may easily to be infected by SARS‐CoV‐2. The expression level of ACE2 was related to the age, and the mid‐aged with higher positive rate than young men testicular cells. Taken together, this research provides a biological background of the potential route for infection of SARS‐CoV‐2 and may enable rapid deciphering male‐related reproductive disorders induced by COVID‐19.

The purpose of this study was to describe the distinct regional distribution patterns of expression of the α7 and α4 subunits of nicotinic acetylcholine receptors (nAChRs) and their left-right lateralisation in the rat hippocampus during the first 2 weeks of postnatal (P) development. Eighteen male pups were randomly divided into three groups: P0, P7, and P14. After removing the newborn brains, real-time polymerase chain reaction, western blot, and immunohistochemistry techniques were used to evaluate expression of the receptors. Results indicated that the expression profile of these receptors were time- and spatially dependent. A significant increase was observed in the distribution of α7 and α4 nAChR subunits in the developing rat hippocampus from P0 to P7 (p

The recent emergence of COVID‐19 has resulted in a worldwide crisis, with large populations locked down and transportation links severed. While approximately 80% of infected individuals have minimal symptoms, around 15–20% need to be hospitalized, greatly stressing global healthcare systems. As of March 10, the death rate appears to be about 3.4%, although this number is highly stratified among different populations. Here, we focus on those individuals who have been exposed to nicotine prior to their exposure to the virus. We predict that these individuals are ‘primed’ to be at higher risk because nicotine can directly impact the putative receptor for the virus (ACE2) and lead to deleterious signaling in lung epithelial cells., Coronavirus disease 2019 (COVID‐19), an infectious respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), originated in Wuhan in China in December 2019. Within months, the disease had spread globally leading to a worldwide pandemic with devastating consequences for communities and public healthcare services. The degree of severity associated with the disease varies widely among different populations. Here, James Olds and Nadine Kabbini discuss the role of smoking as a primary risk factor for severe infection. Drawing on the conclusions of studies of an earlier outbreak (SARS) and analysis of recent data on COVID‐19, the authors propose that some high‐risk individuals are ‘primed’ because of exposure to nicotine, which can directly impact the putative receptor for the virus (ACE2) in lung epithelial cells.

BACKGROUND: The beet armyworm, Spodoptera exigua, is a serious agricultural pest that is primarily controlled using chemical insecticides. Recently, resistance to the insecticide spinosad has been described in S. exigua field populations. To date, there has been no functional evidence proving the involvement of the nicotinic acetylcholine receptor (nAChR) α6 mutation in spinosad resistance in S. exigua. RESULTS: In this study, using the CRISPR/Cas9 genome‐editing system, a homozygous strain (Seα6‐KO) with approximately 1760‐bp deletion within Seα6 in S. exigua causing a premature truncation of Seα6 was successfully constructed. Insecticide bioassays showed that Seα6‐KO exhibited 373‐fold higher resistance to spinosad and 850‐fold higher resistance to spinetoram compared to WH‐S strain with the same genetic background but showed no significant change in susceptibility to emamectin benzoate and chlorantraniliprole. Genetic analysis revealed that Seα6‐KO is inherited as an incompletely recessive trait. CONCLUSION: The results clearly demonstrated the functional role of Seα6 in resistance to spinosyn insecticides and provide an example of using genome editing to verify a target premature truncation associated with resistance.

Adrenal chromaffin cells release neurotransmitters in response to stress and may be involved in conditions such as post-traumatic stress and anxiety disorders. Neurotransmitter release is triggered, in part, by activation of nicotinic acetylcholine receptors (nAChRs). However, despite decades of use as a model system for studying exocytosis, the nAChR subtypes involved have not been pharmacologically identified. Quantitative real-time PCR of rat adrenal medulla revealed an abundance of mRNAs for α3, α7, β2, and β4 subunits. Whole-cell patch-clamp electrophysiology of chromaffin cells and subtype-selective ligands were used to probe for nAChRs derived from the mRNAs found in adrenal medulla. A novel conopeptide antagonist, PeIA-5469, was created that is highly selective for α3β2 over other nAChR subtypes heterologously expressed in Xenopus laevis oocytes. Experiments using PeIA-5469 and the α3β4-selective α-conotoxin TxID revealed that rat adrenal medulla contain two populations of chromaffin cells that express either α3β4 nAChRs alone or α3β4 together with the α3β2β4 subtype. Conclusions were derived from observations that acetylcholine-gated currents in some cells were sensitive to inhibition by PeIA-5469 and TxID, while in other cells, currents were sensitive only to TxID. Expression of functional α7 nAChRs was determined using three α7-selective ligands: the agonist PNU282987, the positive allosteric modulator PNU120596, and the antagonist α-conotoxin [V11L,V16D]ArIB. The results of these studies identify for the first time the expression of α3β2β4 nAChRs as well as functional α7 nAChRs by rat adrenal chromaffin cells.

