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Thomas L Lentz - One of the best experts on this subject based on the ideXlab platform.
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differential binding of nicotine and alpha bungarotoxin to residues 173 204 of the nicotinic acetylcholine receptor alpha 1 subunit
Biochemistry, 1995Co-Authors: Thomas L LentzAbstract:The binding of the agonist L-[3H]nicotine and the competitive antagonist alpha-[125I]bungarotoxin to synthetic peptides comprising residues 173-227 of the Torpedo nicotinic acetylcholine receptor alpha subunit were compared using a solid phase-assay. Equilibrium saturation binding of [3H]nicotine to peptide 173-204 revealed a minor binding component with an apparent KD of 1.9 nM and a major component with a KD of 1.6 microM. Nicotine bound to alpha subunit peptides 173-204, 181-198, and 194-204 and less well to 179-192 and 186-196, and it did not bind to 173-180 and 205-227. Alpha-Bungarotoxin bound to peptides 173-204 and 186-196 and less well to 179-192 and 181-198, and it did not bind to 173-180, 194-204, and 205-227. Agonists (nicotine, suberyldicholine, carbamylcholine, and cytisine) effectively competed [3H]nicotine binding to the 173-204 peptide but competed alpha-[125I]bungarotoxin binding at millimolar concentration and with loss of rank order of potency. The competitive antagonists Alpha-Bungarotoxin, alpha-cobratoxin, and d-tubocurarine effectively blocked alpha-[125I]bungarotoxin binding but competed [3H]nicotine binding only at millimolar concentration. These results indicate that nicotine and Alpha-Bungarotoxin preferentially bind to different determinants within residues 173-204. Alternatively, nicotine and Alpha-Bungarotoxin could bind to different conformations of the peptide. Both agents appear to interact with common residues, most likely Tyr 190 and Cys 192, in the region of Cys 192 so that there is overlap of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
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substitution of torpedo acetylcholine receptor alpha 1 subunit residues with snake alpha 1 and rat nerve alpha 3 subunit residues in recombinant fusion proteins effect on alpha bungarotoxin binding
Biochemistry, 1992Co-Authors: Vijaya Chaturvedi, Diana L Donnellyroberts, Thomas L LentzAbstract:A fusion protein consisting of the TrpE protein and residues 166-211 of the Torpedo acetylcholine receptor alpha 1 subunit was produced in Escherichia coli using a pATH10 expression vector. Residues in the Torpedo sequence were changed by means of oligonucleotide-directed mutagenesis to residues present in snake alpha 1 subunit and rat nerve alpha 3 subunit which do not bind Alpha-Bungarotoxin. The fusion protein of the Torpedo sequence bound 125I-Alpha-Bungarotoxin with high affinity (IC50 = 2.5 x 10(-8) M from competition with unlabeled toxin, KD = 2.3 x 10(-8) M from equilibrium saturation binding data). Mutation of three Torpedo residues to snake residues, W184F, K185W, and W187S, had no effect on binding. Conversion of two additional Torpedo residues to snake, T191S and P194L, reduced Alpha-Bungarotoxin binding to undetectable levels. The P194L mutation alone abolished toxin binding. Mutation of three Torpedo alpha 1 residues to neuronal alpha 3-subunit residues, W187E, Y189K, and T191N, also abolished detectable Alpha-Bungarotoxin binding. Conversion of Try-189 to Asn which is present in the snake sequence (Y189N) abolished toxin binding. It is concluded that in the sequence of the alpha subunit of Torpedo encompassing Cys-192 and Cys-193, Try-189 and Pro-194 are important determinants of Alpha-Bungarotoxin binding. Tyr-189 may interact directly with cationic groups or participate in aromatic-aromatic interactions while Pro-194 may be necessary to maintain a conformation conductive to neurotoxin binding.
David B. Sattelle - One of the best experts on this subject based on the ideXlab platform.
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actions of nitromethylenes on an α bungarotoxin sensitive neuronal nicotinic acetylcholine receptor
Neuropharmacology, 1995Co-Authors: Steven D. Buckingham, Sarah C. R. Lummis, M. L. Balk, P Jewess, David B. SattelleAbstract:Nine nitromethylene analogues were tested for their actions on insect neuronal nicotinic acetylcholine receptors (nAChRs). Microelectrode recordings were used to study the actions of nitromethylenes on the cell body of an identified cockroach (Periplaneta americana) motor neurone, the fast coxal depressor (Df) in the metathoracic ganglion. Six nitromethylenes showed potent nAChR agonist actions; others were without nAChR agonist actions. Five nitromethylenes competitively displaced bound [125I]-Alpha-Bungarotoxin from cockroach nervous system membranes. The rank orders of potency for the compounds determined by their depolarizing actions and their ability to displace [125I]-Alpha-Bungarotoxin binding were similar. These findings, together with toxicity data obtained on the insects, Nephotettix cinciteps and Nilaparvata lugens, support the hypothesis that insect nAChRs are molecular targets of nitromethylene insecticides. Structure-activity relationships of the nitromethylenes suggest that optimal activity at neuronal nAChRs requires the presence of an electron-withdrawing component in the region of the aryl substituent and an electron-donating component at the 3' position of the imidazolidine ring.
