nAChR Function Research Articles

α7 Nicotinic acetylcholine receptors regulate translocation of HIF-1α to the cell nucleus and mitochondria upon hypoxia

Nicotinic acetylcholine receptors (nAChRs), initially characterized as ligand-gated ion channels mediating fast synaptic transmission, are now found in many non-excitable cells and mitochondria where they function in ion-independent manner and regulate vital cellular processes like apoptosis, proliferation, cytokine secretion. Here we show that the nAChRs of α7 subtype are present in the nuclei of liver cells and astrocytoma U373 cell line. As shown by lectin ELISA, the nuclear α7 nAChRs are mature glycoproteins that follow the standard rout of post-translational modifications in Golgi; however, their glycosylation profile is non-identical to that of mitochondrial nAChRs. They are exposed on the outer nuclear membrane and are found in combination with lamin B1. The nuclear α7 nAChRs are up-regulated in liver within 1 h after partial hepatectomy and in H2O2-treated U373 cells. As shown both in silico and experimentally, the α7 nAChR interacts with hypoxia-inducible factor HIF-1α and this interaction is impaired by α7-selective agonists PNU282987 and choline or type 2 positive allosteric modulator PNU120596, which prevent HIF-1α accumulation in the nuclei. Similarly, HIF-1α interacts with mitochondrial α7 nAChRs in U373 cells treated with dimethyloxalylglycine. It is concluded that functional α7 nAChRs influence HIF-1α translocation into the nucleus and mitochondria upon hypoxia.

Design and structure optimization of novel butenolide derivatives as low bee-toxicity candidates

The discovery of new neonicotinoid alternatives with high efficiency against pests and eco-safety toward bees has become an increasingly urgent need in recent years. The relationship between the oil-water partition coefficient (logP) and the bee-toxicity of commercial nicotinic acetylcholine receptor (nAChR) competitive modulators was explored in this work. Compared compounds with a high bee-toxicity, compounds with low bee-toxicity were found to have higher logP values. Therefore, two series of novel butenolide derivatives with different hydrophobic groups were designed and synthesized by including different hydrophobic groups. The insecticidal activity study showed that compounds I-01 (LC50=112.96 μg/mL), II-05 (LC50=124.68 μg/mL), and II-11 (LC50=87.89 μg/mL) were with higher insecticidal activity against Aphis glycines and lower calculated logP values among all synthesized compounds. Molecular dynamics simulations indicated that three butenolide compounds formed some hydrogen bonding or π-π stacking interactions with insect acetylcholine binding protein (AChBP). In addition, the bee-toxicity bioassay also showed that compounds I-01, II-05 and II-11 were with much lower contact bee-toxicity than imidacloprid. The possible mechanism of the bee toxicity of the novel butenolide compounds was explored based on the nAChR function subunit AmeIα8/ratβ2. This study will provide rational guidance on novel butenolide compounds as low-bee-toxicity candidates based on their molecular hydrophobic properties.

Rare Missense Variants of the Human β4 Subunit Alter Nicotinic α3β4 Receptor Plasma Membrane Localisation.

α3β4 nicotinic acetylcholine receptors (nARs) are pentameric ligand-gated cation channels that function in peripheral tissue and in the peripheral and central nervous systems, where they are critical mediators of ganglionic synaptic transmission and modulators of reward-related behaviours. In the pentamer, two α3β4 subunit couples provide ligand-binding sites, and the fifth single (accessory) subunit (α3 or β4) regulates receptor trafficking from the endoplasmic reticulum to the cell surface. A number of rare missense variants of the human β4 subunit have recently been linked to nicotine dependence and/or sporadic amyotrophic lateral sclerosis, and altered responses to nicotine have been reported for these variants; however, it is unknown whether the effects of mutations depend on the subunit within the ligand-binding couples and/or on the fifth subunit. Here, by expressing single populations of pentameric receptors with fixed stoichiometry in cultured cells, we investigated the effect of β4 variants in the fifth position on the assembly and surface exposure of α3β4 nAChRs. The results demonstrate that the missense mutations in the accessory subunit alone, despite not affecting the assembly of α3β4 receptors, alter their trafficking and surface localisation. Thus, altered trafficking of an otherwise functional nAChR may underlie the pathogenic effects of these mutations.

Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR.Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.

Alkaloid ligands enable function of homomeric human α10 nicotinic acetylcholine receptors.