BACKGROUND AND PURPOSE: Animal toxins have contributed significantly to our understanding of the neurobiology of receptors and ion channels. We studied the venom of the coral snake Micrurus fulvius fulvius and identified and characterized the structure and pharmacology of a new homodimeric neurotoxin, fulditoxin, that exhibited novel pharmacology at nicotinic ACh receptors (nAChRs). EXPERIMENTAL APPROACH: Fulditoxin was isolated by chromatography, chemically synthesized, its structure determined by X‐ray crystallography, and its pharmacological actions on nAChRs characterized by organ bath assays and two‐electrode voltage clamp electrophysiology. KEY RESULTS: Fulditoxin's distinct 1.95‐Å quaternary structure revealed two short‐chain three‐finger α‐neurotoxins (α‐3FNTxs) non‐covalently bound by hydrophobic interactions and an ability to bind metal and form tetrameric complexes, not reported previously for three‐finger proteins. Although fulditoxin lacked all conserved amino acids canonically important for inhibiting nAChRs, it produced postsynaptic neuromuscular blockade of chick muscle at nanomolar concentrations, comparable to the prototypical α‐bungarotoxin. This neuromuscular blockade was completely reversible, which is unusual for snake α‐3FNTxs. Fulditoxin, therefore, interacts with nAChRs by utilizing a different pharmacophore. Unlike short‐chain α‐3FNTxs that bind only to muscle nAChRs, fulditoxin utilizes dimerization to expand its pharmacological targets to include human neuronal α4β2, α7, and α3β2 nAChRs which it blocked with IC(50) values of 1.8, 7, and 12 μM respectively. CONCLUSIONS AND IMPLICATIONS: Based on its distinct quaternary structure and unusual pharmacology, we named this new class of dimeric Micrurus neurotoxins represented by fulditoxin as Σ‐neurotoxins, which offers greater insight into understanding the interactions between nAChRs and peptide antagonists.

Recent studies have showed that α5 nicotinic acetylcholine receptor (α5‐nAChR) is closely associated with nicotine‐related lung cancer. Our previous studies also demonstrated that α5‐nAChR mediates nicotine‐induced lung carcinogenesis. However, the mechanism by which α5‐nAChR functions in lung carcinogenesis remains to be elucidated. Jab1/Csn5 is a key regulatory factor in smoking‐induced lung cancer. In this study, we explored the underlying mechanisms linking the α5‐nAChR‐Jab1/Csn5 axis with lung cancer epithelial‐mesenchymal transition (EMT) and metastasis, which may provide potential therapeutic targets for future lung cancer treatments. Our results demonstrated that the expression of α5‐nAChR was correlated with the expression of Jab1/Csn5 in lung cancer tissues and lung cancer cells. α5‐nAChR expression is associated with Jab1/Csn5 expression in lung tumour xenografts in mice. In vitro, the expression of α5‐nAChR mediated Stat3 and Jab1/Csn5 expression, significantly regulating the expression of the EMT markers, N‐cadherin and Vimentin. In addition, the down‐regulation of α5‐nAChR or/and Stat3 reduced Jab1/Csn5 expression, while the silencing of α5‐nAChR or Jab1/Csn5 inhibited the migration and invasion of NSCLC cells. Mechanistically, α5‐nAChR contributes to EMT and metastasis by regulating Stat3‐Jab1/Csn5 signalling in NSCLC, suggesting that α5‐nAChR may be a potential target in NSCLC diagnosis and immunotherapy.

The roles of α5-nicotinic acetylcholine receptors (α5-nAChRs) in various types of solid cancer have been reported; however, its role in melanoma remains unknown. We knocked down α5-nAChR expression in melanoma cells to investigate the role of α5-nAChR in the proliferation, migration, and invasion of melanoma cells, and its effect on downstream signaling pathways. Using immunohistochemical analysis, we determined that α5-nAChR expression is significantly increased in human melanoma tissues and cell lines compared with normal human skin tissues. Knocking down α5-nAChR expression in melanoma cells in culture significantly inhibited the proliferation, migration, and invasiveness of melanoma cell lines. Specifically, knockdown of α5-nAChR inhibited PI3K-AKT and ERK1/2 signaling activity. Moreover, we confirmed that the Notch1 signaling pathway is the downstream target of α5-nAChR in melanoma. Our findings suggest that α5-nAChR plays a critical role in melanoma development and progression, and that targeting α5-nAChR may be a strategy for melanoma treatment.

As a major metabolite of kynurenine in the oxidative metabolism of tryptophan, kynurenic acid is of considerable biological and clinical importance as an endogenous antagonist of glutamate in the central nervous system. It is most active as an antagonist at receptors sensitive to N‐methyl‐D‐aspartate (NMDA) which regulate neuronal excitability and plasticity, brain development and behaviour. It is also thought to play a causative role in hypo‐glutamatergic conditions such as schizophrenia, and a protective role in several neurodegenerative disorders, notably Huntington’s disease. An additional hypothesis, that kynurenic acid could block nicotinic receptors for acetylcholine in the central nervous system has been proposed as an alternative mechanism of action of kynurenate. However, the evidence for this alternative mechanism is highly controversial, partly because at least eight earlier studies concluded that kynurenic acid blocked NMDA receptors but not nicotinic receptors and five subsequent, independent studies designed to repeat the results have failed to do so. Many studies considered to support the alternative ‘nicotinic’ hypothesis have been based on the use of analogs of kynurenate such as 7‐chloro‐kynurenic acid, or putatively nicotinic modulators such as galantamine, but a detailed analysis of the pharmacology of these compounds suggests that the results have often been misinterpreted, especially since the pharmacology of galantamine itself has been disputed. This review examines the evidence in detail, with the conclusion that there is no confirmed, reliable evidence for an antagonist activity of kynurenic acid at nicotinic receptors. Therefore, since there is overwhelming evidence for kynurenate acting at ionotropic glutamate receptors, especially NMDAR glutamate and glycine sites, with some activity at GPR35 sites and Aryl Hydrocarbon Receptors, results with kynurenic acid should be interpreted only in terms of these confirmed sites of action., Kynurenic acid has well‐established activity as an antagonist at glutamate receptors, especially the glutamate and glycine co‐agonist binding sites, with possibly relevant actions on Aryl Hydrocarbon Receptors and the GPR35 binding site. A single proposal that it also acts on acetylcholine nicotinic receptors is analysed and discussed in the light of at least 12 failures to reproduce the activity, and a wide range of pharmacological arguments. It is concluded that there is no confirmed, reliable evidence for any effect at nicotinic acetylcholine receptors.

