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Daniel Bertrand - One of the best experts on this subject based on the ideXlab platform.
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molecular blueprint of allosteric binding sites in a homologue of the agonist binding domain of the α7 Nicotinic Acetylcholine Receptor
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Radovan Spurny, Sarah Debaveye, A Farinha, Ken Veys, Thomas Gossas, John R Atack, Sonia Bertrand, Daniel Bertrand, Helena U Danielson, Gary TresadernAbstract:The α7 Nicotinic Acetylcholine Receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 Nicotinic Acetylcholine Receptor and Acetylcholine binding protein, termed α7-AChBP. This chimeric Receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the Receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate Receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential.
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azemiopsin from azemiops feae viper venom a novel polypeptide ligand of Nicotinic Acetylcholine Receptor
Journal of Biological Chemistry, 2012Co-Authors: Yuri N Utkin, Daniel Bertrand, Christoph Weise, Igor E Kasheverov, T V Andreeva, Elena V Kryukova, M N Zhmak, Vladislav G Starkov, Ngoc Anh Hoang, Joachim RamerstorferAbstract:Abstract Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino-acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a β-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo Nicotinic Acetylcholine Receptor (nAChR) (IC50 0.18±0.03 μM) and with lower efficiency to human α7 nAChR (IC50 22±2 μM). It dose-dependently blocked Acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1β1ϵδ) than the fetal form (α1β1γδ), EC50 being 0.44±0.1 μM and 1.56±0.37 μM, respectively. The peptide had no effect on GABAA (α1β3γ2 or α2β3γ2) Receptors at concentration up to 100 μM or on 5-HT3 Receptors at concentration up to 10 μM. Ala scanning showed that amino-acid residues at positions 3-6, 8-11 and 13-14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges.
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structural determinants of selective alpha conotoxin binding to a Nicotinic Acetylcholine Receptor homolog achbp
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Chris Ulens, Daniel Bertrand, Ron C Hogg, Patrick H N Celie, Victor I Tsetlin, August B Smit, Titia K SixmaAbstract:The Nicotinic Acetylcholine Receptor (nAChR) is the prototype member of the superfamily of pentameric ligand-gated ion channels. How the extracellular ligand-binding domain coordinates selective binding of ligand molecules to different subtypes of the Receptor is unknown at the structural level. Here, we present the 2.2-A crystal structure of a homolog of the ligand-binding domain of the nAChR, Aplysia californica AChBP (Ac-AChBP), in complex with α-conotoxin ImI. This conotoxin is unique in its selectivity toward the neuronal α3β2 and α7 nAChR, a feature that is reflected in its selective binding to Ac-AChBP compared with other AChBP homologs. We observe a network of interactions between the residues of the ligand-binding site and the toxin, in which ImI Arg-7 and Trp-10 play a key role. The toxin also forms interactions in the ligand-binding site that were not seen in the complex of Ac-AChBP with PnIA(A10L D14K), a conotoxin variant that lacks binding selectivity to AChBP homologs. In combination with electrophysiological recordings obtained by using the wild-type α7 nAChR and L247T mutant, we show that conotoxin ImI inhibits ion conduction by stabilizing the Receptor in a desensitized conformation. Comparison of the Ac-AChBP–ImI crystal structure with existing AChBP structures offers structural insight into the extent of flexibility of the interface loops and how their movement may couple ligand binding to channel gating in the context of a nAChR.