In the nervous system, nicotinic acetylcholine receptors (nAChRs) rapidly transduce a chemical signal into one that is electrical via ligand-gated ion flux through the central channel of the receptor. However, some nAChR subunits are expressed by non-excitable cells where signal transduction apparently occurs through non-ionic mechanisms. One such nAChR subunit, α10, is present in a discreet subset of immune cells and has been implicated in pathologies including cancer, neuropathic pain, and chronic inflammation. Longstanding convention holds that human α10 subunits require co-assembly with α9 subunits for function. Here we assessed whether cholinergic ligands can enable or uncover ionic functions from homomeric α10 nAChRs. Xenopus laevis oocytes expressing human α10 subunits were exposed to a panel of ligands and examined for receptor activation using voltage-clamp electrophysiology. Functional expression of human α10 nAChRs was achieved by exposing the oocytes to the alkaloids strychnine, brucine, or methyllycaconitine. Furthermore, acute exposure to the alkaloid ligands significantly enhanced ionic responses. Acetylcholine-gated currents mediated by α10 nAChRs were potently inhibited by the snake toxins α-bungarotoxin and α-cobratoxin but not by α-conotoxins that target α9 and α9α10 nAChRs. Our findings indicate that human α10 homomers are expressed in oocytes and exposure to certain ligands can enable ionic functions. To our knowledge, this is the first demonstration that human α10 subunits can assemble as functional homomeric nAChRs. These findings have potential implications for receptor regulatory-mechanisms and will enable structural, functional, and further pharmacological characterization of human α10 nAChRs.

A Functional Interaction Between Y674-R685 Region of the SARS-CoV-2 Spike Protein and the Human α7 Nicotinic Receptor.

The α7 nicotinic acetylcholine receptor (nAChR) is present in neuronal and non-neuronal cells and has anti-inflammatory actions. Molecular dynamics simulations suggested that α7 nAChR interacts with a region of the SARS-CoV-2 spike protein (S), and a potential contribution of nAChRs to COVID-19 pathophysiology has been proposed. We applied whole-cell and single-channel recordings to determine whether a peptide corresponding to the Y674-R685 region of the S protein can directly affect α7 nAChR function. The S fragment exerts a dual effect on α7. It activates α7 nAChRs in the presence of positive allosteric modulators, in line with our previous molecular dynamics simulations showing favourable binding of this accessible region of the S protein to the nAChR agonist binding site. The S fragment also exerts a negative modulation of α7, which is evidenced by a profound concentration-dependent decrease in the durations of openings and activation episodes of potentiated channels and in the amplitude of macroscopic responses elicited by ACh. Our study identifies a potential functional interaction between α7 nAChR and a region of the S protein, thus providing molecular foundations for further exploring the involvement of nAChRs in COVID-19 pathophysiology.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12035-022-02947-8.

Mice lacking α4 nicotinic acetylcholine receptors are protected against alcohol-associated liver injury.

Chronic heavy alcohol consumption is a major risk factor for the development of liver steatosis, fibrosis, and cirrhosis, but the mechanisms by which alcohol causes liver damage remain incompletely elucidated. This group has reported that α4 nicotinic acetylcholine receptors (α4 nAChRs) act as sensors for alcohol in lung cells. This study tested the hypothesis that α4 nAChRs mediate the effects of alcohol in the liver. Expression of acetylcholine receptor subunits in mouse liver was determined by RNA sequencing (RNA-seq). α4 nAChR knockout (α4 KO) mice were generated in C57BL/6J mice by introducing a mutation encoding an early stop codon in exon 4 of Chrna4, the gene encoding the α4 subunit of the nAChR. The presence of the inactivating mutation was established by polymerase chain reaction and genomic sequencing, and the lack of α4 nAChR function was confirmed in primary fibroblasts isolated from the α4 KO mice. Wild-type (WT) and α4 KO mice were fed the Lieber-DeCarli diet (with 36% of calories from alcohol) or pair fed an isocaloric maltose-dextrin control diet for a 6-week period that included a ramping up phase of increasing dietary alcohol. Chrna4 was the most abundantly expressed nAChR subunit gene in mouse livers. After 6 weeks of alcohol exposure, WT mice had elevated serum transaminases and their livers showed increased fat accumulation, decreased Sirt1 protein levels, and accumulation of markers of oxidative stress and inflammation including Cyp2E1, Nos2, Sod1, Slc7a11, TNFα, and PAI1. All these responses to alcohol were either absent or significantly attenuated in α4 KO animals. Together, these observations support the conclusion that activation of α4 nAChRs by alcohol or one of its metabolites is one of the initial events promoting the accumulation of excess fat and expression of inflammatory mediators. Thus, α4 nAChRs may represent viable targets for intervention in chronic alcohol-related liver disease.