Tumor cells modulate and are modulated by their microenvironments, which include the nervous system. Accumulating evidence links the overexpression and activity of nicotinic and muscarinic cholinergic receptor subtypes to tumorigenesis in breast, ovarian, prostate, gastric, pancreatic, and head and neck cancers. Nicotinic and muscarinic receptors have downstream factors are associated with angiogenesis, cell proliferation and migration, antiapoptotic signaling, and survival. Clinical trials analyzing the efficacy of various therapies targeting cholinergic signaling or downstream pathways of acetylcholine have shed promising light on novel cancer therapeutics. Although the evidence for cholinergic signaling involvement in tumor development is substantial, a more detailed understanding of the acetylcholine-induced mechanisms of tumorigenesis remains to be unlocked. Such an understanding would enable the development of clinical applications ranging from the identification of novel biomarkers to the utilization of existing drugs to modulate cholinergic signaling to the development of novel cancer therapies, as discussed in this review.

BACKGROUND Insect desensitizing nicotinic acetylcholine (nAChD) receptors are desensitized by low concentrations of agonists, including neonicotinoid insecticides, but are essentially insensitive to spinosyns, while non-desensitizing nicotinic acetylcholine (nAChN) receptors are selectively activated by spinosyns and relatively insensitive to neonicotinoids. RESULTS The single-electrode voltage-clamp technique was used to measure the actions of newer nicotinic insecticides dinotefuran, sulfoxaflor, triflumezopyrim, spinetoram and GS-ω/k-hexatoxin-Hv1a on cockroach neuronal nAChD and nAChN currents. Like imidacloprid and clothianidin, newer orthosteric nicotinic agonist insecticides dinotefuran and sulfoxaflor act by desensitizing nAChD receptors. The mesoionic insecticide triflumezopyrim selectively inhibited nAChD current with an half maximal inhibitory concentration (IC50 ) of 1.2 nmol L-1 , with no activation. Unlike other Group 4 insecticides, it did not activate nAChN current, but inhibited it with an IC50 of 3.8 μmol L-1 , indicating that the compound is a true antagonist. Spinosad and the spinosyn-derived insecticide spinetoram potently and selectively activated nAChN receptors. GS-ω/k-hexatoxin-Hv1a had no effect on nAChN currents and it had a complex action on nAChD currents, inhibiting at sub-nanomolar concentrations and causing some activation and enhancement of ACh-evoked currents at 30 nmol L-1 and above. Some cells express GS-ω/k-hexatoxin-Hv1a-resistant nAChD receptors. CONCLUSIONS Nicotinic acetylcholine receptor competitive modulators (IRAC Group 4) and nicotinic acetylcholine receptor allosteric modulators, site II (hexatoxins, IRAC Group 32) are selective for nAChD receptors, while nicotinic acetylcholine receptor allosteric modulators, site I (spinosyns, IRAC Group 5) are selective for nAChN receptors. It is proposed that IRAC Groups 5 and 32 be re-named non-desensitizing nicotinic acetylcholine receptor allosteric modulators and desensitizing nicotinic acetylcholine receptor allosteric modulators, respectively. © 2021 Society of Chemical Industry.

Introduction Muscle fiber denervation increases with age, yet studies at the tissue level are sparse due to the challenging nature of establishing the relative role of regeneration and denervation. Methods Muscle biopsies were obtained from the vastus lateralis of 70 healthy men (aged 72 ± 6 years; range, 65-94). Messenger RNA (mRNA) levels of acetylcholine receptors (AchR) were measured, and sections were stained for embryonic myosin, neonatal myosin (MHCn ), and neural cell adhesion molecule (NCAM). Results Embryonic myosin+ fibers were rare, while MHCn + and NCAM+ fibers were observed in all samples. Age (range, 65-94 years) was negatively associated with AchRγ mRNA. Discussion Muscle from healthy older individuals expressed developmental myosins to varying degrees but more than has been previously reported for young individuals. Along with the AchR correlations, we propose that these findings support the presence of neuromuscular junction destabilization, denervation, and reinnervation in aging human skeletal muscle.