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Actions of vesamicol on an Alpha-Bungarotoxin-sensitive neuronal nicotinic acetylcholine receptor.
The Journal of experimental biology, 1993Co-Authors: Steven D. Buckingham, Sarah C. R. Lummis, M. L. Balk, M. Schroeder, David B. SattelleAbstract:Electrophysiology and binding studies were used to determine the actions of vesamicol [2-(4-phenylpiperidino)cyclohexanol (AH5183)] on an Alpha-Bungarotoxin-sensitive, neuronal nicotinic acetylcholine receptor in the nervous system of the cockroach, Periplaneta americana. Electrophysiological studies on an identified motor neurone revealed a reversible blocking action of (+/-)-vesamicol on the response to ionophoretically applied acetylcholine with an IC50 value of 8.0 x 10(-6) mol1(-1). The block was weakly voltage-dependent over the membrane potential range of -50 mV to -90 mV, and appeared to be non-competitive. No difference in potency was observed between the resolved stereoisomers. (+/-)-Vesamicol was found to suppress specific binding of 125I-labelled Alpha-Bungarotoxin to cockroach nervous tissue with an IC50 value of 5.1 x 10(-3) mol1(-1) and an estimated Hill coefficient of 0.73. Differences in the Hill coefficients were found when the resolved stereoisomers were tested separately. These data provide the first demonstration of a blocking action by vesamicol of a neuronal nicotinic acetylcholine receptor.
Steeve Thany - One of the best experts on this subject based on the ideXlab platform.
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Neonicotinoid Binding, Toxicity and Expression of Nicotinic Acetylcholine Receptor Subunits in the Aphid Acyrthosiphon pisum
PLoS ONE, 2014Co-Authors: Emiliane Taillebois, Steeve Thany, Abdelhamid Beloula, Sophie Quinchard, Stéphanie Jaubert-possamai, Antoine Daguin, Denis Servent, Denis Tagu, Helene Tricoire-leignelAbstract:Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [H-3]-IMI (Kd of 0.16 +/- 0.04 nM and 41.7 +/- 5.9 nM) and for the nicotinic antagonist [I-125]-alpha- bungarotoxin (Kd of 0.008 +/- 0.002 nM and 1.135 +/- 0.213 nM). Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [I-125]-Alpha-Bungarotoxin binding sites while CLT affinity was similar for both [I-125]-Alpha-Bungarotoxin and [H-3]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 mu g/ml) and TMX (LC50 = 0.034 mu g/ml) were more toxic than CLT (LC50 = 0.118 mu g/ml). The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisum alpha 10 and Apisum beta 1 expressions levels, whereas Apisum beta 2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies
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Quinuclidine compounds differently act as agonists of Kenyon cell nicotinic acetylcholine receptors and induced distinct effect on insect ganglionic depolarizations
INVERTEBRATE NEUROSCIENCE, 2013Co-Authors: Monique Mathe-allainmat, Jacques Lebreton, Daniel Swale, Xavier Leray, Yassine Benzidane, Jeffrey R. Bloomquist, Steeve ThanyAbstract:We have recently demonstrated that a new quinuclidine benzamide compound named LMA10203 acted as an agonist of insect nicotinic acetylcholine receptors. Its specific pharmacological profile on cockroach dorsal unpaired median neurons (DUM) helped to identify Alpha-Bungarotoxin-insensitive nAChR2 receptors. In the present study, we tested its effect on cockroach Kenyon cells. We found that it induced an inward current demonstrating that it bounds to nicotinic acetylcholine receptors expressed on Kenyon cells. Interestingly, LMA10203-induced currents were completely blocked by the nicotinic antagonist Alpha-Bungarotoxin. We suggested that LMA10203 effect occurred through the activation of Alpha-Bungarotoxin-sensitive receptors and did not involve Alpha-Bungarotoxin-insensitive nAChR2, previously identified in DUM neurons. In addition, we have synthesized two new compounds, LMA10210 and LMA10211, and compared their effects on Kenyon cells. These compounds were members of the 3-quinuclidinyl benzamide or benzoate families. Interestingly, 1 mM LMA10210 was not able to induce an inward current on Kenyon cells compared to LMA10211. Similarly, we did not find any significant effect of LMA10210 on cockroach ganglionic depolarization, whereas these three compounds were able to induce an effect on the central nervous system of the third instar M. domestica larvae. Our data suggested that these three compounds could bind to distinct cockroach nicotinic acetylcholine receptors.