Nicotinic receptors modulate antitumor therapy response in triple negative breast cancer cells.

BACKGROUNDTriple negative breast cancer is more aggressive than other breast cancer subtypes and constitutes a public health problem worldwide since it has high morbidity and mortality due to the lack of defined therapeutic targets. Resistance to chemotherapy complicates the course of patients’ treatment. Several authors have highlighted the participation of nicotinic acetylcholine receptors (nAChR) in the modulation of conventional chemotherapy treatment in cancers of the airways. However, in breast cancer, less is known about the effect of nAChR activation by nicotine on chemotherapy treatment in smoking patients.AIMTo investigate the effect of nicotine on paclitaxel treatment and the signaling pathways involved in human breast MDA-MB-231 tumor cells.METHODSCells were treated with paclitaxel alone or in combination with nicotine, administered for one or three 48-h cycles. The effect of the addition of nicotine (at a concentration similar to that found in passive smokers’ blood) on the treatment with paclitaxel (at a therapeutic concentration) was determined using the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The signaling mediators involved in this effect were determined using selective inhibitors. We also investigated nAChR expression, and ATP “binding cassette” G2 drug transporter (ABCG2) expression and its modulation by the different treatments with Western blot. The effect of the treatments on apoptosis induction was determined by flow cytometry using annexin-V and 7AAD markers.RESULTSOur results confirmed that treatment with paclitaxel reduced MDA-MB-231 cell viability in a concentration-dependent manner and that the presence of nicotine reversed the cytotoxic effect induced by paclitaxel by involving the expression of functional α7 and α9 nAChRs in these cells. The action of nicotine on paclitaxel treatment was linked to modulation of the protein kinase C, mitogen-activated protein kinase, extracellular signal-regulated kinase, and NF-κB signaling pathways, and to an up-regulation of ABCG2 protein expression. We also detected that nicotine significantly reduced the increase in cell apoptosis induced by paclitaxel treatment. Moreover, the presence of nicotine reduced the efficacy of paclitaxel treatment administered in three cycles to MDA-MB-231 tumor cells.CONCLUSIONOur findings point to nAChRs as responsible for the decrease in the chemotherapeutic effect of paclitaxel in triple negative tumors. Thus, nAChRs should be considered as targets in smoking patients.

Mutation of the α5 nicotinic acetylcholine receptor subunit increases ethanol and nicotine consumption in adolescence and impacts adult drug consumption

Alcohol and nicotine are commonly used during adolescence, establishing long-lasting neuroplastic alterations that influence subsequent drug use and abuse. Drinking- and smoking-related traits have been extensively associated with variation in CHRNA5 – the gene that encodes the α5 subunit of neuronal nicotinic acetylcholine receptors (nAChRs). The single nucleotide polymorphism (SNP) rs16969968 in CHRNA5 encodes an amino acid substitution (D398N) that alters the function and pharmacokinetics of α5-containing nAChR. When expressed in rodents, this variant results in increased ethanol and nicotine operant self-administration. How disruption of α5-containing nAChRs influences adolescent ethanol and nicotine intake, and how it modulates interactions between these drugs has not been previously explored. In the present study, we examined volitional ethanol and nicotine consumption in adolescent mice (post-natal day 30–43) of both sexes with mutated (SNP) or lacking (KO) the α5 nAChR subunit. The effect of adolescent alcohol or nicotine exposure on home cage consumption of the opposite drug in adulthood and its modulation by Chrna5 mutation and sex were examined. During adolescence, we found that α5 nAChR disruption increases nicotine intake in mice of both sexes, but the effect on alcohol intake was only observed in females. The sex-specific increase in alcohol consumption in α5 SNP and KO was replicated in adulthood. The effect of adolescent alcohol or nicotine exposure on subsequent intake of the opposite drug in adulthood is modulated by sex and Chrna5 mutation. These observations suggest sex differences in the genetic architecture of alcohol dependence, and modulators of alcohol and nicotine interactions.

