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Accueil > Agenda > Les séminaires Jean Roche > Première identification d’un canal bactérien pH-sensible de la famille des (...)

Première identification d’un canal bactérien pH-sensible (...)

Lundi 2 octobre 2006, salle Lissitzky.

Bibliographie

*1 : *J Physiol Paris. 2006 Mar-May ;99(2-3):162-71. Epub 2006 Feb 3.

Related Articles, <http://www.ncbi.nlm.nih.gov/entrez/...> Compound via MeSH, <http://www.ncbi.nlm.nih.gov/entrez/...> Substance via MeSH, <http://www.ncbi.nlm.nih.gov/entrez/...> Books, <http://www.ncbi.nlm.nih.gov/entrez/...> LinkOut <http://www.ncbi.nlm.nih.gov/entrez/...>

Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *Nicotine enhances intracellular nicotinic receptor maturation : a novel mechanism of neural plasticity ?*

*Corringer PJ* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Sallette J* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Changeux JP* <http://www.ncbi.nlm.nih.gov/entrez/...> .

Unit of receptor and Cognition, Pasteur Institute, 25 rue du docteur Roux, 75724 Paris Cedex 15, France. pjcorrin pasteur.fr

Nicotine addiction, the primary cause of tobacco consumption, is mediated through nicotine binding to brain nicotinic acetylcholine receptor (nAChRs). Upon chronic exposure, nicotine elicits a cascade of events, starting with nAChR activation and desensitization, followed by a long term up-regulation that corresponds to an increase in the number of the high affinity nAChRs, a paradoxical process that occurs in the brain of smokers. Recent investigation of the maturation and trafficking of the major brain alpha4beta2 nAChR demonstrates that up-regulation is initiated in the endoplasmic reticulum soon after protein translation. The data thus far accumulated provide evidence that nicotine elicits up-regulation by promoting maturation of nAChR precursors that would otherwise be degraded. This "maturational enhancer" action of nicotine probably contributes to the long term effect of chronic nicotine, and suggests a novel mechanism of neuronal plasticity through an yet unknown endogenous substance which would modulate the receptor expression under physiological conditions.

Publication Types :

· Review

PMID : 16458492 [PubMed - indexed for MEDLINE]


*2 : *Neuron. 2005 May 19 ;46(4):595-607.

Related Articles, <http://www.ncbi.nlm.nih.gov/entrez/...> Gene, <http://www.ncbi.nlm.nih.gov/entrez/...> Gene (GeneRIF), <http://www.ncbi.nlm.nih.gov/entrez/...> Nucleotide (RefSeq), <http://www.ncbi.nlm.nih.gov/entrez/...> Compound via MeSH, <http://www.ncbi.nlm.nih.gov/entrez/...> < a CLASS="dblinks" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=pubmed_Abstract&db=pubmed&cmd=Display&dopt=pubmed_pcsubstance_mesh&from_uid=15944128">Substance via MeSH, Protein (RefSeq), <http://www.ncbi.nlm.nih.gov/entrez/...> UniGene, <http://www.ncbi.nlm.nih.gov/entrez/...> Nucleotide, <http://www.ncbi.nlm.nih.gov/entrez/...> Protein, <http://www.ncbi.nlm.nih.gov/entrez/...> GEO Profiles, <http://w%0A%20ww.ncbi.nlm.nih.gov/e...> Books, <http://www.ncbi.nlm.nih.gov/entrez/...> LinkOut <http://www.ncbi.nlm.nih.gov/entrez/...>

Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *Nicotine upregulates its own receptors through enhanced intracellular maturation.*

*Sallette J* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Pons S* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Devillers-Thiery A* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Soudant M* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Prado de Carvalho L* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Changeux JP* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Corringer PJ* <http://www.ncbi.nlm.nih.gov/entrez/...> .

CNRS URA D2182 Recepteurs et Cognition, Institut Pasteur, Paris, France.