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chronic exposure to nicotine upregulates the human α4β2 Nicotinic Acetylcholine Receptor function
The Journal of Neuroscience, 2001Co-Authors: Bruno Buisson, Daniel BertrandAbstract:Widely expressed in the brain, the α4β2 Nicotinic Acetylcholine Receptor (nAChR) is proposed to play a major role in the mechanisms that lead to and maintain nicotine addiction. Using the patch-clamp technique and pharmacological protocols, we examined the consequences of long-term exposure to 0.1–10 μm nicotine in K-177 cells expressing the major human brain α4β2 Receptor. The Acetylcholine dose–response curves are biphasic and revealed both a high- and a low-affinity component with apparent EC 50 values of 1.6 and 62 μm. Ratios of Receptors in the high- and low-affinity components are 25 and 75%, respectively. Chronic exposure to nicotine or Nicotinic antagonists [dihydro-β-erytroidine (DHβE) or methyllycaconitine (MLA)] increases the fraction of high-affinity Receptors up to 70%. Upregulated Acetylcholine-evoked currents increase by twofold or more and are less sensitive to desensitization. Functional upregulation is independent of protein synthesis as shown by the lack of effect of 20 μmcycloheximide. Single-channel currents recorded with 100 nmAcetylcholine show predominantly high conductances (38.8 and 43.4 pS), whereas additional smaller conductances (16.7 and 23.5 pS) were observed with 30 μm Acetylcholine. In addition, long-term exposure to dihydro-β-erytroidine increases up to three times the frequency of channel openings. These data indicate, in contrast to previous studies, that human α4β2 nAChRs are functionally upregulated by chronic nicotine exposure.
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a neuronal Nicotinic Acetylcholine Receptor subunit α7 is developmentally regulated and forms a homo oligomeric channel blocked by α btx
Neuron, 1990Co-Authors: Sabine Couturier, Sonia Bertrand, Daniel Bertrand, Jeanmarc Matter, Mariaclemencia Hernandez, Neil Millar, Soledad Valera, Thomas Barkas, Marc BallivetAbstract:Abstract cDNA and genomic clones encoding α7, a novel neuronal Nicotinic Acetylcholine Receptor (nAChR) α subunit, were isolated and sequenced. The mature α7 protein (479 residues) has moderate homology with all other α and non-α nAChR subunits and probably assumes the same transmembrane topology. α7 transcripts transiently accumulate in the developing optic tectum between E5 and E16. They are present in both the deep and the superficial layers of E12 tectum. In Xenopus oocytes, the α7 protein assembles into a homo-oligomeric channel responding to Acetylcholine and nicotine. The α7 channel desensitizes very rapidly, rectifies strongly above −20 mV, and is blocked by α-bungarotoxin. A bacterial fusion protein encompassing residues 124–239 of α7 binds labeled α-bungarotoxin. We conclude that α-bungarotoxin binding proteins in the vertebrate nervous system can function as nAChRs.
Arthur L Beaudet - One of the best experts on this subject based on the ideXlab platform.
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megacystis mydriasis and ion channel defect in mice lacking the α3 neuronal Nicotinic Acetylcholine Receptor
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Wei Xu, James W Patrick, Avi Orrurtreger, Shari Gelber, Dawna L Armstrong, Richard A Lewis, Chingnan Ou, Lorna W Role, Mariella De Biasi, Arthur L BeaudetAbstract:Abstract The α3 subunit of the neuronal Nicotinic Acetylcholine Receptor is widely expressed in autonomic ganglia and in some parts of the brain. The α3 subunit can form heteromultimeric ion channels with other α subunits and with β2 and β4 subunits, but its function in vivo is poorly understood. We prepared a null mutation for the α3 gene by deletion of exon 5 and found that homozygous (−/−) mice lacked detectable mRNA on Northern blotting. The −/− mice survive to birth but have impaired growth and increased mortality before and after weaning. The −/− mice have extreme bladder enlargement, dribbling urination, bladder infection, urinary stones, and widely dilated ocular pupils that do not contract in response to light. Detailed histological studies of −/− mice revealed no significant abnormalities in brain or peripheral tissues except urinary bladder, where inflammation was prominent. Ganglion cells and axons were present in bladder and bowel. Bladder strips from −/− mice failed to contract in response to 0.1 mM nicotine, but did contract in response to electrical field stimulation or carbamoylcholine. The number of Acetylcholine-activated single-channel currents was severely reduced in the neurons of superior cervical ganglia in −/− mice with five physiologically distinguishable Nicotinic Acetylcholine Receptor subtypes with different conductance and kinetic properties in wild-type mice, all of which were reduced in −/− mice. The findings in the α3-null mice suggest that this subunit is an essential component of the Nicotinic Receptors mediating normal function of the autonomic nervous system. The phenotype in −/− mice may be similar to the rare human genetic disorder of megacystis–microcolon–intestinal hypoperistalsis syndrome.