A tonic nicotinic brake controls spike timing in striatal spiny projection neurons.

Striatal spiny projection neurons (SPNs) transform convergent excitatory corticostriatal inputs into an inhibitory signal that shapes basal ganglia output. This process is fine-tuned by striatal GABAergic interneurons (GINs), which receive overlapping cortical inputs and mediate rapid corticostriatal feedforward inhibition of SPNs. Adding another level of control, cholinergic interneurons (CINs), which are also vigorously activated by corticostriatal excitation, can disynaptically inhibit SPNs by activating α4β2 nicotinic acetylcholine receptors (nAChRs) on various GINs. Measurements of this disynaptic inhibitory pathway, however, indicate that it is too slow to compete with direct GIN-mediated feedforward inhibition. Moreover, functional nAChRs are also present on populations of GINs that respond only weakly to phasic activation of CINs, such as parvalbumin-positive fast-spiking interneurons (PV-FSIs), making the overall role of nAChRs in shaping striatal synaptic integration unclear. Using acute striatal slices from mice we show that upon synchronous optogenetic activation of corticostriatal projections blockade of α4β2 nAChRs shortened SPN spike latencies and increased postsynaptic depolarizations. The nAChR-dependent inhibition was mediated by downstream GABA release, and data suggest that the GABA source was not limited to GINs that respond strongly to phasic CIN activation. In particular, the observed decrease in spike latency caused by nAChR blockade was associated with a diminished frequency of spontaneous inhibitory postsynaptic currents in SPNs, a parallel hyperpolarization of PV-FSIs, and was occluded by pharmacologically preventing cortical activation of PV-FSIs. Taken together, we describe a role for tonic (as opposed to phasic) activation of nAChRs in striatal function. We conclude that tonic activation of nAChRs by CINs maintains a GABAergic brake on cortically-driven striatal output by 'priming' feedforward inhibition, a process that may shape SPN spike timing, striatal processing, and synaptic plasticity.

Comparison of the Anti-inflammatory Properties of Two Nicotinic Acetylcholine Receptor Ligands, Phosphocholine and pCF3-diEPP.

Activation of nicotinic acetylcholine receptors (nAChRs) expressed by innate immune cells can attenuate pro-inflammatory responses. Silent nAChR agonists, which down-modulate inflammation but have little or no ionotropic activity, are of outstanding clinical interest for the prevention and therapy of numerous inflammatory diseases. Here, we compare two silent nAChR agonists, phosphocholine, which is known to interact with nAChR subunits α7, α9, and α10, and pCF3-N,N-diethyl-N′-phenyl-piperazine (pCF3-diEPP), a previously identified α7 nAChR silent agonist, regarding their anti-inflammatory properties and their effects on ionotropic nAChR functions. The lipopolysaccharide (LPS)-induced release of interleukin (IL)-6 by primary murine macrophages was inhibited by pCF3-diEPP, while phosphocholine was ineffective presumably because of instability. In human whole blood cultures pCF3-diEPP inhibited the LPS-induced secretion of IL-6, TNF-α and IL-1β. The ATP-mediated release of IL-1β by LPS-primed human peripheral blood mononuclear leukocytes, monocytic THP-1 cells and THP-1-derived M1-like macrophages was reduced by both phosphocholine and femtomolar concentrations of pCF3-diEPP. These effects were sensitive to mecamylamine and to conopeptides RgIA4 and [V11L; V16D]ArIB, suggesting the involvement of nAChR subunits α7, α9 and/or α10. In two-electrode voltage-clamp measurements pCF3-diEPP functioned as a partial agonist and a strong desensitizer of classical human α9 and α9α10 nAChRs. Interestingly, pCF3-diEPP was more effective as an ionotropic agonist at these nAChRs than at α7 nAChR. In conclusion, phosphocholine and pCF3-diEPP are potent agonists at unconventional nAChRs expressed by monocytic and macrophage-like cells. pCF3-diEPP inhibits the LPS-induced release of pro-inflammatory cytokines, while phosphocholine is ineffective. However, both agonists signal via nAChR subunits α7, α9 and/or α10 to efficiently down-modulate the ATP-induced release of IL-1β. Compared to phosphocholine, pCF3-diEPP is expected to have better pharmacological properties. Thus, low concentrations of pCF3-diEPP may be a therapeutic option for the treatment of inflammatory diseases including trauma-induced sterile inflammation.

