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Seven cell lines expressing native and transfected nicotinic receptor subtypes were evaluated functionally by using fluorescent assays based on membrane potential and calcium dynamics with "no-wash" dye systems. Both assays provided the same rank orders of potency for (+/-)-epibatidine, 2S-(-)-nicotine, 7R,9S-(-)-cytisine, and 1,1-dimethyl-4-phenyl...
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We describe an electrophysiological preparation of the neuromuscular junction of the nematode C. elegans, which adds to its considerable genetic and genomic resources. Mutant analysis, pharmacology and patch-clamp recording showed that the body wall muscles of wild-type animals expressed a GABA receptor and two acetylcholine receptors. The muscle G...
Evaluation of the discriminative stimulus effects of drugs is a useful procedure for identification of receptor mediation of in vivo drug effects. This assay can be enhanced when the stimulus effects of different doses of agonist are evaluated. In the present study, rats were trained to discriminate small or large doses of nicotine from saline, and...
Attention depends on cholinergic stimulation of nicotinic and muscarinic acetylcholine receptors in the medial prefrontal cortex. Pyramidal neurons in layer VI of this region express cholinergic receptors of both families and play an important role in attention through their feedback projections to the thalamus. Here, we investigate how nicotinic a...
Rats learned to lever-press for microinjections of the cholinergic agonist carbachol (30-500 pmol per infusion) or the acetylcholinesterase inhibitor neostigmine (7.5-75 pmol per infusion) into the posterior ventral tegmental area (VTA) of the brain. Intracranial carbachol self-administration was site-specific. Carbachol was not reliably self-admin...
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... As previously described by our lab and others, these assays were performed using a protocol that sequentially measures receptor activation and inhibition. 25,30 In this assay, solutions of compounds (0.3 nM to 100 μM) are added to a 96-well plate containing cells preincubated with an intracellular calcium dye (Calcium 6, Molecular Devices) using a robotic pipettor integrated into the plate reader (FlexStation 3 Multi-Mode Microplate Reader, Molecular Devices). Subsequent increases in fluorescence intensity correspond to increases in intracellular [Ca 2+ ] arising from channel opening. ...
Growing evidence suggests that inhibition of the α3β4 nicotinic acetylcholine receptor (nAChR) represents a promising therapeutic strategy to treat cocaine use disorder. Recently, aristoquinoline (1), an alkaloid from Aristotelia chilensis, was identified as an α3β4-selective nAChR inhibitor. Here, we prepared 22 derivatives of 1 and evaluated their ability to inhibit the α3β4 nAChR. These studies revealed structure-activity trends and several compounds with increased potency compared to 1 with few off-target liabilities. Additional mechanistic studies indicated that these compounds inhibit the α3β4 nAChR non-competitively, but do not act as channel blockers, suggesting they are negative allosteric modulators. Finally, using a cocaine-primed reinstatement paradigm, we demonstrated that 1 significantly attenuates drug-seeking behavior in an animal model of cocaine relapse. The results from these studies further support a role for the α3β4 nAChR in the addictive properties of cocaine and highlight the possible utility of aristoquinoline derivatives in treating cocaine use disorder.
... A promising approach using immortalised TE671 cells expressing the foetal muscle-type nAChR [30] and a membrane potential dye to report receptor activation [31] has been used to investigate the activity of a Lcα-NTx isolated from black mamba (Dendroaspis polylepis) venom [32]. ...
... The following method was adapted from Fitch et al. [31] and Wang et al. [32] and, as in section 2.2, all reagents were acquired from Gibco, Thermo Fisher Scientific, Paisley, UK, unless stated otherwise. One vial of FLIPR membrane potential dye (Component A, Explorer Kit Blue, R8042, Molecular Devices, San Jose, CA, USA) was dissolved in 36 mL assay buffer to create the dye solution. ...