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mice deficient in the α7 neuronal Nicotinic Acetylcholine Receptor lack α bungarotoxin binding sites and hippocampal fast Nicotinic currents
The Journal of Neuroscience, 1997Co-Authors: Avi Orrurtreger, James W Patrick, Mariella De Biasi, Finn M Goldner, Mayuko Saeki, Isabel Lorenzo, Leah Goldberg, John A Dani, Arthur L BeaudetAbstract:The α7 subunit of the neuronal Nicotinic Acetylcholine Receptor (nAChR) is abundantly expressed in hippocampus and is implicated in modulating neurotransmitter release and in binding α-bungarotoxin (α-BGT). A null mutation for the α7 subunit was prepared by deleting the last three exons of the gene. Mice homozygous for the null mutation lack detectable mRNA, but the mice are viable and anatomically normal. Neuropathological examination of the brain revealed normal structure and cell layering, including normal cortical barrel fields; histochemical assessment of the hippocampus was also normal. Autoradiography with [3H]nicotine revealed no detectable abnormalities of high-affinity nicotine binding sites, but there was an absence of high-affinity [125I]α-BGT sites. Null mice also lack rapidly desensitizing, methyllycaconitine-sensitive, Nicotinic currents that are present in hippocampal neurons. The results of this study indicate that the α-BGT binding sites are equivalent to the α7-containing nAChRs that mediate fast, desensitizing Nicotinic currents in the hippocampus. These mice demonstrate that the α7 subunit is not essential for normal development or for apparently normal neurological function, but the mice may prove to have subtle phenotypic abnormalities and will be valuable in defining the functional role of this gene product in vivo.
M.j Choi - One of the best experts on this subject based on the ideXlab platform.
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genomic organization and partial duplication of the human α7 neuronal Nicotinic Acetylcholine Receptor gene chrna7
Genomics, 1998Co-Authors: Jeremy Gault, C Drebing, J. Meriwether, J. Hopkins, S. Jacobs, R Berger, M Robinson, T. Moore, Judith Logel, M.j ChoiAbstract:Abstract The human α7 neuronal Nicotinic Acetylcholine Receptor gene (HGMW-approved symbol CHRNA7) has been characterized from genomic clones. The gene is similar in structure to the chick α7 gene with 10 exons and conserved splice junction positions. The size of the human gene is estimated to be larger than 75 kb. A putative promoter 5′ of the translation start in exon 1 has been cloned and sequenced. The promoter region lacks a TATA box and has a high GC content (77%). Consensus Sp1, AP-2, Egr-1, and CREB transcription factor binding sites appear to be conserved between bovine and human genes. The α7 nAChR gene was found to be partially duplicated, with both loci mapping to the chromosome 15q13 region. A yeast artificial chromosome contig was constructed over a genetic distance of 5 cM that includes both α7 loci and the region between them. Four novel exons are described, located in genomic clones containing the partially duplicated gene. The duplicated sequences, including the novel exons, are expressed in human brain.
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Genomic organization and partial duplication of the human alpha7 neuronal Nicotinic Acetylcholine Receptor gene (CHRNA7).
Genomics, 1998Co-Authors: Jeremy Gault, C Drebing, J Logel, J. Meriwether, J. Hopkins, S. Jacobs, R Berger, M Robinson, T. Moore, M.j ChoiAbstract:The human alpha7 neuronal Nicotinic Acetylcholine Receptor gene (HGMW-approved symbol CHRNA7) has been characterized from genomic clones. The gene is similar in structure to the chick alpha7 gene with 10 exons and conserved splice junction positions. The size of the human gene is estimated to be larger than 75 kb. A putative promoter 5' of the translation start in exon 1 has been cloned and sequenced. The promoter region lacks a TATA box and has a high GC content (77%). Consensus Sp1, AP-2, Egr-1, and CREB transcription factor binding sites appear to be conserved between bovine and human genes. The alpha7 nAChR gene was found to be partially duplicated, with both loci mapping to the chromosome 15q13 region. A yeast artificial chromosome contig was constructed over a genetic distance of 5 cM that includes both alpha7 loci and the region between them. Four novel exons are described, located in genomic clones containing the partially duplicated gene. The duplicated sequences, including the novel exons, are expressed in human brain.