A Novel α4/7-Conotoxin QuIA Selectively Inhibits α3β2 and α6/α3β4 Nicotinic Acetylcholine Receptor Subtypes with High Efficacy.

α6β4 nAChR is expressed in the peripheral and central nervous systems and is associated with pain, addiction, and movement disorders. Natural α-conotoxins (α-CTxs) can effectively block different nAChR subtypes with higher efficacy and selectivity. However, the research on α6β4 nAChR is relatively poor, partly because of the lack of available target-specific α-CTxs. In this study, we synthesized a novel α-4/7 conotoxin QuIA that was found from Conus quercinus. We investigated the efficacy of this peptide to different nAChR subtypes using a two-electrode voltage-clamp technique. Remarkably, we found α-QuIA inhibited the neuronal α3β2 and α6/α3β4 nAChR subtypes with significantly high affinity (IC50 was 55.7 nM and 90.68 nM, respectively), and did not block other nAChR subtypes even at a high concentration of 10 μM. In contrast, most α-CTxs have been determined so far to effectively block the α6/α3β4 nAChR subtype while also maintaining a similar higher efficacy against the closely related α6β2β3 and/or α3β4 subtypes, which are different from QuIA. In conclusion, α-QuIA is a novel α4/7-CTx, which has the potential to develop as an effective neuropharmacology tool to detect the function of α6β4 nAChR.

The actions of neonicotinoid insecticides on nicotinic acetylcholine subunits Ldα1 and Ldα8 of Leptinotarsa decemlineata and assembled receptors.

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 72h 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 (Imax ) varied. For Ldα1/Ldα8/rβ2, a reduction of Lda8 cRNA resulted in increased sensitivity to IMI and decreased sensitivity to TMX. The expression of Ldα1 and Ldα8 significantly decreased in adults by 82.12% and 47.02%, respectively, while Ldα8 was significantly upregulated by 2.44 times in 4th instar larvae after exposure to TMX. We infer that Ldα1 and Ldα8 together play an important role in the sensitivity of L. decemlineata to neonicotinoids.

Neurobiological Mechanisms of Nicotine Reward and Aversion.

Neuronal nicotinic acetylcholine receptors (nAChRs) regulate the rewarding actions of nicotine contained in tobacco that establish and maintain the smoking habit. nAChRs also regulate the aversive properties of nicotine, sensitivity to which decreases tobacco use and protects against tobacco use disorder. These opposing behavioral actions of nicotine reflect nAChR expression in brain reward and aversion circuits. nAChRs containing α4 and β2 subunits are responsible for the high-affinity nicotine binding sites in the brain and are densely expressed by reward-relevant neurons, most notably dopaminergic, GABAergic, and glutamatergic neurons in the ventral tegmental area. High-affinity nAChRs can incorporate additional subunits, including β3, α6, or α5 subunits, with the resulting nAChR subtypes playing discrete and dissociable roles in the stimulatory actions of nicotine on brain dopamine transmission. nAChRs in brain dopamine circuits also participate in aversive reactions to nicotine and the negative affective state experienced during nicotine withdrawal. nAChRs containing α3 and β4 subunits are responsible for the low-affinity nicotine binding sites in the brain and are enriched in brain sites involved in aversion, including the medial habenula, interpeduncular nucleus, and nucleus of the solitary tract, brain sites in which α5 nAChR subunits are also expressed. These aversion-related brain sites regulate nicotine avoidance behaviors, and genetic variation that modifies the function of nAChRs in these sites increases vulnerability to tobacco dependence and smoking-related diseases. Here, we review the molecular, cellular, and circuit-level mechanisms through which nicotine elicits reward and aversion and the adaptations in these processes that drive the development of nicotine dependence. SIGNIFICANCE STATEMENT: Tobacco use disorder in the form of habitual cigarette smoking or regular use of other tobacco-related products is a major cause of death and disease worldwide. This article reviews the actions of nicotine in the brain that contribute to tobacco use disorder.

Synthesis of full-length homodimer αD-VxXXB that targets human α7 nicotinic acetylcholine receptors.