Chronic exposure to nicotine elicits upregulation of high-affinity nicotinic receptors in the smoker’s brain. To address the molecular mechanism of upregulation, we transfected HEK293 cells with human alpha4beta2 receptors and traced the subunits throughout their intracellular biosynthesis, using metabolic labeling and immunoprecipitation techniques. We show that high-mannose glycosylated subunits mature and assemble into pentamers in the endoplasmic reticulum and that only pentameric receptors reach the cell surface following carbohydrate processing. Nicotine is shown to act inside the cell and to increase the amount of beta subunits immunoprecipitated by the conformation-dependent mAb290, indicating that nicotine enhances a critical step in the intracellular maturation of these receptors. This effect, which also takes place at concentrations of nicotine found in the blood of smokers upon expression of alpha4beta2 in SH-SY5Y neuroblastoma cells, may play a crucial role in nicotine addiction and possibly implement a model of neural plasticity.

PMID : 15944128 [PubMed - indexed for MEDLINE]


*3 : *Biophys J. 2005 Jun ;88(6):3954-65. Epub 2005 Apr 1.

Related Articles, <http://www.ncbi.nlm.nih.gov/entrez/...> Cited Articles, <http://www.ncbi.nlm.nih.gov/entrez/...> Free in PMC, <http://www.pubmedcentral.gov/articl...> Cited in PMC, <http://www.pubmedcentral.gov/tocren...> Books, <http://www.ncbi.nlm.nih.gov/entrez/...> LinkOut <http://www.ncbi.nlm.nih.gov/entrez/...>

Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *Normal mode analysis suggests a quaternary twist model for the nicotinic receptor gating mechanism.*

*Taly A* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Delarue M* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Grutter T* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Nilges M* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Le Novere N* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Corringer PJ* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Changeux JP* <http://www.ncbi.nlm.nih.gov/entrez/...> .

Recepteurs et Cognition, Unite de Recherche Associee (URA) Centre National de la Recherche Scientifique 2182, Institut Pasteur, Paris, France.

We present a three-dimensional model of the homopentameric alpha7 nicotinic acetylcholine receptor (nAChR), that includes the extracellular and membrane domains, developed by comparative modeling on the basis of : 1), the x-ray crystal structure of the snail acetylcholine binding protein, an homolog of the extracellular domain of nAChRs ; and 2), cryo-electron microscopy data of the membrane domain collected on Torpedo marmorata nAChRs. We performed normal mode analysis on the complete three-dimensional model to explore protein flexibility. Among the first 10 lowest frequency modes, only the first mode produces a structural reorganization compatible with channel gating : a wide opening of the channel pore caused by a concerted symmetrical quaternary twist motion of the protein with opposing rotations of the upper (extracellular) and lower (transmembrane) domains. Still, significant reorganizations are observed within each subunit, that involve their bending at the domain interface, an increase of angle between the two beta-sheets composing the extracellular domain, the internal beta-sheet being significantly correlated to the movement of the M2 alpha-helical segment. This global symmetrical twist motion of the pentameric protein complex, which resembles the opening transition of other multimeric ion channels, reasonably accounts for the available experimental data and thus likely describes the nAChR gating process.

PMID : 15805177 [PubMed - indexed for MEDLINE]


*4 : *J Biol Chem. 2004 Apr 30 ;279(18):18767-75. Epub 2004 Feb 5.

Related Articles, <http://www.ncbi.nlm.nih.gov/entrez/...> Gene, <http://www.ncbi.nlm.nih.gov/entrez/...> Gene (GeneRIF), <http://www.ncbi.nlm.nih.gov/entrez/...> HomoloGene, <http://www.ncbi.nlm.nih.gov/entrez/...> Nucleotide (RefSeq), <http://www.ncbi.nlm.nih.gov/entrez/...> Compound via MeSH, <http://www.ncbi.nlm.nih.gov/entrez/...> Substance via MeSH, <http://www.ncbi.nlm.nih.gov/entrez/...> Protein (RefSeq), <http://www.ncbi.nlm.nih.gov/entrez/...> UniGene, <http://www.ncbi.nlm.nih.gov/entrez/...> Nucleotide, <http://www.ncbi.nlm.nih.gov/entrez/...> Protein, <http://www.ncbi.nlm.nih.gov/ht%0A%2...> GEO Profiles, <http://www.ncbi.nlm.nih.gov/entrez/...> Cited in PMC, <http://www.pubmedcentral.gov/tocren...> Books, <http://www.ncbi.nlm.nih.gov/entrez/...> LinkOut <http://www.ncbi.nlm.nih.gov/entrez/...>

Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *An extracellular protein microdomain controls up-regulation of neuronal nicotinic acetylcholine receptors by nicotine.*

*Sallette J* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Bohler S* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Benoit P* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Soudant M* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Pons S* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Le Novere N* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Changeux JP* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Corringer PJ* <http://www.ncbi.nlm.nih.gov/entrez/...> .