... In this study, we developed a cell-based assay to investigate venom toxin activity on muscle-type nAChR activation and explored its capability to detect inhibition by various toxin-inhibiting molecules. For validation, we first quantified the effects of known nAChR agonists and antagonists to ensure that their observed effects were consistent with other validated experimental techniques, and that any modifications made to previously published approaches [31,32] did not affect the assay (Fig. 2). The time to peak and decay of fluorescent responses during the recording time ( Fig. 2A) were consistent to those observed in other studies employing the same experimental approach [61], though differences were observed when comparing outcomes with electrophysiology approaches. ...
Snakebite envenoming is a neglected tropical disease that causes over 100,000 deaths annually. Envenomings result in variable pathologies, but systemic neurotoxicity is among the most serious and is currently only treated with difficult to access and variably efficacious commercial antivenoms. Venom-induced neurotoxicity is often caused by α-neurotoxins antagonising the muscle-type nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel. Discovery of therapeutics targeting α-neurotoxins is hampered by relying on binding assays that do not reveal restoration of receptor activity or more costly and/or lower throughput electrophysiology-based approaches. Here, we report the validation of a screening assay for nAChR activation using immortalised TE671 cells expressing the γ-subunit containing muscle-type nAChR and a fluorescent dye that reports changes in cell membrane potential. Assay validation using traditional nAChR agonists and antagonists, which either activate or block ion fluxes, was consistent with previous studies. We then characterised antagonism of the nAChR by a variety of elapid snake venoms that cause muscle paralysis in snakebite victims, before defining the toxin-inhibiting activities of commercial antivenoms, and new types of snakebite therapeutic candidates, namely monoclonal antibodies, decoy receptors, and small molecules. Our findings show robust evidence of assay uniformity across 96-well plates and highlight the amenability of this approach for the future discovery of new snakebite therapeutics via screening campaigns. The described assay therefore represents a useful first-step approach for identifying α-neurotoxins and their inhibitors in the context of snakebite envenoming, and it should provide wider value for studying modulators of nAChR activity from other sources.
... [Ca 2+ ] mt uptake was determined according to the general protocol, using 5 mM caffeine (Sigma Aldrich, #C0750) plus 10 μM epibatidine (Sigma Aldrich, #E1145) to trigger calcium release from the sarcoplasmic reticulum. Caffeine and epibatidine are used to evoke cytosolic and hence [Ca 2+ ] mt rise, by discharging the sarcoplasmic reticulum pool [31,42,43]. ...
... In this primary cell model, we used AAV-mediated delivery of an MCU-shRNA to knockdown MCU (MCU-kd) (Fig. 4B), and functionally validated that MCU-kd inhibits [Ca 2+ ] mt uptake after stimulation of Ca 2+ release from the sarcoplasmic reticulum with the ryanodine receptor agonists [58]. We used caffeine plus epibatidine to evoke cytosolic and hence [Ca 2+ ] mt rise, by discharging the sarcoplasmic reticulum pool [31,42,43] ( Fig. 4C). Knockdown of MCU strongly decreased [Ca 2+ ] mt uptake after stimulation in myotubes (Fig. 4D). ...
Regulation of mitochondrial redox balance is emerging as a key event for cell signaling in both physiological and pathological conditions. However, the link between the mitochondrial redox state and the modulation of these conditions remains poorly defined. Here, we discovered that activation of the evolutionary conserved mitochondrial calcium uniporter (MCU) modulates mitochondrial redox state. By using mitochondria-targeted redox and calcium sensors and genetic MCU-ablated models, we provide evidence of the causality between MCU activation and net reduction of mitochondrial (but not cytosolic) redox state. Redox modulation of redox-sensitive groups via MCU stimulation is required for maintaining respiratory capacity in primary human myotubes and C. elegans, and boosts mobility in worms. The same benefits are obtained bypassing MCU via direct pharmacological reduction of mitochondrial proteins. Collectively, our results demonstrate that MCU regulates mitochondria redox balance and that this process is required to promote the MCU-dependent effects on mitochondrial respiration and mobility.