Luis Ulloa - One of the best experts on this subject based on the ideXlab platform.
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the alpha7 Nicotinic Acetylcholine Receptor as a pharmacological target for inflammation
British Journal of Pharmacology, 2009Co-Authors: W J De Jonge, Luis UlloaAbstract:The physiological regulation of the immune system encompasses comprehensive anti-inflammatory mechanisms that can be harnessed for the treatment of infectious and inflammatory disorders. Recent studies indicate that the vagal nerve, involved in control of heart rate, hormone secretion and gastrointestinal motility, is also an immunomodulator. In experimental models of inflammatory diseases, vagal nerve stimulation attenuates the production of proinflammatory cytokines and inhibits the inflammatory process. Acetylcholine, the principal neurotransmitter of the vagal nerve, controls immune cell functions via the alpha7 Nicotinic Acetylcholine Receptor (alpha7nAChR). From a pharmacological perspective, Nicotinic agonists are more efficient than Acetylcholine at inhibiting the inflammatory signaling and the production of proinflammatory cytokines. This 'Nicotinic anti-inflammatory pathway' may have clinical implications as treatment with Nicotinic agonists can modulate the production of proinflammatory cytokines from immune cells. Nicotine has been tested in clinical trials as a treatment for inflammatory diseases such as ulcerative colitis, but the therapeutic potential of this mechanism is limited by the collateral toxicity of nicotine. Here, we review the recent advances that support the design of more specific Receptor-selective Nicotinic agonists that have anti-inflammatory effects while eluding its collateral toxicity.
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Nicotinic Acetylcholine Receptor alpha7 subunit is an essential regulator of inflammation
Nature, 2003Co-Authors: Hong Wang, Man Yu, Mahendar Ochani, C A Amella, Mahira Tanovic, Seenu Susarla, Jianhua Li, Haichao Wang, Huan Yang, Luis UlloaAbstract:Excessive inflammation and tumour-necrosis factor (TNF) synthesis cause morbidity and mortality in diverse human diseases including endotoxaemia, sepsis, rheumatoid arthritis and inflammatory bowel disease1,2,3,4. Highly conserved, endogenous mechanisms normally regulate the magnitude of innate immune responses and prevent excessive inflammation. The nervous system, through the vagus nerve, can inhibit significantly and rapidly the release of macrophage TNF, and attenuate systemic inflammatory responses5,6,7. This physiological mechanism, termed the ‘cholinergic anti-inflammatory pathway’5 has major implications in immunology and in therapeutics; however, the identity of the essential macrophage Acetylcholine-mediated (cholinergic) Receptor that responds to vagus nerve signals was previously unknown. Here we report that the Nicotinic Acetylcholine Receptor α7 subunit is required for Acetylcholine inhibition of macrophage TNF release. Electrical stimulation of the vagus nerve inhibits TNF synthesis in wild-type mice, but fails to inhibit TNF synthesis in α7-deficient mice. Thus, the Nicotinic Acetylcholine Receptor α7 subunit is essential for inhibiting cytokine synthesis by the cholinergic anti-inflammatory pathway.
Jean-pierre Changeux - One of the best experts on this subject based on the ideXlab platform.
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the Nicotinic Acetylcholine Receptor the founding father of the pentameric ligand gated ion channel superfamily
Journal of Biological Chemistry, 2012Co-Authors: Jean-pierre ChangeuxAbstract:A critical event in the history of biological chemistry was the chemical identification of the first neurotransmitter Receptor, the Nicotinic Acetylcholine Receptor. Disciplines as diverse as electrophysiology, pharmacology, and biochemistry joined together in a unified and rational manner with the common goal of successfully identifying the molecular device that converts a chemical signal into an electrical one in the nervous system. The Nicotinic Receptor has become the founding father of a broad family of pentameric membrane Receptors, paving the way for their identification, including that of the GABAA Receptors.