αD-Conotoxin VxXXB is a pseudo-homodimer that allosterically inhibits nicotinic acetylcholine receptors (nAChRs) with high potency and selectivity. However, challenges in synthesizing αD-conotoxins have hindered further structure-function studies on this novel class of peptides. To address this gap, we synthesized and characterized its C-terminal domain (CTD) and N-terminal domain (NTD). The CTD inhibited α7 nAChRs (IC50 of 23 nM, measured via FLIPR assays) and bound at the acetylcholine binding protein (Ls-AChBP) through an allosteric binding mode determined from radioligand binding assays. The anti-parallel dimeric NTD synthesised via a regioselective strategy also inhibited α7 nAChRs but with reduced potency (IC50 of 30 μM). The α-ketoacid-hydroxylamine (KAHA) method generated CTD linked to the NTD (VxXXB-NC; α7 IC50 of 27 nM) and full-length synthetic VxXXB variant (α7 IC50 of 11 nM), while the three other native chemical ligation approaches proved unsuccessful. This work underpins further characterisation of the structural components contributing to αD-conotoxin affinity, selectivity and allosteric inhibition of nAChR function that may prove useful in the development of new treatments for nAChR-related disorders.

N-acetylcholine receptors regulate cytokines expression and neutrophils recruitment via MAPK/ERK signaling in zebrafish

N-acetylcholine receptors (AChRs) are mainly distributed in the postsynaptic membrane and have been widely studied for their control of muscle contraction by regulating neural action potentials. However, the influences of AChRs on immune responses and potential mechanisms remain unclear. Here, we used the advantages of live imaging of zebrafish to explore the regulation process of AChRs on inflammatory responses. Pharmacologically activating of the receptor, we found that the expression of pro-inflammatory cytokines il-1β, il-6, tnf-α and il-8 was significantly up-regulated and neutrophil migration to injury sites was also significantly increased. However, these phenomena were reversed under antagonism of the receptor activity. Results showed that interfering with nAChRs functions did not significantly affect zebrafish motion behavior. Results also showed that activation and antagonism of nAChRs function could regulate the phosphorylation of ERK protein respectively. We further demonstrated that ERK participated in the regulation of AChRs in cytokines expression and neutrophils migration in zebrafish. This study preliminarily revealed the roles of AChRs in inflammatory processes and their potential mechanism, providing additional evidence of peripheral immune regulation by cholinergic receptors.

Developmental age and biological sex influence muscarinic receptor function and neuron morphology within layer VI of the medial prefrontal cortex.

Acetylcholine (ACh) neurotransmission within the medial prefrontal cortex (mPFC) plays an important modulatory role to support mPFC-dependent cognitive functions. This role is mediated by ACh activation of its nicotinic (nAChR) and muscarinic (mAChR) classes of receptors, which are both present on mPFC layer VI pyramidal neurons. While the expression and function of nAChRs have been characterized thoroughly for rodent mPFC layer VI neurons during postnatal development, mAChRs have not been characterized in detail. We employed whole-cell electrophysiology with biocytin filling to demonstrate that mAChR function is greater during the juvenile period of development than in adulthood for both sexes. Pharmacological experiments suggest that each of the M1, M2, and M3 mAChR subtypes contributes to ACh responses in these neurons in a sex-dependent manner. Analysis of dendrite morphology identified effects of age more often in males, as the amount of dendrite matter was greatest during the juvenile period. Interestingly, a number of positive correlations were identified between the magnitude of ACh/mAChR responses and dendrite morphology in juvenile mice that were not present in adulthood. To our knowledge, this work describes the first detailed characterization of mAChR function and its correlation with neuron morphology within layer VI of the mPFC.

A Functional Human-on-a-Chip Autoimmune Disease Model of Myasthenia Gravis for Development of Therapeutics.