... Our experimental protocol was based on the initial study of Fitch et al. [33]. Cells were seeded at a density of roughly 5 × 10 4 to 10 5 cells/well in 96-well flat-bottom black wall culture plates coated with 50 µg /mL poly-D-lysine hydrobromide (Sigma-Aldrich, 70-150 kDa) and grown overnight in 100 µL culture medium. ...
Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond. In spite of its size, it acts as an antagonist at insect and mammalian nicotinic acetylcholine receptors (nAChRs). The functional importance of the disulfide bond was assessed by determining the effects of inserting a methylene group between the two sulfur atoms, creating dimethylaminodithiane (DMA-DT). We also assessed the effect of methylating the NTX and DMA-DT dimethylamino groups on binding to three vertebrate nAChRs. Radioligand receptor binding experiments were carried out using washed membranes from rat brain and fish (Torpedo) electric organ; [3H]-cytisine displacement was used to assess binding to the predominantly high affinity alpha4beta2 nAChRs and [125I]-alpha-bungarotoxin displacement was used to measure binding of NTX and analogs to the alpha7 and skeletal muscle type nAChRs. While the two quaternary nitrogen analogs, relative to their respective tertiary amines, displayed lower α4β2 nAChR binding affinities, both displayed much higher affinities for the Torpedo muscle nAChR and rat alpha7 brain receptors than their respective tertiary amine forms. The binding affinities of DMA-DT for the three nAChRs were lower than those of NTX and MeNTX. An AChBP mutant lacking the C loop disulfide bond that would potentially react with the NTX disulfide bond displayed an NTX affinity very similar to the parent AChBP. Inhibition of [3H]-epibatidine binding to the AChBPs was not affected by exposure to NTX or MeNTX for up to 24 hr prior to addition of the radioligand. Thus, the disulfide bond of NTX is not required to react with the vicinal disulfide in the AChBP C loop for inhibition of [3H]-epibatidine binding. However, a reversible disulfide interchange reaction of NTX with nAChRs might still occur, especially under reducing conditions. Labeled MeNTX, because it can be readily prepared with high specific radioactivity and possesses relatively high affinity for the nAChR-rich Torpedo nAChR, would be a useful probe to detect and identify any nereistoxin adducts.
... This gave an EC50 of 19 nM (pEC50 = 7.73 ± 0.06 M). This is lower than the EC50 of 3.4 µM reported in Fitch et al. (2003) for rat α4β2 nACh receptors using similar methods. This was as expected, firstly because the L9'A decreases EC50 by ~40 fold (Fonck et al., 2005;Tapper et al., 2004), and secondly, the HEK293 cell used in Fitch et al. (2003) were stably transfected ...
... This is lower than the EC50 of 3.4 µM reported in Fitch et al. (2003) for rat α4β2 nACh receptors using similar methods. This was as expected, firstly because the L9'A decreases EC50 by ~40 fold (Fonck et al., 2005;Tapper et al., 2004), and secondly, the HEK293 cell used in Fitch et al. (2003) were stably transfected ...