URA CNRS D2182 Recepteurs et Cognition, Institut Pasteur, 25 ru e du Docteur Roux, 75724 Paris Cedex 15, France.

In smoker’s brain, rodent brain, and in cultured cells expressing nicotinic receptors, chronic nicotine treatment induces an increase in the total number of high affinity receptors for acetylcholine and nicotine, a process referred to as up-regulation. Up-regulation induced by 1 mm nicotine reaches 6-fold for alpha3beta2 nicotinic receptors transiently expressed in HEK 293 cells, whereas it is much smaller for alpha3beta4 receptors, offering a rationale to investigate the molecular mechanism underlying up-regulation. In this expression system binding sites are mainly intracellular, as shown by [(3)H]epibatidine binding experiments and competition with the impermeant ligand carbamylcholine. Systematic analysis of beta2/beta4 chimeras demonstrates the following. (i) The extracellular domain critically contributes to up-regulation. (ii) Only residues belonging to two beta2 segments, 74-89 and 106-115, confer up-regulation to beta4, mainly by decreasing the amount of binding sites in the absence of nicotine ; on an atomic three-dimensional model of the alpha3beta2 receptor these amino acids form a compact microdomain that mainly contributes to the subunit interface and also faces the acetylcholine binding site. (iii) The beta4 microdomain is sufficient to confer to beta2 a beta4-like up-regulation. (iv) This microdomain makes an equivalent contribution to the up-regulation differences between alpha4beta2 and alpha4beta4. We propose that nicotine, by binding to immature oligomers, elicits a conformational reorganization of the microdomain, strengthening the interaction between adjacent subunits and, thus, facilitating maturation processes toward high affinity receptors. This mechanism may be central to nicotine addiction, since alpha4beta2 is the subtype exhibiting the highest degree of up-regulation in the brain.

PMID : 14764595 [PubMed - indexed for MEDLINE]


*5 : *EMBO J. 2003 May 1 ;22(9):1990-2003.

Related Articles, <http://www.ncbi.nlm.nih.gov/entrez/...> Compound via MeSH, <http://www.ncbi.nlm.nih.gov/entrez/...> Substance via MeSH, <http://www.ncbi.nlm.nih.gov/entrez/...> Cited Articles, <http://www.ncbi.nlm.nih.gov/entrez/...> Free in PMC, <http://www.pubmedcentral.gov/articl...> Cited in PMC, <http://www.pubmedcentral.go%0A%20v/...> Books, <http://www.ncbi.nlm.nih.gov/entrez/...> LinkOut <http://www.ncbi.nlm.nih.gov/entrez/...>

Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *An H-bond between two residues from different loops of the acetylcholine binding site contributes to the activation mechanism of nicotinic receptors.*

*Grutter T* <http://www.ncbi.n%0A%20lm.nih.gov/e...> , *Prado de Carvalho L* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Le Novere N* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Corringer PJ* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Edelstein S* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Ch angeux JP* <http://www.ncbi.nlm.nih.gov/entrez/...> .

Institut Pasteur, URA 2182 CNRS ’Recepteurs et Cognition’, Departement des Biotechnologies, 25 rue du Dr Roux, 75724 Paris cedex 15, France, grutter pasteur.fr or changeux pasteur.fr