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Nornicotine application on cockroach dorsal unpaired median neurons induces two distinct ionic currents: Implications of different nicotinic acetylcholine receptors
Neuroscience Letters, 2012Co-Authors: Delphine Calas-list, Olivier List, Steeve ThanyAbstract:The goal of the present study is to examine the agonist action of nornicotine on insect nicotinic acetylcholine receptors. Using patch-clamp techniques on cockroach dorsal unpaired median neurons, we demonstrated that nornicotine induced two distinct ionic currents named types 1 and 2. We found that Alpha-Bungarotoxin induced a rapid desensitization of type 1 currents whereas type 2 was completely blocked. Interestingly, types 1 and 2 currents were not blocked by the muscarinic antagonist, pirenzepine but by co-application of 1 μM pirenzepine and 0.5 μM Alpha-Bungarotoxin, suggesting that muscarinic receptors modulated nornicotine-induced current amplitudes. In addition, type 1 current amplitudes were strongly reduced by 20 μM d-tubocurarine and 5 μM mecamylamine which blocked the previously identified Alpha-Bungarotoxin-insensitive nAChR1 and nAChR2 receptors. Co-application of Alpha-Bungarotoxin with d-tubocurarine or mecamylamine completely blocked all ionic currents. We propose that types 1 and 2 currents are associated to several nicotinic receptors subtypes, including nAChR1 and nAChR2 receptors. Finally, we conclude that nornicotine could be used as an agonist to identify distinct insect nicotinic receptors.
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A fluorinated quinuclidine benzamide named LMA 10203 acts as an agonist of insect nicotinic acetylcholine receptors
INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, 2012Co-Authors: Monique Mathe-allainmat, Beatrice Bodereau-dubois, Bruno Lapied, Jacques Lebreton, Steeve ThanyAbstract:In the the present study, we take advantage of the fact that cockroach dorsal unpaired median neurons express different nicotinic acetylcholine receptor subtypes to demonstrate that simple quinuclidine benzamides such as the 2-fluorinated benzamide LMA 10203, could act as an agonist of cockroach Alpha-Bungarotoxin-insensitive nicotinic acetylcholine receptor subtype, called nAChR2. Indeed, 1 mM LMA 10203 induced ionic currents which were partially blocked by 0.5 mu M Alpha-Bungarotoxin and methyllycaconitine and completely blocked by 5 mu M mecamylamine. Moreover, the current voltage curve revealed that the ionic current induced by LMA 10203 increased from -30 mV to +20 mV confirming that it acted as an agonist of Alpha-Bungarotoxin-insensitive nAChR2. In addition, 1 mM LMA 10203 induced a depolarization of the sixth abdominal ganglion and this neuroexcitatory activity was completely blocked by 5 mu M mecamylamine. These data suggest that nAChR2 was also expressed at the postsynaptic level on the synapse between the cercal afferent nerve and the giant interneurons. Interestingly, despite LMA 10203 being an agonist of cockroach nicotinic receptors, it had a poor insecticidal activity. We conclude that LMA 10203 could be used as an interesting compound to identify specific insect nAChR subtypes. (C) 2012 Elsevier Ltd. All rights reserved.
Cecilia Gotti - One of the best experts on this subject based on the ideXlab platform.