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abnormal functional organization in the dorsal lateral geniculate nucleus of mice lacking the β2 subunit of the Nicotinic Acetylcholine Receptor
Neuron, 2003Co-Authors: Matthew S Grubb, Jean-pierre Changeux, Francesco M Rossi, Ian ThompsonAbstract:Abstract Spontaneous activity patterns in the developing retina appear important for the functional organization of the visual system. We show here that an absence of early retinal waves in mice lacking the β2 subunit of the Nicotinic Acetylcholine Receptor (nAChR) is associated with both gain and loss of functional organization in the dorsal lateral geniculate nucleus (dLGN). Anatomical studies show normal gross retinotopy in the β2 −/− dLGN but suggest reduced topographic precision in the retinogeniculate projection. Physiological recordings reveal normal topography in the dorsoventral visual axis but a lack of fine-scale mapping in the nasotemporal visual plane. In contrast, unlike wild-type mice, on- and off-center cells in the β2 −/− dLGN are spatially segregated. The presence of the β2 subunit of the nAChR in the CNS is therefore important for normal functional organization in the retinogeniculate projection.
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requirement of the Nicotinic Acetylcholine Receptor β2 subunit for the anatomical and functional development of the visual system
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Francesco M Rossi, Lisa M. Marubio, Tommaso Pizzorusso, Vittorio Porciatti, Lamberto Maffei, Jean-pierre ChangeuxAbstract:In the mammalian visual system the formation of eye-specific layers at the thalamic level depends on retinal waves of spontaneous activity, which rely on Nicotinic Acetylcholine Receptor activation. We found that in mutant mice lacking the β2 subunit of the neuronal Nicotinic Receptor, but not in mice lacking the α4 subunit, retinofugal projections do not segregate into eye-specific areas, both in the dorso-lateral geniculate nucleus and in the superior colliculus. Moreover, β2−/− mice show an expansion of the binocular subfield of the primary visual cortex and a decrease in visual acuity at the cortical level but not in the retina. We conclude that the β2 subunit of the Nicotinic Acetylcholine Receptor is necessary for the anatomical and functional development of the visual system.
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Nicotinic Acetylcholine Receptor knockout mice as animal models for studying Receptor function
European Journal of Pharmacology, 2000Co-Authors: Lisa M. Marubio, Jean-pierre ChangeuxAbstract:Nicotinic Acetylcholine Receptors are pentameric ligand-gated ion channels, which are involved in a wide range of neuronal functions. During the past decade, a large number of Nicotinic Acetylcholine Receptor subunits have been cloned and showed a discreet yet overlapping distribution pattern. Recently, several groups have produced mutant mice lacking specific Nicotinic Acetylcholine Receptor subunits. In this review, we focus on how the study of these knockout mouse models has advanced our understanding of the role individual Nicotinic Acetylcholine Receptor subunits play in the function and composition of endogenous Receptors and the diverse pharmacological actions of nicotine in the mammalian nervous system.
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Implication of a multisubunit Ets-related transcription factor in synaptic expression of the Nicotinic Acetylcholine Receptor
EMBO Journal, 1998Co-Authors: L. Schaeffer, Nathalie Duclert, Monique Huchet-dymanus, Jean-pierre ChangeuxAbstract:In adult muscle, transcription of the Nicotinic Acetylcholine Receptor (AChR) is restricted to the nuclei located at the neuromuscular junction. The N-box, a new promoter element, was identified recently and shown to contribute to this compartmentalized synaptic expression of the AChR delta- and epsilon-subunits. We demonstrate that the N-box mediates transcriptional activation in cultured myotubes and identify the transcription factor that binds to the N-box as a heterooligomer in myotubes and adult muscle. The GABP (GA-binding protein) alpha-subunit belongs to the Ets family of transcription factors, whereas the beta-subunit shares homology with IkappaB and Drosophila Notch protein. GABP binding specificity to mutated N-box in vitro strictly parallels the sequence requirement for beta-galactosidase targeting to the endplate in vivo. In situ hybridization studies reveal that the mRNAs of both GABP subunits are abundant in mouse diaphragm, with preferential expression of the alpha-subunit at motor endplates. In addition, heregulin increases GABPalpha protein levels and regulates phosphorylation of both subunits in cultured chick myotubes. Finally, dominant-negative mutants of either GABPalpha or GABPbeta block heregulin-elicited transcriptional activation of the AChR delta and epsilon genes. These findings establish the expected connection with a presynaptic trophic factor whose release contributes to the accumulation of AChR subunit mRNAs at the motor endplate.