The molecular mechanisms of nicotinic receptor activation are still largely unknown. The crystallographic structure of the acetylcholine binding protein (AChBP) reveals a single H-bond between two different acetylcholine binding loops. Within these homologous loops we systematically introduced alpha4 residues into the alpha7/5HT(3) chimeric receptor and found that the single point mutations G152K (loop B) and P193I (loop C) displayed a non-additive increase of equilibrium binding affinity for several agonists compared with the double mutant G152K/P193I. In whole-cell patch-clamp recordings, G152K, P193I and G152K/P193I mutants displayed an increase up to 5-fold in acetylcholine potency with a large decrease of the apparent Hill coefficients (significantly smaller than one). Concomitantly, the G152K/P193I mutant showed a dramatic loss of high-affinity alpha-bungarotoxin binding (100-fold decrease), thus pinpointing a new contact area for the toxin. Fitting the data with an allosteric-kinetic model, together with molecular dynamic simulations, suggests that the presence of the inter-backbone H-bond between positions 152 and 193, revealed in alpha4 and in alpha7 double mutant but not in alpha7, coincides with a large stabilization of both open and desensitized states of nicotinic receptors.

PMID : 12727867 [PubMed - indexed for MEDLINE]


*6 : *Eur J Neurosci. 2004 Feb ;19(4):855-62. Related Articles, <http://www.ncbi.nlm.nih.gov/entrez/...> Gene, <http://www.ncbi.nlm.nih.gov/entrez/...> Gene (GeneRIF), <http://www.ncbi.nlm.nih.gov/entrez/...> GENSAT, <http://www.ncbi.nlm.nih.gov/entrez/...> HomoloGene, <http://www.ncbi.nlm.nih.gov/entrez/...> Nucleotide (RefSeq), <http://www.n%20cbi.nlm.nih.gov/entr...> Protein (RefSeq), <http://www.ncbi.nlm.nih.gov/entrez/...> UniGene, <http://www.ncbi.nlm.nih.gov/entrez/...> Nucleotide, <http://www.ncbi.nlm.nih.gov/entrez/...> Protein, <http://www.ncbi.nlm.nih.gov/entrez/...> GEO Profiles, <http://www.ncbi.nlm.nih.gov/entrez/...> Books, <http://www.ncbi.nlm.nih.gov/entrez/...> LinkOut <http://www.ncbi.nlm.nih.gov/entrez/...>

Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *Distinct subcellular targeting of fluorescent nicotinic alpha 3 beta 4 and serotoninergic 5-HT3A receptors in hippocampal neurons.*

*Grailhe R* <http://www.ncbi.nlm.nih.gov/entrez/...> , *de Carvalho LP* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Paas Y* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Le Poupon C* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Soudant M* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Bregestovski P* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Changeux JP* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Corringer PJ* <http://www.ncbi.nlm.nih.gov/entrez/...> .

Recepteurs et Cognition, Unite de recherche associee D1284, CNRS, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, France. regis grailhe.com

The nicotinic acetylcholine receptors (nAChRs) and the 5-HT3 serotonin receptor subtype belong to a superfamily of neurotransmitter-gated ion channels involved in fast synaptic communication throughout the nervous system. Their trafficking to the neuron plasmalemma, as well as their targeting to specific subcellular compartments, is critical for understanding their physiological role. In order to investigate the cellular distribution of these receptors, we tagged the N-termini of alpha3beta4-nAChR subunits and the 5-HT3AR subunit with cyan and yellow fluorescent proteins (CFP, YFP). The fusion subunits were coexpressed in human embryonic kidney (HEK-293) cells, where they assemble into functional receptor channels, as well as in primary cultures of hippocampal neurons. Fluorescence microscopy of living cells revealed that the heteropentameric alpha3CFP-beta4 and YFP-alpha3beta4 receptors are mainly distributed in the endoplasmic reticulum, while the homopentameric YFP-5-HT3A receptor was localized both to the plasma membrane and within intracellular compartments. Moreover, the YFP-5-HT3A receptor was found to be targeted to the micropodia in HEK-293 cells and to the dendritic spines in hippocampal neurons, where it could be accessed by extracellularly applied specific fluorescent probes. The efficient targeting of the YFP-5-HT3A to the cytoplasmic membrane is in line with the large serotonin-elicited currents (nA range) measured by whole-cell voltage-clamp recordings in transfected HEK-293 cells. In contrast, alpha3beta4-nAChRs expressed in the same cells yielded weaker ACh-evoked responses. Taken together, the fluorescent and electrophysiological studies presented here demonstrate the predominant intracellular location of alpha3beta4-nACh receptors and the predominant expression of the 5-HT3AR in dendritic surface loci.