Pentameric ligand-gated ion channels (pLGICs) are expressed throughout the human nervous system, and contribute to a range of muscle, gut, and neurological functions. Elucidating their mechanism of action and how it might be modulated would improve our understanding of the nervous system, and contribute to building tools to treat diseases arising from dysregulated pLGICs. The outermost lipid-facing transmembrane helix (M4) and the lipids surrounding it have recently emerged as important factors in pLGIC function. To investigate the role of the M4 helix in cation-selective mammalian pLGICs, I studied the effects of mutations in the M4 helices of the 5-HT3A and α4β2 nACh receptors. I used a membrane potential-sensitive fluorescent dye, two-electrode voltage clamp and manual patch-clamp for functional characterisation of mutant receptors in HEK293 cells and Xenopus oocytes, and radioligand binding and immunofluorescence to assess ligand binding and receptor expression. I show that 1 out of 28 alanine mutations in the 5-HT3AR M4 and 8 out of28 double alanine mutations in the α4β2 nAChR abolish receptor function in HEK cells without ablating ligand binding, indicating that the M4 helices of these cation-selective pLGICs are involved in, and can modulate, receptor function. I explored the mechanism of action of these key M4 residues by characterising prospective interaction partners, and identified a potential chain of interactions going from the outermost M4 helix all the way to the channel pore. I also show that eight of the nine 5-HT3AR and α4β2 nAChR mutants that showed ligand binding but no receptor function in HEK cells, showed WT-like function when expressed in Xenopus oocytes. In addition, M4 mutations that altered the function of receptors expressed in HEK cells had different effects on receptors expressed in oocytes. Together this shows that the role of the M4 helix in cation-selective pLGIC function depends on the expression system. For comparison, I investigated another peripheral helix in the 5-HT3AR; the N-terminal helix, which rests above the extracellular domain of mammalian pLGICs, and showed that it is important for correct receptor expression. Overall, this work shows that the M4 helix of cation-selective pLGICs is an attractive target for receptor modulation by small-molecule binding, as this helix is both accessible, poorly conserved between pLGICs, and intimately involved in receptor function. It has also laid the groundwork for further understanding the functional mechanism of pLGICs, especially the interactions of the M4 helices and with the rest of the transmembrane helical bundle. Finally, it has highlighted the dependency on the expression system of both pLGIC function and of the role of the M4 helix, and emphasises the need to understand the native environment of these receptors and how that modulates function.
... A quantification of the nicotine signaling yielded a pEC 50 value of 5.9 (Fig. 1b). This finding is in line with other published datasets on human nicotinic receptors determined by other techniques, confirming the applicability of Ca 2+ -imaging used here: e.g., pEC 50 values of 5.5-6.1 (EC 50 : 0.9-3.5 µM) have been reported for human α4β2 nAChRs using patch clamp (Buisson et al. 1996;Wu et al. 2006;Chen et al. 2018), Ca 2+ -imaging (Chavez-Noriega et al. 2000;Capelli et al. 2011) and membrane potential fluorescence (Fitch et al. 2003). Patch clamp recordings with human α6/3β2β3 (Armstrong et al. 2017;Chen et al. 2018) or α4β4 (Wu et al. 2006) nAChRs showed pEC 50 values of ~ 5.9. ...
... Patch clamp recordings with human α6/3β2β3 (Armstrong et al. 2017;Chen et al. 2018) or α4β4 (Wu et al. 2006) nAChRs showed pEC 50 values of ~ 5.9. In this context, it is noteworthy that muscular (nonneuronal) nAChR (α1β1δγ/ε) have clearly lower nicotine affinities (Fitch et al. 2003;Capelli et al. 2011) of around 25 µM (pEC 50 : ~ 4.6). ...
... A similar set of data (pEC 50 : 6.1) was obtained for a third nicotinic agonist, varenicline ( Fig. 1b) (Mihalak et al. 2006). Again, these data were in good agreement with data obtained by several other functional test platforms (Chavez-Noriega et al. 2000;Fitch et al. 2003;Coe et al. 2005;Wu et al. 2006;Chatterjee et al. 2011;Arias et al. 2015). ...