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subunit composition of nicotinic receptors in monkey striatum effect of treatments with 1 methyl 4 phenyl 1 2 3 6 tetrahydropyridine or l dopa
Molecular Pharmacology, 2005Co-Authors: Maryka Quik, Francesco Clementi, Silvia Vailati, Tanuja Bordia, Jennifer M Kulak, Hong Fan, Michael J Mcintosh, Cecilia GottiAbstract:Nicotinic acetylcholine receptors (nAChRs) represent an important modulator of striatal function both under normal conditions and in pathological states such as Parkinson's disease. Because different nAChR subtypes may have unique functions, immunoprecipitation and ligand binding studies were done to identify their subunit composition. As in the rodent, alpha2, alpha4, alpha6, beta2, and beta3 nAChR subunit immunoreactivity was identified in monkey striatum. However, distinct from the rodent, the present results also revealed the novel presence of alpha3 nAChR subunit-immunoreactivity in this same region, but not that for alpha5 and beta4. Relatively high levels of alpha2 and alpha3 subunits were also identified in monkey cortex, in addition to alpha4 and beta2. Experiments were next done to determine whether striatal subunit expression was changed with nigrostriatal damage. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment decreased alpha6 and beta3 subunit immunoreactivity by approximately 80% in parallel with the dopamine transporter, suggesting that they are predominantly expressed on nigrostriatal dopaminergic projections. In contrast, alpha3, alpha4, and beta2 subunit immunoreactivity was decreased approximately 50%, whereas alpha2 was not changed. These data, together with those from dual immunoprecipitation and radioligand binding studies ([(3)H]cytisine, (125)I-Alpha-Bungarotoxin, and (125)I-alpha-conotoxin MII) suggest the following: that alpha6beta2beta3, alpha6alpha4beta2beta3, and alpha3beta2* nAChR subtypes are present on dopaminergic terminals and that the alpha4beta2 subtype is localized on both dopaminergic and nondopaminergic neurons, whereas alpha2beta2* and alpha7 receptors are localized on nondopaminergic cells in monkey striatum. Overall, these results suggest that drugs targeting non-alpha7 nicotinic receptors may be useful in the treatment of disorders characterized by nigrostriatal dopaminergic damage, such as Parkinson's disease.
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pharmacology and biophysical properties of α7 and α7 α8 α bungarotoxin receptor subtypes immunopurified from the chick optic lobe
European Journal of Neuroscience, 1994Co-Authors: Cecilia Gotti, Francesco Clementi, Wolfgang Hanke, K Maury, Milena Moretti, M Ballivet, Daniel BertrandAbstract:Two chick optic lobe Alpha-Bungarotoxin receptor subtypes (alpha 7 and alpha 7-alpha 8) were immunopurified using polyclonal antibodies raised against synthetic peptides of chick alpha 7 and alpha 8 Alpha-Bungarotoxin receptor subunits. The alpha 7 subtype contained the M(r) 57,000 alpha 7 subunit, and represented 60-70% of the Alpha-Bungarotoxin receptors; the alpha 7-alpha 8 subtype contained the M(r) 57,000 alpha 7 and alpha 8 subunits, and represented only 20-25% of the receptors. Both subtypes also had an additional M(r) 52,000 subunit. The affinity of these subtypes for Alpha-Bungarotoxin as well as antagonists was similar. However, the alpha 7-alpha 8 subtype displayed consistently higher affinities for agonists. When reconstituted in planar lipid bilayers, the alpha 7-alpha 8 subtype displayed several conductance states of 10-50 pS; the alpha 7 subtype had only one conductance state of 45 pS. The alpha 7-alpha 8 subtype was activated by lower agonist concentrations than the alpha 7 subtype. When expressed in Xenopus oocytes, the alpha 8 subunit formed functional homomeric receptors that desensitized rapidly. These channels were blocked by Alpha-Bungarotoxin and displayed a higher affinity for agonists than the alpha 7 homomeric receptor. Taken together, these data indicate that at least two Alpha-Bungarotoxin subtypes are present in the chick optic lobe. They operate as ligand-gated channels and display different agonist sensitivities and kinetics/conductance properties.
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A functional α-bungarotoxin receptor is present in chick cerebellum: Purification and characterization
Neuroscience, 1992Co-Authors: Cecilia Gotti, Milena Moretti, W. Hanke, W.-r. Schlue, L. Briscini, Francesco ClementiAbstract:It has recently been demonstrated that Alpha-Bungarotoxin receptors, which behave as functional nicotinic receptors, are present in chick CNS. In this paper, we report the purification and characterization of a functional Alpha-Bungarotoxin receptor from chick cerebellum, a nervous tissue in which a clear inhibition of induced nicotine effects has been reported in vivo. This receptor contains at least three subunits of apparent mol. wt 52,000, 57,000 and 67,000. The use of monoclonal antibodies specific for the alpha 7 subunit demonstrated that 75% of the molecules present in our purified preparation belong to the alpha 7 subtype and that this antibody labels the 57,000 band in western blot, thus indicating that this is the toxin binding subunit. Reconstruction experiments in planar lipid bilayers show that this Alpha-Bungarotoxin receptor forms a cation selective channel whose opening is blocked by d-tubocurarine. Binding experiments on immobilized receptors over an Alpha-Bungarotoxin-Sepharose affinity column show that the ligand binding subunit is present in vivo in two copies per receptor. Immunological, pharmacological and functional experiments show that this purified receptor is very similar, but not identical, to the previously characterized chick optic lobe receptor, thus indicating the heterogeneity of these Alpha-Bungarotoxin receptors in the CNS.