BACKGROUND AND PURPOSE: The heteromeric α4β2 nicotinic acetylcholine receptor (nAChR) is abundant in the human brain and is associated with a range of CNS disorders. This nAChR subtype has been recently crystallised in a conformation that was proposed to represent a desensitised state. Here, we investigated the conformational transition mechanism of this nAChR from a desensitised to a closed/resting state. EXPERIMENTAL APPROACH: The competitive antagonist dihydro‐β‐erythroidine (DHβE) was modelled by replacement of the agonist nicotine in the α4β2 nAChR experimental structure. DHβE is used both in vitro and in vivo for its ability to block α4β2 nAChRs. This system was studied by three molecular dynamics simulations with a combined simulation time of 2.6 μs. Electrophysiological studies of mutated receptors were performed to validate the simulation results. KEY RESULTS: The relative positions of the extracellular and transmembrane domains in the models are distinct from those of the desensitised state structure and are compatible with experimental structures of Cys‐loop receptors captured in a closed/resting state. CONCLUSIONS AND IMPLICATIONS: Our model suggests that the side chains of α4 L257 (9′) and α4 L264 (16′) are the main constrictions in the transmembrane pore. The involvement of position 9′ in channel gating is well established, but position 16′ was only previously identified as a gate for the bacterial channels, ELIC and GLIC. L257 but not L264 was found to influence the slow component of desensitisation. The structure of the antagonist‐bound state proposed here should be valuable for the development of therapeutic or insecticide compounds.

The insecticidal effects of ω-hexatoxin-Hv1a, κ-hexatoxin-Hv1c and ω/κ-hexatoxin-Hv1h are currently attributed to action at calcium and potassium channels. By characterizing the binding of these toxins to neuronal membranes, we show that they have more potent effects as positive allosteric modulators (PAMs) of insect nicotinic acetylcholine receptors (nAChRs), consistent with their neuroexcitatory toxicology. Alanine scanning analysis of ω-hexatoxin-Hv1a reveals a structure-activity relationship for binding that mirrors that for insecticidal activity. Spinosyn A does not compete with ω-hexatoxin-Hv-1a for binding, and we show that these two PAMs have distinct pharmacology of binding indicating that they act at different receptor populations. These toxins represent valuable tools for the characterization of insect nAChRs and for the development of more selective agrochemicals.

Ageing attenuates muscarinic-mediated sweating. However, whether ageing also impairs nicotinic-mediated sweating remains unclear. Further, despite the known sex-related differences in peripheral sweat gland function, it remains unclear whether age-related modifications of muscarinic and nicotinic-mediated sweating, if any, are similar between men and women. We assessed local sweating in young and older healthy men and women (n = 11, each group) at two dorsal forearm skin sites receiving either: (a) methacholine (muscarinic receptor agonist, 5 doses: 0.0125, 0.25, 5, 100, 2000 mmol/L) or (b) nicotine (nicotinic receptor agonist, 5 doses: 1.2, 3.6, 11, 33, 100 mmol/L) via intradermal microdialysis. Age-related reductions in methacholine-induced sweating were observed at low-to-moderate doses (0.0125-5 mmol/L; all P ≤ 0.05) in men, whereas a reduction was only evident at the highest methacholine dose (2000 mmol/L; P ≤ 0.05) in women. No effect of ageing was observed for nicotine-induced sweating (all P > 0.26 for main effects of age, dose and all interactions). We showed that while healthy ageing attenuates low-to-moderate levels of muscarinic-mediated sweating in men, reductions are only observed at high levels of muscarinic-mediated sweating in women. However, healthy ageing does not modulate nicotinic-mediated sweating in either men or women.

Background To investigate the gene expression profile of a set of candidate genes for a better understanding of the molecular mechanism underlying the pathogenesis of thymoma with or without myasthenia gravis. Methods Thymoma patients and thymoma patients with myasthenia gravis were analyzed using microarray profiling to identify significant changes in gene expression of autoimmune regulator pathway genes including AIRE, IL-7R, CHRNA3, SYMD1, THRA, and CAV3. Results Across all of our samples, we found that 1484 mRNAs were upregulated and 770 were downregulated in thymoma patients compared with thymoma with myasthenia gravis patients. Gene ontology and pathway analysis revealed that a large number of genes participated in cellular functions for humoral immune response, sequence-specific DNA binding RNA polymerase II transcription factor activity, positive regulation of gene expression, regulation of neuron projection development, extracellular ligand-gated ion channel activity, positive regulation of striated muscle cell differentiation, and regulation of nuclear factor-kappaB import into the nucleus. Conclusion Our results revealed genetic differences between thymomas and myasthenia gravis, and identified the key candidate genes/pathways for molecular mechanism.