Neonicotinoid pesticides, originally developed to target the insect nervous system, have been reported to interact with human receptors and to activate rodent neurons. Therefore, we evaluated in how far these compounds may trigger signaling in human neurons, and thus, affect the human adult or developing nervous system. We used SH-SY5Y neuroblastoma cells as established model of nicotinic acetylcholine receptor (nAChR) signaling. In parallel, we profiled dopaminergic neurons, generated from LUHMES neuronal precursor cells, as novel system to study nAChR activation in human post-mitotic neurons. Changes of the free intracellular Ca ²⁺ concentration ([Ca ²⁺ ] i ) were used as readout, and key findings were confirmed by patch clamp recordings. Nicotine triggered typical neuronal signaling responses that were blocked by antagonists, such as tubocurarine and mecamylamine. Pharmacological approaches suggested a functional expression of α7 and non-α7 nAChRs on LUHMES cells. In this novel test system, the neonicotinoids acetamiprid, imidacloprid, clothianidin and thiacloprid, but not thiamethoxam and dinotefuran, triggered [Ca ²⁺ ] i signaling at 10–100 µM. Strong synergy of the active neonicotinoids (at low micromolar concentrations) with the α7 nAChR-positive allosteric modulator PNU-120596 was observed in LUHMES and SH-SY5Y cells, and specific antagonists fully inhibited such signaling. To provide a third line of evidence for neonicotinoid signaling via nAChR, we studied cross-desensitization: pretreatment of LUHMES and SH-SY5Y cells with active neonicotinoids (at 1–10 µM) blunted the signaling response of nicotine. The pesticides (at 3–30 µM) also blunted the response to the non-α7 agonist ABT 594 in LUHMES cells. These data show that human neuronal cells are functionally affected by low micromolar concentrations of several neonicotinoids. An effect of such signals on nervous system development is a toxicological concern.
... Particularly high signals were obtained in differentiated cells for the α4, α7, and β2 chains, but also some other cholinergic components showed gene expression. For instance, ACh esterase (AChE) and the muscarinic AChR4 were up-regulated during the differentiation process confirming the applicability of Ca 2+ -imaging used here: e.g., pEC50 values of 5.5-6.1 (EC50: 0.9-3.5 μM) have been reported for human α4β2 nAChRs using patch clamp (Buisson et al., 1996;Chen et al., 2018;Wu et al., 2006), Ca 2+ -imaging (Capelli et al., 2011;Chavez-Noriega et al., 2000) and membrane potential fluorescence (Fitch et al., 2003). Patch clamp recordings with human α6/3β2β3 (Armstrong et al., 2017;Chen et al., 2018) or α4β4 (Wu et al., 2006) nAChRs showed pEC50 values of ~ 5.9. ...
... Patch clamp recordings with human α6/3β2β3 (Armstrong et al., 2017;Chen et al., 2018) or α4β4 (Wu et al., 2006) nAChRs showed pEC50 values of ~ 5.9. In this context, it is noteworthy that muscular (nonneuronal) nAChR (α1β1δγ/ε) have clearly lower nicotine affinities (Capelli et al., 2011;Fitch et al., 2003) of around 25 μM (pEC50: ~ 4.6). ...
... A similar set of data (pEC50: 6.1) was obtained for a third nicotinic agonist, varenicline MS2_1b) (Mihalak et al., 2006). Again, these data were in good agreement with data obtained by several other functional test platforms (Arias et al., 2015;Chatterjee et al., 2011;Chavez-Noriega et al., 2000;Coe et al., 2005;Fitch et al., 2003;Wu et al., 2006). ...
... Our experimental protocol was based on the initial study of Fitch et al. [56]. Cells were seeded at a density of roughly 5 × 10 4 to 10 5 cells/well in 96-well flat-bottom black wall culture plates coated with 50 µg /ml poly-d-lysine hydrobromide (Sigma-Aldrich, 70-150 kDa) and grown overnight in 100 µL culture medium. ...