PMID : 15009132 [PubMed - indexed for MEDLINE]

Bonjour, Voici la biblio de l’intervenant du Lundi 2 octobre le docteur Pierre-Jean Corringer. Celle-ci a été validée par Pascale Marchot Bonne lecture,

Christine Estève tel : 04-91-69-89-52

Docteur Pierre-Jean CORRINGER

Institut Pasteur, Paris.

“ Première identification d’un canal bactérien pH-sensible de la famille des récepteurs nicotiniques de l’acétylcholine.”

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1 : J Physiol Paris. 2006 Mar-May ;99(2-3):162-71. Epub 2006 Feb 3. Related Articles, Compound via MeSH, Substance via MeSH, Books, LinkOut

Nicotine enhances intracellular nicotinic receptor maturation : a novel mechanism of neural plasticity ?

Corringer PJ, Sallette J, Changeux JP.

Unit of receptor and Cognition, Pasteur Institute, 25 rue du docteur Roux, 75724 Paris Cedex 15, France. pjcorrin pasteur.fr

Nicotine addiction, the primary cause of tobacco consumption, is mediated through nicotine binding to brain nicotinic acetylcholine receptor (nAChRs). Upon chronic exposure, nicotine elicits a cascade of events, starting with nAChR activation and desensitization, followed by a long term up-regulation that corresponds to an increase in the number of the high affinity nAChRs, a paradoxical process that occurs in the brain of smokers. Recent investigation of the maturation and trafficking of the major brain alpha4beta2 nAChR demonstrates that up-regulation is initiated in the endoplasmic reticulum soon after protein translation. The data thus far accumulated provide evidence that nicotine elicits up-regulation by promoting maturation of nAChR precursors that would otherwise be degraded. This "maturational enhancer" action of nicotine probably contributes to the long term effect of chronic nicotine, and suggests a novel mechanism of neuronal plasticity through an yet unknown endogenous substance which would modulate the receptor expression under physiological conditions.

Publication Types :

· Review

PMID : 16458492 [PubMed - indexed for MEDLINE]


2 : Neuron. 2005 May 19 ;46(4):595-607. Related Articles, Gene, Gene (GeneRIF), Nucleotide (RefSeq), Compound via MeSH, < a CLASS="dblinks" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=pubmed_Abstract&db=pubmed&cmd=Display&dopt=pubmed_pcsubstance_mesh&from_uid=15944128">Substance via MeSH, Protein (RefSeq), UniGene, Nucleotide, Protein, GEO Profiles, Books, LinkOut

Nicotine upregulates its own receptors through enhanced intracellular maturation.

Sallette J, Pons S, Devillers-Thiery A, Soudant M, Prado de Carvalho L, Changeux JP, Corringer PJ.

CNRS URA D2182 Recepteurs et Cognition, Institut Pasteur, Paris, France.

Chronic exposure to nicotine elicits upregulation of high-affinity nicotinic receptors in the smoker’s brain. To address the molecular mechanism of upregulation, we transfected HEK293 cells with human alpha4beta2 receptors and traced the subunits throughout their intracellular biosynthesis, using metabolic labeling and immunoprecipitation techniques. We show that high-mannose glycosylated subunits mature and assemble into pentamers in the endoplasmic reticulum and that only pentameric receptors reach the cell surface following carbohydrate processing. Nicotine is shown to act inside the cell and to increase the amount of beta subunits immunoprecipitated by the conformation-dependent mAb290, indicating that nicotine enhances a critical step in the intracellular maturation of these receptors. This effect, which also takes place at concentrations of nicotine found in the blood of smokers upon expression of alpha4beta2 in SH-SY5Y neuroblastoma cells, may play a crucial role in nicotine addiction and possibly implement a model of neural plasticity.

PMID : 15944128 [PubMed - indexed for MEDLINE]


3 : Biophys J. 2005 Jun ;88(6):3954-65. Epub 2005 Apr 1. Related Articles, Cited Articles, Free in PMC, Cited in PMC, Books, LinkOut

Normal mode analysis suggests a quaternary twist model for the nicotinic receptor gating mechanism.

Taly A, Delarue M, Grutter T, Nilges M, Le Novere N, Corringer PJ, Changeux JP.