The current standard therapy to counteract organophosphate intoxication is not effective in equal measure against all types of organophosphorus compounds (OPCs), as the outcome of oxime-induced reactivation of inactivated acetylcholinesterase (AChE) strongly depends on the particular OPC. In case the reactivation is insufficient, acetylcholine concentrations that rise to pathophysiological levels force the nicotinic acetylcholine receptor (nAChR) into a desensitized state and hence a functionally inactive state. As a consequence, neurotransmission is irreversibly disrupted at the neuromuscular junction. Previous electrophysiological studies identified the symmetric bispyridinium compound 1,1 '-(propane-1,3-diyl)bis[4-(tert-butyl)pyridin-1-ium] diiodide (MB327) as a re-sensitizer of the desensitized nAChR. MB327 is thereby capable of restoring the functional activity. Very recently, in silico modeling studies suggested non-symmetric derivatives of MB327 as potential re-sensitizers with enhanced binding affinity and thus possible enhanced efficacy. In this study, 26 novel non-symmetric bispyridinium compounds and related derivatives were synthesized. For the synthesis of the highly polar target compounds in sufficient quantities, newly developed and highly efficient two-step procedures were used. Compounds were characterized in terms of their binding affinity toward the MB327 binding site at the nAChR using recently developed mass spectrometry (MS) Binding Assays. Regarding structure-affinity relationships at the MB327 binding site, the presence of two quaternary aromatic nitrogen centers as well as pyridinium systems with a tert-butyl group at the 4-position or a NMe2 group at the 3- or 4-positions appeared to be beneficial for high binding affinities.

Methyl palmitate (MP) is a fatty acid methyl ester. Our recent study indicated that adrenergic nerve-dependent functional sympathetic-sensory nerve interactions were abolished by MP in mesenteric arteries. However, the effect of MP on perivascular nerves and cerebral blood flow remains unclear. In this study, the increase in basilar arterial blood flow (BABF) after the topical application of nicotinic acetylcholine receptor agonists was measured using laser Doppler flowmetry in anesthetized rats. The choline (a selective α7-nicotinic acetylcholine receptor agonist)-induced increase in BABF was abolished by tetrodotoxin (a neurotoxin), NG -nitro-L-arginine (a nonselective NO synthase inhibitor), α-bungarotoxin (a selective α7-nicotinic acetylcholine receptor inhibitor), and chronic sympathetic denervation. In addition, the nicotine (a nicotinic acetylcholine receptor agonist)-induced increase in BABF was inhibited by MP in a concentration-dependent manner. The acetylcholine-induced increase in BABF was not affected by MP. The myography results revealed that nicotine-induced vasorelaxation was significantly inhibited by MP, but was reversed by chelerythrine (a protein kinase C inhibitor). MP-induced vasodilation was significantly greater in BA rings without endothelium compared to those with endothelium. Meanwhile, MP did not affect baseline BABF. Our results indicate that MP acts as a neuromodulator in the cerebral circulation where it activates the PKC pathway and causes a diminished nicotine-induced increase in blood flow in the brainstem, and that the vasorelaxation effect of MP may play a minor role.

Cigarette smoking is the single most important risk factor for the development of cardiovascular diseases (CVDs). However, the role of nicotine, the addictive component of all tobacco products, in the development of CVD is incompletely understood. Although increased public awareness of the harms of cigarette smoking has successfully led to a decline in its prevalence, the use of electronic cigarettes (e-cig) or electronic nicotine delivery system has increased dramatically in recent years because of the perception that these products are safe. This review summarizes our current knowledge of the expression and function of the nicotinic acetylcholine receptors in the cardiovascular system and the impact of nicotine exposure on cardiovascular health, with a focus on nicotine-induced vascular dysfunction. Nicotine alters vasoreactivity through endothelium-dependent and/or endothelium-independent mechanisms, leading to clinical manifestations in both cigarette smokers and e-cig users. In addition, nicotine induces vascular remodelling through its effects on proliferation, migration and matrix production of both vascular endothelial and vascular smooth muscle cells. The purpose of this review is to identify critical knowledge gaps regarding the effects of nicotine on the vasculature and to stimulate continued nicotine research.

Neonicotinoids are selective modulators of insect nicotinic acetylcholine receptors (nAChRs). These widely deployed insecticides interact with the orthosteric sites of nAChRs, not only to activate nAChRs on their own, but also to block the desensitizing component of nAChR responses. To date recombinant vertebrate or insect/vertebrate hybrid nAChRs have been deployed to understand the mechanism of selectivity and diversity of neonicotinoid actions as well as to show that both α/α and α/non-α interfaces are involved in the interactions with neonicotinoids. However, many of the fine details of insecticide interactions with sites on nAChRs remain to be resolved. The breakthrough of functional expression of insect nAChRs allows such questions to be addressed, not only for neonicotinoids but for other insecticides targeting insect nAChRs. © 2020 Society of Chemical Industry.

Acetylcholine (ACh) signaling in the hippocampus, is important for behaviors related to learning, memory and stress. In this study, we investigated the role of two ACh receptor subtypes previously shown to be involved in fear and anxiety, the M1 mAChR and the α2 nAChR, in mediating the effects of hippocampal ACh on stress-related behaviors. Adeno-associated viral vectors containing short-hairpin RNAs targeting M1 or α2 were infused into the hippocampus of male C57BL/6J mice, and behavior in a number of paradigms related to stress responses and fear learning was evaluated. There were no robust effects of hippocampal M1 mAChR or α2 nAChR knockdown in the light/dark box, tail suspension, forced swim or novelty-suppressed feeding tests. However, effects on fear learning were observed in both knockdown groups. Short term learning was intact immediately after training in all groups of mice, but both the M1 and α2 hippocampal knock down resulted in impaired cued fear conditioning 24 hours after training. In addition, there was a trend for a deficit in contextual memory the M1 mAChR knockdown (KD) group 24 hours after training. These results suggest that α2 nicotinic and M1 muscarinic ACh receptors in the hippocampus contribute to fear learning and could be relevant targets to modify brain circuits involved in stress-induced reactivity to associated cues.