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purification and characterization of an alpha bungarotoxin receptor that forms a functional nicotinic channel
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Cecilia Gotti, A. Esparis Ogando, Wolfgang Hanke, Milena Moretti, R Schlue, Francesco ClementiAbstract:Neither the structure nor the function of Alpha-Bungarotoxin (alpha Bgtx) binding molecules in the nervous system have yet been completely defined, although it is known that some of these molecules are related to cation channels and some are not. Using an improved method of affinity chromatography, we have isolated a toxin binding molecule from chicken optic lobe that contains at least three subunits with apparent Mr values of 52,000, 57,000, and 67,000. The Mr 57,000 subunit binds alpha Bgtx and seems to be present in two copies per receptor. The receptor is recognized by antibodies raised against the alpha Bgtx receptors of human neuroblastoma cells, fetal calf muscle, and chicken optic lobe but not by antibodies raised against Torpedo acetylcholine receptor, the serum of myasthenic patients, or monoclonal antibody, 35. 125I-labeled alpha Bgtx binding to the isolated receptor is blocked, with the same potency, by nicotinic agonists and antagonists, such as nicotine, neuronal bungarotoxin and, d-tubocurarine. When reconstituted in a planar lipid bilayer, the purified alpha Bgtx receptor forms cationic channels with a conductance of 50 pS. These channels are activated in a dose-dependent manner by carbamylcholine and blocked by d-tubocurarine.
Steven D. Buckingham - One of the best experts on this subject based on the ideXlab platform.
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actions of nitromethylenes on an α bungarotoxin sensitive neuronal nicotinic acetylcholine receptor
Neuropharmacology, 1995Co-Authors: Steven D. Buckingham, Sarah C. R. Lummis, M. L. Balk, P Jewess, David B. SattelleAbstract:Nine nitromethylene analogues were tested for their actions on insect neuronal nicotinic acetylcholine receptors (nAChRs). Microelectrode recordings were used to study the actions of nitromethylenes on the cell body of an identified cockroach (Periplaneta americana) motor neurone, the fast coxal depressor (Df) in the metathoracic ganglion. Six nitromethylenes showed potent nAChR agonist actions; others were without nAChR agonist actions. Five nitromethylenes competitively displaced bound [125I]-Alpha-Bungarotoxin from cockroach nervous system membranes. The rank orders of potency for the compounds determined by their depolarizing actions and their ability to displace [125I]-Alpha-Bungarotoxin binding were similar. These findings, together with toxicity data obtained on the insects, Nephotettix cinciteps and Nilaparvata lugens, support the hypothesis that insect nAChRs are molecular targets of nitromethylene insecticides. Structure-activity relationships of the nitromethylenes suggest that optimal activity at neuronal nAChRs requires the presence of an electron-withdrawing component in the region of the aryl substituent and an electron-donating component at the 3' position of the imidazolidine ring.
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Actions of vesamicol on an Alpha-Bungarotoxin-sensitive neuronal nicotinic acetylcholine receptor.
The Journal of experimental biology, 1993Co-Authors: Steven D. Buckingham, Sarah C. R. Lummis, M. L. Balk, M. Schroeder, David B. SattelleAbstract:Electrophysiology and binding studies were used to determine the actions of vesamicol [2-(4-phenylpiperidino)cyclohexanol (AH5183)] on an Alpha-Bungarotoxin-sensitive, neuronal nicotinic acetylcholine receptor in the nervous system of the cockroach, Periplaneta americana. Electrophysiological studies on an identified motor neurone revealed a reversible blocking action of (+/-)-vesamicol on the response to ionophoretically applied acetylcholine with an IC50 value of 8.0 x 10(-6) mol1(-1). The block was weakly voltage-dependent over the membrane potential range of -50 mV to -90 mV, and appeared to be non-competitive. No difference in potency was observed between the resolved stereoisomers. (+/-)-Vesamicol was found to suppress specific binding of 125I-labelled Alpha-Bungarotoxin to cockroach nervous tissue with an IC50 value of 5.1 x 10(-3) mol1(-1) and an estimated Hill coefficient of 0.73. Differences in the Hill coefficients were found when the resolved stereoisomers were tested separately. These data provide the first demonstration of a blocking action by vesamicol of a neuronal nicotinic acetylcholine receptor.