Nicotine Upregulates Its Own Receptors through Enhanced Intracellular Maturation

and Pierre Jean Corringer * The neuronal nAChRs subject to upregulation are CNRS URA D2182 Récepteurs et Cognition pseudosymmetrical heteropentameric ion channels re-Institut Pasteur sulting from the assembly of principal (α2, α3, α4, α6) 25 rue du Dr. Roux and complementary (β2, β4) subunits. At the concentra-75724 Paris Cedex 15 tions of nicotine found in the brain of smokers (low France micromolar range), the highly expressed α4β2 receptors display the highest level of upregulation (Nguyen et al., 2003). Since receptors containing the β2 subunits are the most important for nicotine self-administration Summary in mice (Picciotto et al., 1998), it is likely that their upregulation contributes to the long-term effect of Chronic exposure to nicotine elicits upregulation of chronic nicotine and potentially to nicotine addiction high-affinity nicotinic receptors in the smoker’s brain

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

International audienceIn 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

Nicotine upregulates its own receptors through enhanced intracellular maturation.

International audienceChronic 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

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

International audienceThe 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

OBSLAG 2018 - volet eutrophisation. Lagunes méditerranéennes (période 2013-2018). Etat DCE de la colonne d'eau et du phytoplancton, tendance et variabilité des indicateurs

Cette étude est réalisée dans le cadre du projet OBSLAG (OBServatoire des LAGunes) et porte sur les 10 masses d’eau lagunaires méditerranéennes situées à l’aval des cours d’eau suivis depuis 2015 par le « réseau flux » de l'Agence de l’Eau Rhône Méditerranée Corse. Le rapport correspond aux actions réalisées en 2018 dans le cadre du volet « eutrophisation » d’OBSLAG, répondant aux objectifs suivants : (1) réaliser un suivi estival en 2018 du phytoplancton et de la physico-chimie de l’eau et évaluer l’état DCE de ces compartiments pour la période 2013-2018 sur les 10 masses d’eau lagunaires du suivi « réseau flux », regroupant 13 lagunes poly-euhalines. Ce suivi permet de renforcer la fréquence des suivis DCE sur la colonne d’eau et le phytoplancton, qui ne sont réalisés qu’un été sur deux depuis 2015 ; (2) appliquer des indicateurs plus sensibles que les indicateurs DCE, mettant en évidence, sur les périodes d’évaluation, les tendances d’évolution et la variabilité de l’état du phytoplancton et de la colonne d’eau, compartiments les plus réactifs des écosystèmes lagunaires. Les suivis réalisés au cours de l’été 2018 sur les 10 masses d’eau lagunaires ont montré que le bon état DCE est atteint pour la période 2013-2018 pour 7 masses d’eau en ce qui concerne les paramètres physico-chimiques de la colonne d’eau et pour 4 masses d’eau en ce qui concerne le compartiment phytoplancton. Quatre masses d’eau atteignent le bon état pour les deux compartiments colonne d’eau et phytoplancton (La Palme, Bages-Sigean, Ayrolle et Thau). L’application de méthodes statistiques adaptées (bootstrap, tests de Mann Kendall et de Kruskal-Wallis) sur les données acquises de 2013 à 2018 ont permis : · d’évaluer la confiance accompagnant les diagnostics DCE des masses d’eau, · de mettre en évidence une tendance à l’augmentation des concentrations en nutriments sur 4 masses d’eau et une tendance à la diminution sur deux masses d’eau, · de mettre en évidence une tendance à l’augmentation des abondances de phytoplancton sur une masse d’eau et une tendance à la diminution sur une masse d’eau. Les indicateurs fournis permettent ainsi de détecter des changements de façon précoce et d’attirer l’attention sur des nouveaux signes de dégradation ou de restauration de l’état des lagunes.