Background Benign childhood epilepsy with centrotemporal spikes (BECTS) or benign rolandic epilepsy is the most common epileptic syndrome in school‐age children. Genetics is an important factor in BECTS pathogenesis, and A was identified in three patients with BECTS in a pedigree. Carbamazepine, which should be carefully used in BECTS, was observed to be effective in the treatment of our atypical BECTS proband based on the molecular diagnosis of CHRNA4. Conclusion This is the first study on CHRNA4 variant in BECTS, which widened the genetic spectrum of BECTS and contributed to precise medicine in BECTS., Our research is the first report of CHRNA4 variant in BECTS. Carbamazepine which should be carefully used in BECTS was observed to be effective in the treatment of the disease based on molecular diagnosis.

The prevalence of alcohol use disorders (AUDs) has steadily increased in the United States over the last 30 years. Alcohol acts on multiple receptor systems including the nicotinic acetylcholine receptors (nAChRs), which are known to mediate alcohol consumption and reward. We previously reported that the preclinical drug sazetidine-A, a nAChR agonist and desensitizer, reduces alcohol consumption without affecting nicotine consumption in C57BL/6J mice. Here, we found that sazetidine-A enhances the expression of alcohol aversion without affecting the expression or acquisition of conditioned alcohol reward in C57BL/6J mice. Microinjection of sazetidine-A into the ventral midbrain targeting the ventral tegmental area (VTA) reduced binge alcohol consumption, implicating this region in mediating the effects of sazetidine-A. Furthermore, the sazetidine-A-induced reduction in alcohol consumption was mediated by non-α4 containing nAChRs, as sazetidine-A reduced binge alcohol consumption in both α4 knock-out and wild-type mice. Finally, we found that in mice pretreated with sazetidine-A, alcohol induced Fos transcript in Th-, but not Gad2-expressing neurons in the VTA as measured by increased Fos transcript expression. In summary, we find that sazetidine-A enhances the expression of alcohol aversion, which may underlie the reduction in alcohol consumption induced by sazetidine-A. Elucidating the identity of non-α4 nAChRs in alcohol aversion mechanisms will provide a better understanding the complex role of nAChRs in alcohol addiction and potentially reveal novel drug targets to treat AUDs.

Micro‐RNAs (miRNAs) are highly evolutionarily conserved short‐length/noncoding RNA molecules that modulate a wide range of cellular functions in many cell types by regulating the expression of a variety of targeted genes. miRNAs have also recently emerged as key regulators of neuronal genes mediating the effects of psychostimulant drugs and memory‐related neuroplasticity processes. Smoking is a predominant addictive behaviour associated with millions of deaths worldwide, and nicotine is a potent natural psychoactive agonist of cholinergic receptors, highly abundant in cigarettes. The influence of miRNAs modulation on cholinergic signalling in the nervous system remains however poorly explored. Using miRNA knockout mice and biochemical, electrophysiological and pharmacological approaches, we examined the effects of miR‐132/212 gene disruption on the levels of hippocampal nicotinic acetylcholine receptors, total ERK and phosphorylated ERK (pERK) and MeCP2 protein levels, and studied the impact of nicotine stimulation on hippocampal synaptic transmission and synaptic depression and strengthening. miR‐132/212 deletion significantly altered α7‐nAChR and pERK protein levels, but not total ERK or MeCP2, and resulted in both exacerbated synaptic depression and virtually abolished memory‐related synaptic strengthening upon nicotine stimulation. These observations reveal a functional miRNAs/nicotinergic signalling interplay critical for nicotinic‐receptor expression and neuroplasticity in brain structures relevant for drug addiction and learning and memory functions., Micro‐RNAs are key regulators of neuronal genes mediating the effects of psychostimulant drugs and neuroplasticity. Using miRNA knockout mice and biochemical, electrophysiological and pharmacological approaches, this work describes that miR‐132/212 gene deletion alters molecular elements from the cholinergic signalling pathway (α7‐nAChR and pERK protein levels) and not only exacerbates synaptic depression but also virtually abolishes memory‐related synaptic strengthening upon nicotine stimulation.

Synaptic strength depends on the number of cell-surface neurotransmitter receptors in dynamic equilibrium with intracellular pools. Dysregulation of this homeostatic balance occurs, for example in myasthenia gravis, an autoimmune disease characterized by a decrease in the number of postsynaptic nicotinic acetylcholine receptors (nAChRs). Monoclonal antibody mAb35 mimics this effect. Here we use STORM nanoscopy to characterize the individual and ensemble dynamics of monoclonal antibody-crosslinked receptors in the clonal cell line CHO-K1/A5, which robustly expresses adult muscle-type nAChRs. Antibody labeling of live cells results in 80% receptor immobilization. The remaining mobile fraction exhibits a heterogeneous combination of Brownian and anomalous diffusion. Single-molecule trajectories exhibit a two-state switching behavior between free Brownian walks and anticorrelated walks within confinement areas. The latter act as permeable fences (~34 nm radius, ~400 ms lifetime). Dynamic clustering, trapping, and immobilization also occur in larger nanocluster zones (120–180 nm radius) with longer lifetimes (11 ± 1 s), in a strongly cholesterol-sensitive manner. Cholesterol depletion increases the size of the clustering phenomenon; cholesterol enrichment has the opposite effect. The disclosed high proportion of monoclonal antibody-crosslinked immobile receptors, together with their anomalous, cholesterol-sensitive diffusion and clustering, provides new insights into the antibody-enhanced antigenic modulation that leads to physiopathological internalization and degradation of receptors in myasthenia. (Figure presented.). Fil: Mosqueira, Alejo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina Fil: Camino, Pablo A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina Fil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina

Pterygium is a triangular-shaped hyperplastic growth, characterized by conjunctivalization, inflammation, and connective tissue remodeling. Our previous meta-analysis found that cigarette smoking is associated with a reduced risk of pterygium. Yet, the biological effect of cigarette smoke components on pterygium has not been studied. Here we reported the proliferation and migration properties of human primary pterygium cells with continuous exposure to nicotine and cotinine. Human primary pterygium cells predominantly expressed the α5, β1, and γ subunits of the nicotinic acetylcholine receptor. Continuous exposure to the mixture of 0.15 μM nicotine and 2 μM cotinine retarded pterygium cell proliferation by 16.04% (P = 0.009) and hindered their migration by 11.93% ( P = 0.039), without affecting cell apoptosis. SNAIL and α-smooth muscle actin protein expression was significantly downregulated in pterygium cells treated with 0.15 μM nicotine-2 μM cotinine mixture by 1.33- ( P = 0.036) and 1.31-fold ( P = 0.001), respectively. Besides, the 0.15 μM nicotine-2 μM cotinine mixture also reduced matrix metalloproteinase (MMP)-1 and MMP-9 expressions in pterygium cells by 1.56- ( P = 0.043) and 1.27-fold ( P = 0.012), respectively. In summary, this study revealed that continuous exposure of nicotine and cotinine inhibited human primary pterygium cell proliferation and migration in vitro by reducing epithelial-to-mesenchymal transition and MMP protein expression, partially explaining the lower incidence of pterygium in cigarette smokers.

Breast cancer (BC) is the most common cancer affecting women worldwide and has been associated with active tobacco smoking. Low levels of nicotine (Nic) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have been detected in cases of second-hand smoke (SHS). However, the correlation between SHS and BC risk remains controversial. In this study, we investigated whether the physiological SHS achievable dose of Nic and tobacco specific nitrosamine, NNK act together to induce breast carcinogenesis using an in vitro breast cell carcinogenesis model. Immortalized non-tumorigenic breast epithelial cell line, HBL-100 used for a time-course assay, was exposed to very low levels of either Nic or NNK, or both. The time-course assay consisted of 23 cycles of nitrosamines treatment. In each cycle, HBL-100 cells were exposed to 1pM of Nic and/or 100 femtM of NNK for 48 hours. Cells were passaged every 3 days and harvested after 10, 15, and 23 cycles. Our results demonstrated that the tumorigenicity of HBL-100, defined by soft agar colony forming, proliferation, migration and invasion abilities, was enhanced by co-exposure to physiologically SHS achievable doses of Nic and NNK. In addition, α9-nAChR signaling activation, which plays an important role in cellular proliferation and cell survival, was also observed. Importantly, an increase in stemness properties including the prevalence of CD44+/CD24- cells, increase Nanog expression and mammosphere-forming ability were also observed. Our results indicate that chronic and long term exposure to environmental tobacco smoke, may induce breast cell carcinogenesis even at extremely low doses.

The amygdala is involved in processing incoming information about rewarding stimuli and emotions that denote danger such as anxiety and fear. Bi-directional neural connections between basolateral amygdala (BLA) and brain regions such as nucleus accumbens, prefrontal cortex, hippocampus, and hindbrain regions regulate motivation, cognition, and responses to stress. Altered local regulation of BLA excitability is pivotal to the behavioral disturbances characteristic of posttraumatic stress disorder, and relapse to drug use induced by stress. Herein, we review the physiological regulation of BLA by cholinergic inputs, emphasizing the role of BLA nicotinic receptors. We review BLA-dependent effects of nicotine on cognition, motivated behaviors, and emotional states, including memory, taking and seeking drugs, and anxiety and fear in humans and animal models. The alterations in BLA activity observed in animal studies inform human behavioral and brain imaging research by enabling a more exact understanding of altered BLA function. Converging evidence indicates that cholinergic signaling from basal forebrain projections to local nicotinic receptors is an important physiological regulator of BLA and that nicotine alters BLA function. In essence, BLA is necessary for behavioral responses to stimuli that evoke anxiety and fear; reinstatement of cue-induced drug seeking; responding to second-order cues conditioned to abused drugs; reacquisition of amplified nicotine self-administration due to chronic stress during abstinence; and to promote responding for natural reward.