Recepteurs et Cognition, Unite de Recherche Associee (URA) Centre National de la Recherche Scientifique 2182, Institut Pasteur, Paris, France.

We present a three-dimensional model of the homopentameric alpha7 nicotinic acetylcholine receptor (nAChR), that includes the extracellular and membrane domains, developed by comparative modeling on the basis of : 1), the x-ray crystal structure of the snail acetylcholine binding protein, an homolog of the extracellular domain of nAChRs ; and 2), cryo-electron microscopy data of the membrane domain collected on Torpedo marmorata nAChRs. We performed normal mode analysis on the complete three-dimensional model to explore protein flexibility. Among the first 10 lowest frequency modes, only the first mode produces a structural reorganization compatible with channel gating : a wide opening of the channel pore caused by a concerted symmetrical quaternary twist motion of the protein with opposing rotations of the upper (extracellular) and lower (transmembrane) domains. Still, significant reorganizations are observed within each subunit, that involve their bending at the domain interface, an increase of angle between the two beta-sheets composing the extracellular domain, the internal beta-sheet being significantly correlated to the movement of the M2 alpha-helical segment. This global symmetrical twist motion of the pentameric protein complex, which resembles the opening transition of other multimeric ion channels, reasonably accounts for the available experimental data and thus likely describes the nAChR gating process.

PMID : 15805177 [PubMed - indexed for MEDLINE]


4 : J Biol Chem. 2004 Apr 30 ;279(18):18767-75. Epub 2004 Feb 5. Related Articles, Gene, Gene (GeneRIF), HomoloGene, Nucleotide (RefSeq), Compound via MeSH, Substance via MeSH, Protein (RefSeq), UniGene, Nucleotide, Protein, GEO Profiles, Cited in PMC, Books, LinkOut

An extracellular protein microdomain controls up-regulation of neuronal nicotinic acetylcholine receptors by nicotine.

Sallette J, Bohler S, Benoit P, Soudant M, Pons S, Le Novere N, Changeux JP, Corringer PJ.

URA CNRS D2182 Recepteurs et Cognition, Institut Pasteur, 25 ru e du Docteur Roux, 75724 Paris Cedex 15, France.

In smoker’s brain, rodent brain, and in cultured cells expressing nicotinic receptors, chronic nicotine treatment induces an increase in the total number of high affinity receptors for acetylcholine and nicotine, a process referred to as up-regulation. Up-regulation induced by 1 mm nicotine reaches 6-fold for alpha3beta2 nicotinic receptors transiently expressed in HEK 293 cells, whereas it is much smaller for alpha3beta4 receptors, offering a rationale to investigate the molecular mechanism underlying up-regulation. In this expression system binding sites are mainly intracellular, as shown by [(3)H]epibatidine binding experiments and competition with the impermeant ligand carbamylcholine. Systematic analysis of beta2/beta4 chimeras demonstrates the following. (i) The extracellular domain critically contributes to up-regulation. (ii) Only residues belonging to two beta2 segments, 74-89 and 106-115, confer up-regulation to beta4, mainly by decreasing the amount of binding sites in the absence of nicotine ; on an atomic three-dimensional model of the alpha3beta2 receptor these amino acids form a compact microdomain that mainly contributes to the subunit interface and also faces the acetylcholine binding site. (iii) The beta4 microdomain is sufficient to confer to beta2 a beta4-like up-regulation. (iv) This microdomain makes an equivalent contribution to the up-regulation differences between alpha4beta2 and alpha4beta4. We propose that nicotine, by binding to immature oligomers, elicits a conformational reorganization of the microdomain, strengthening the interaction between adjacent subunits and, thus, facilitating maturation processes toward high affinity receptors. This mechanism may be central to nicotine addiction, since alpha4beta2 is the subtype exhibiting the highest degree of up-regulation in the brain.

PMID : 14764595 [PubMed - indexed for MEDLINE]


5 : EMBO J. 2003 May 1 ;22(9):1990-2003. Related Articles, Compound via MeSH, Substance via MeSH, Cited Articles, Free in PMC, Cited in PMC, Books, LinkOut

An H-bond between two residues from different loops of the acetylcholine binding site contributes to the activation mechanism of nicotinic receptors.