Three major forms of the nicotinic agonist toxin anabaseine (cyclic iminium, cyclic imine and the monocationic open-chain ammonium-ketone) co-exist in almost equal concentrations at physiological pH. We asked the question: Which of these forms is pharmacologically active? First, we investigated the pH dependence of anabaseine inhibition of [3H]-methylcarbamylcholine binding at rat brain α4β2 nicotinic acetylcholine receptors (nAChRs). These experiments indicated that one or both monocationic forms interact with the orthosteric binding site for ACh. However, since they occur at equal concentrations near physiological pH, we employed another approach, preparing a stable analog of each form and examining its agonist activities and binding affinities at several vertebrate brain and neuromuscular nAChRs. Only 2-(3-pyridyl)-1,4,5,6-tetrahydropyrimidine monohydrogen chloride (PTHP), the cyclic iminium analog, displayed nAChR potencies and binding affinities similar to anabaseine. The cyclic imine analog 2,3′-bipyridyl and the open-chain ammonium-ketone analog 5-methylamino-1-(3-pyridyl)-1-pentanone (MAPP), displayed ≤1% of the activity predicted if the one form was solely active. The lower potency of weakly basic 2,3′-bipyridyl can be explained by the presence of a small concentration of its monocationic form. Since the open chain ammonium-ketone monocationic form of anabaseine has some structural similarity to the neurotransmitter GABA, we also tested the ability of anabaseine and its 1,2-dehydropyrrolidinyl analog myosmine to activate a mammalian GABAA receptor, but no activity was detected. We conclude that the monocationic cyclic iminium is the form which avidly binds and activates vertebrate nAChRs.
... Previous work with recombinant h5-HT3A receptors has shown vortioxetine to induce a rapidly desensitizing inward current with a peak response around 65% of that of 5-HT Cadia et al., 2013;Lummis et al., 2011;Price et al., 2008;Price and Lummis, 2005;Sullivan et al., 2006;Thompson et al., 2006a). The assay is based on measurement of agonist-evoked changes in cell membrane potential by use of a fluorescent dye that is sensitive to the membrane field potential (Fitch et al., 2003). We receptors (Brady et al., 2001;Hope et al., 1996;Miyake et al., 1995); thus corroborating previous assessments of the usefulness of membrane potential assay for pharmacological characterization of recombinant 5-HT3 receptors Price and Lummis, 2005). ...
5-HT3 receptors are ligand-gated ion channels that mediate neurotransmission by serotonin in the central nervous system. Pharmacological inhibition of 5-HT3 receptor activity has therapeutic potential in several psychiatric diseases, including depression and anxiety. The recently approved multimodal antidepressant vortioxetine has potent inhibitory activity at 5-HT3 receptors. Vortioxetine has an inhibitory mechanism that differs from classical 5-HT3 receptor competitive antagonists despite being believed to bind in the same binding site. Specifically, vortioxetine shows partial agonist activity followed by a persistent and insurmountable inhibition. We have investigated the binding mode of vortioxetine at the human 5-HT3A receptor through computational and in vitro experiments to provide insight into the molecular mechanisms behind the unique pharmacological profile of the drug. We find that vortioxetine bind in a manner different from currently known 5-HT3A orthosteric ligands. Specifically, while the binding pattern of vortioxetine mimics some aspects of both the setron class of competitive antagonists and 5-HT with regards to interactions with residues of the aromatic box motif in the orthosteric binding site, vortioxetine also form interactions with residues not previously described to be important for the binding of either setrons or 5-HT such as Thr176 and Val202 on loop B and F, respectively. Our results expand the framework for understanding how orthosteric ligands drive 5-HT3 receptor function, which is of importance for the potential future development of novel classes of 5-HT3 receptor antagonists.
... Antibodies' antagonistic effects toward nAChRs were measured in accordance with previously described methods (Arias et al. 2010;Fitch et al. 2003). For the antagonistic activity assay, DB40 cells (2 × 10 4 cells/well) were grown overnight in 96-well black flat plates coated with Poly-D-Lysine (Corning). ...
Many myasthenia gravis (MG) patients have auto-antibodies against the nicotinic acetylcholine receptor (nAChR), and monoclonal antibodies against the main immunogenic region (MIR) of nAChR can induce experimental autoimmune MG (EAMG). We investigated whether Fab fragment of MIR antibody (Fab35) could block the pathogenicity of polyclonal antibodies. Fab35 partially inhibited nAChR downmodulation, blocked EAMG serum-induced binding of polyclonal antibodies and complement deposition in vitro. Moreover, Fab35 did not ameliorate the EAMG serum-induced EAMG phenotype in rats. These results suggested that the EAMG serum possessed several different pathogenic antibodies that might be sufficient to induce the EAMG phenotype.