Background Neonicotinoids, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) used to control Nilaparvata lugens, a major rice insect pest. High imidacloprid resistance has been reported in N. lugens both in the laboratory and in the field. Cycloxaprid (CYC), an oxa-bridged cis-nitromethylene neonicotinoid, showed high insecticidal activity against N. lugens and low cross-resistance in imidacloprid-resistant strains and field populations. Results Binding studies demonstrated that imidacloprid has two binding sites with different affinities (Kd = 3.18 ± 0.43 pm and 1.78 ± 0.19 nm) in N. lugens nAChRs. CYC was poor at displacing [3 H]imidacloprid at its high-affinity binding site (Ki = 159.38 ± 20.43 nm), but quite efficient at the low-affinity binding site (Ki = 1.27 ± 0.35 nm). These data showed that CYC had overlapping binding sites with imidacloprid only at its low-affinity binding site. Therefore, the low displacement ability of CYC against imidacloprid binding at its high-affinity site could partially explain the low cross-resistance of CYC in imidacloprid-resistant populations. Conclusion The high insecticidal activity, low cross-resistance and different binding properties on insect nAChRs of CYC show that it is a potential insecticide for the control of N. lugens and related insect pests, especially ones with high resistance to neonicotinoids. © 2018 Society of Chemical Industry.

A novel series of 30 symmetric bispyridinium and related N-heteroaromatic bisquaternary salts with a propane-1,3-diyl linker was synthesized and characterized for their binding affinity at the MB327 binding site of nicotinic acetylcholine receptor (nAChR) from Torpedo californica. Compounds targeting this binding site are of particular interest for research into new antidotes against organophosphate poisoning, as therapeutically active 4-tert-butyl-substituted bispyridinium salt MB327 was previously identified as a nAChR re-sensitizer. Efficient access to the target compounds was provided by newly developed methods enabling N-alkylation of sterically hindered or electronically deactivated heterocycles exhibiting a wide variety of functional groups. Determination of binding affinities toward the MB327 binding site at the nAChR, using a recently developed mass spectrometry (MS)-based Binding Assay, revealed that several compounds reached affinities similar to that of MB327 (pK(i)=4.73 +/- 0.03). Notably, the newly prepared lipophilic 4-tert-butyl-3-phenyl-substituted bispyridinium salt PTM0022 (3h) was found to have significantly higher binding affinity, with a pK(i) value of 5.16 +/- 0.07, thus representing considerable progress toward the development of more potent nAChR re-sensitizers.

Nicotinic acetylcholine receptors (nAChRs), prototype members of the cys-loop ligand-gated ion channel family, are key mediators of cholinergic transmission in the central nervous system. Despite their importance, technical gaps exist in our ability to dissect the function of individual subunits in the brain. To overcome these barriers, we designed CRISPR/Cas9 small guide RNA sequences (sgRNAs) for the production of loss-of-function alleles in mouse nAChR genes. These sgRNAs were validated in vitro via deep sequencing. We subsequently targeted candidate nAChR genes in vivo by creating herpes simplex virus (HSV) vectors delivering sgRNAs and Cas9 expression to mouse brain. The production of loss-of-function insertions or deletions (indels) by these 'all-in-one' HSV vectors was confirmed using brain slice patch clamp electrophysiology coupled with pharmacological analysis. Next, we developed a scheme for cell type-specific gene editing in mouse brain. Knockin mice expressing Cas9 in a Cre-dependent manner were validated using viral microinjections and genetic crosses to common Cre-driver mouse lines. We subsequently confirmed functional Cas9 activity by targeting the ubiquitous neuronal protein, NeuN, using adeno-associated virus (AAV) delivery of sgRNAs. Finally, the mouse β2 nAChR gene was successfully targeted in dopamine transporter (DAT)-positive neurons via CRISPR/Cas9. The sgRNA sequences and viral vectors, including our scheme for Cre-dependent gene editing, should be generally useful to the scientific research community. These tools could lead to new discoveries related to the function of nAChRs in neurotransmission and behavioral processes.

Background Cigarette consumption has been identified as the main non‐etiological factor in head and neck cancer (HNC) development. One of the main compounds in cigarettes is nicotine, which binds directly to nicotine acetylcholine receptors (nAchRs) in the body, which are encoded by different genes of the CHRNA family. Polymorphisms in some of these genes have been studied in relation to the risk of HNC and cigarette consumption intensity. The aim of this study was to evaluate whether there were associations between the CHRNA3 (rs578776) and CHRNA5 (rs16969968) polymorphisms and HNC risk and between the polymorphisms and the intensity of cigarette consumption. Methods A total of 1,067 individuals from Heliopolis Hospital in São Paulo were investigated, including 619 patients with HNC and 448 patients without diagnosed tumors. All participants answered a questionnaire about sociodemographic information and cigarette consumption data. The polymorphisms were determined by TaqMan genotyping by real‐time PCR. Results The polymorphisms studied, rs578776 (CHRNA3) and rs16969968 (CHRNA5), did not have an association with HNC risk, but the rs16969968 homozygous genotype was associated with increased cigarette consumption intensity (OR 1.93, 95% CI 1.05–3.58). Conclusion The polymorphism CHRNA5 can be considered an indirect risk factor for neoplasms in these Brazilian samples when cigarette consumption increased., The gene CHRNA5, that encoded the α5 subunit of nicotinic receptor, increased the cigarette consumption intensity in Brazilian sample. Once tobacco consumed is a risk factor to the head and neck cancer development, these polymorphism can be considered an indirect risk factor for these neoplasm.