Grutter T, Prado de Carvalho L, Le Novere N, Corringer PJ, Edelstein S, Ch angeux JP.

Institut Pasteur, URA 2182 CNRS ’Recepteurs et Cognition’, Departement des Biotechnologies, 25 rue du Dr Roux, 75724 Paris cedex 15, France, grutter pasteur.fr or changeux pasteur.fr

The molecular mechanisms of nicotinic receptor activation are still largely unknown. The crystallographic structure of the acetylcholine binding protein (AChBP) reveals a single H-bond between two different acetylcholine binding loops. Within these homologous loops we systematically introduced alpha4 residues into the alpha7/5HT(3) chimeric receptor and found that the single point mutations G152K (loop B) and P193I (loop C) displayed a non-additive increase of equilibrium binding affinity for several agonists compared with the double mutant G152K/P193I. In whole-cell patch-clamp recordings, G152K, P193I and G152K/P193I mutants displayed an increase up to 5-fold in acetylcholine potency with a large decrease of the apparent Hill coefficients (significantly smaller than one). Concomitantly, the G152K/P193I mutant showed a dramatic loss of high-affinity alpha-bungarotoxin binding (100-fold decrease), thus pinpointing a new contact area for the toxin. Fitting the data with an allosteric-kinetic model, together with molecular dynamic simulations, suggests that the presence of the inter-backbone H-bond between positions 152 and 193, revealed in alpha4 and in alpha7 double mutant but not in alpha7, coincides with a large stabilization of both open and desensitized states of nicotinic receptors.

PMID : 12727867 [PubMed - indexed for MEDLINE]


6 : Eur J Neurosci. 2004 Feb ;19(4):855-62. Related Articles, Gene, Gene (GeneRIF), GENSAT, HomoloGene, Nucleotide (RefSeq), Protein (RefSeq), UniGene, Nucleotide, Protein, GEO Profiles, Books, LinkOut

Distinct subcellular targeting of fluorescent nicotinic alpha 3 beta 4 and serotoninergic 5-HT3A receptors in hippocampal neurons.

Grailhe R, de Carvalho LP, Paas Y, Le Poupon C, Soudant M, Bregestovski P, Changeux JP, Corringer PJ.

Recepteurs et Cognition, Unite de recherche associee D1284, CNRS, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, France. regis grailhe.com

The nicotinic acetylcholine receptors (nAChRs) and the 5-HT3 serotonin receptor subtype belong to a superfamily of neurotransmitter-gated ion channels involved in fast synaptic communication throughout the nervous system. Their trafficking to the neuron plasmalemma, as well as their targeting to specific subcellular compartments, is critical for understanding their physiological role. In order to investigate the cellular distribution of these receptors, we tagged the N-termini of alpha3beta4-nAChR subunits and the 5-HT3AR subunit with cyan and yellow fluorescent proteins (CFP, YFP). The fusion subunits were coexpressed in human embryonic kidney (HEK-293) cells, where they assemble into functional receptor channels, as well as in primary cultures of hippocampal neurons. Fluorescence microscopy of living cells revealed that the heteropentameric alpha3CFP-beta4 and YFP-alpha3beta4 receptors are mainly distributed in the endoplasmic reticulum, while the homopentameric YFP-5-HT3A receptor was localized both to the plasma membrane and within intracellular compartments. Moreover, the YFP-5-HT3A receptor was found to be targeted to the micropodia in HEK-293 cells and to the dendritic spines in hippocampal neurons, where it could be accessed by extracellularly applied specific fluorescent probes. The efficient targeting of the YFP-5-HT3A to the cytoplasmic membrane is in line with the large serotonin-elicited currents (nA range) measured by whole-cell voltage-clamp recordings in transfected HEK-293 cells. In contrast, alpha3beta4-nAChRs expressed in the same cells yielded weaker ACh-evoked responses. Taken together, the fluorescent and electrophysiological studies presented here demonstrate the predominant intracellular location of alpha3beta4-nACh receptors and the predominant expression of the 5-HT3AR in dendritic surface loci.

PMID : 15009132 [PubMed - indexed for MEDLINE]

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