Myasthenia gravis (MG) is a chronic and progressive neuromuscular disease where autoantibodies target essential proteins such as the nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction (NMJ) causing muscle fatigue and weakness. Autoantibodies directed against nAChRs are proposed to work by three main pathological mechanisms of receptor disruption: blocking, receptor internalization, and downregulation. Current in vivo models using experimental autoimmune animal models fail to recapitulate the disease pathology and are limited in clinical translatability due to disproportionate disease severity and high animal death rates. The development of a highly sensitive antibody assay that mimics human disease pathology is desirable for clinical advancement and therapeutic development. To address this lack of relevant models, an NMJ platform derived from human iPSC differentiated motoneurons and primary skeletal muscle was used to investigate the ability of an anti-nAChR antibody to induce clinically relevant MG pathology in the serum-free, spatially organized, functionally mature NMJ platform. Treatment of the NMJ model with the anti-nAChR antibody revealed decreasing NMJ stability as measured by the number of NMJs before and after the synchrony stimulation protocol. This decrease in NMJ stability was dose-dependent over a concentration range of 0.01–20 μg/mL. Immunocytochemical (ICC) analysis was used to distinguish between pathological mechanisms of antibody-mediated receptor disruption including blocking, receptor internalization and downregulation. Antibody treatment also activated the complement cascade as indicated by complement protein 3 deposition near the nAChRs. Additionally, complement cascade activation significantly altered other readouts of NMJ function including the NMJ fidelity parameter as measured by the number of muscle contractions missed in response to increasing motoneuron stimulation frequencies. This synchrony readout mimics the clinical phenotype of neurological blocking that results in failure of muscle contractions despite motoneuron stimulations. Taken together, these data indicate the establishment of a relevant disease model of MG that mimics reduction of functional nAChRs at the NMJ, decreased NMJ stability, complement activation and blocking of neuromuscular transmission. This system is the first functional human in vitro model of MG to be used to simulate three potential disease mechanisms as well as to establish a preclinical platform for evaluation of disease modifying treatments (etiology).

Antagonistic Mechanism of α-Conotoxin BuIA toward the Human α3β2 Nicotinic Acetylcholine Receptor

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that are abundantly expressed in the central and peripheral nervous systems, playing an important role in mediating neurotransmitter release and inter-synaptic signaling. Dysfunctional nAChRs are associated with neurological disorders, and studying the structure and function of nAChRs is essential for development of drugs or strategies for treatment of related diseases. α-Conotoxins are selective antagonists of the nAChR and are an important class of drug leads. So far, the antagonistic mechanism of α-conotoxins toward the nAChRs is still unclear. In this study, we built an α3β2 nAChR homology model and investigated its conformational transition mechanism upon binding with a highly potent inhibitor, α-conotoxin BuIA, through μs molecular dynamic simulations and site-directed mutagenesis studies. The results suggested that the α3β2 nAChR underwent global conformational transitions and was stabilized into a closed state with three hydrophobic gates present in the transmembrane domain by BuIA. Finally, the probable antagonistic mechanism of BuIA was proposed. Overall, the closed-state model of the α3β2 nAChR bound with BuIA is not only essential for understanding the antagonistic mechanism of α-conotoxins but also particularly valuable for development of therapeutic inhibitors in future.

Ethanol interaction with α3β4 nicotinic acetylcholine receptors in neurons of the laterodorsal tegmentum.

Nicotinic acetylcholine receptors (nAChRs) play a key role in the rewarding effects of ethanol (EtOH), and while several nAChR subtypes have been implicated, attention has recently shifted to a role for the α3β4 nAChR. The laterodorsal tegmental nucleus (LDTg), a brainstem cholinergic nucleus that sends excitatory projections to the ventral tegmental area, is an Integral part of the brain reward pathway. Here we investigate a potential role for LDTg α3β4 nAChRs in EtOH self-administration and reward. Sprague-Dawley rats were given ad libitum access to a 20% EtOH solution, as part of a two-bottle choice paradigm. Approximately 1week after removal of EtOH access, we measured LDTg α3β4 nAChR current responses to focal application of acetylcholine (ACh), using whole-cell patch clamp electrophysiology recordings in acute brain slices. In addition, we used whole-cell electrophysiology to assess the acute effects of EtOH on the sensitivity of LDTg α3β4 nAChRs. Focal application of ACh onto LDTg neurons resulted in large α3β4 nAChR-mediated inward currents, the magnitude of which showed a positive correlation with levels of EtOH self-administration. In addition, using brain slices taken from EtOH-naïve rats, bath application of EtOH resulted in a moderate potentiation of LDTg α3β4 nAChR sensitivity. Using a rat model, increased α3β4 nAChR function was associated with greater EtOH self-administration, with α3β4 nAChR function also acutely potentiated by EtOH. Assuming that similar findings apply to humans, the α3β4 nAChR could be a therapeutic target in the treatment of EtOH use disorder.