Structural Basis for Potentiation by Alcohols and Anaesthetics in a Ligand-gated Ion Channel

Ethanol alters nerve signalling by interacting with proteins in the central nervous system, particularly pentameric ligand-gated ion channels. A recent series of mutagenesis experiments on Gloeobacter violaceus ligand-gated ion channel, a prokaryotic member of this family, identified a single-site variant that is potentiated by pharmacologically relevant concentrations of ethanol. Here we determine crystal structures of the ethanol-sensitized variant in the absence and presence of ethanol and related modulators, which bind in a transmembrane cavity between channel subunits and may stabilize the open form of the channel. Structural and mutagenesis studies defined overlapping mechanisms of potentiation by alcohols and anaesthetics via the inter-subunit cavity. Furthermore, homology modelling show this cavity to be conserved in human ethanol-sensitive glycine and GABA(A) receptors, and to involve residues previously shown to influence alcohol and anaesthetic action on these proteins. These results suggest a common structural basis for ethanol potentiation of an important class of targets for neurological actions of ethanol

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

International audienceNicotine 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

The dual-gate model for pentameric ligand-gated ion channels activation and desensitization

International audiencePentameric ligand-gated ion channels (pLGICs) mediate fast neurotransmission in the nervous system. Their dysfunction is associated with psychiatric, neurological and neurodegenerative disorders such as schizophrenia, epilepsy and Alzheimer's disease. Understanding their biophysical and pharmacological properties, both at the functional and structural levels, thus holds many therapeutic promises. In addition to their agonist-elicited activation, most pLGICs display another key allosteric property, namely desensitization, in which they enter a shut state refractory to activation upon sustained agonist binding. While the activation mechanisms of several pLGICs have been revealed at near-atomic resolution, the structural foundation of desensitization has long remained elusive. Recent structural and functional data now suggest that the activation and desensitization gates are distinct, and are located at both sides of the ion channel. Such a "dual gate mechanism" accounts for the marked allosteric effects of channel blockers, a feature illustrated herein by theoretical kinetics simulations. Comparison with other classes of ligand- and voltage-gated ion channels show that this dual gate mechanism emerges as a common theme for the desensitization and inactivation properties of structurally unrelated ion channels. This article is protected by copyright. All rights reserved

The diversity of subunit composition in nAChRs: Evolutionary origins, physiologic and pharmacologic consequences

International audienceNicotinic acetylcholine receptors are made up of homologous subunits, which are encoded by a large multigene family. The wide number of receptor oligomers generated display variable pharmacological properties. One of the main questions underlying research in molecular pharmacology resides in the actual role of this diversity. It is generally assumed that the observed differences between the pharmacology of homologous receptors, for instance, the EC(50) for the endogenous agonist, or the kinetics of desensitization, bear some kind of physiologic relevance in vivo. Here we develop the quite challenging point of view that, at least within a given subfamily of nicotinic receptor subunits, the pharmacologic variability observed in vitro would not be directly relevant to the function of receptor proteins in vivo. In vivo responses are not expected to be sensitive to mild differences in affinities, and several examples of functional replacement of one subunit by another have been unravelled by knockout animals. The diversity of subunits might have been conserved through evolution primarily to account for the topologic diversity of subunit distribution patterns, at the cellular and subcellular levels. A quantitative variation of pharmacological properties would be tolerated within a physiologic envelope, as a consequence of a near-neutral genetic drift. Such a "gratuitous" pharmacologic diversity is nevertheless of practical interest for the design of drugs, which would specifically tackle particular receptor oligomers with a defined subunit composition among the multiple nicotinic receptors present in the organism

Structure atomique et transitions allostériques des récepteurs canaux pentamériques (apport de nouveaux homologues bactériens)

La superfamille des récepteurs canaux pentamériques activables par un ligand (pLGIC) comprend les récepteurs nicotiniques de l acétylcholine (nAChR) et de l acide g-amino butyrique (GABA). Ces protéines membranaires assurent la transduction chimio-électrique rapide du signal, jouant ainsi un rôle primordial dans les communications neuronales et sont la cible de nombreuses classes de médicaments. Dans cette thèse nous avons exploité de nouveaux homologues procaryotes de ces récepteurs afin d'obtenir une structure atomique par diffraction des rayons X.Dans un premier temps, nous avons caractérisé fonctionnellement et biochimiquement la protéine GLIC, l'homologue de la cyanobactérie Gloeobacter violaceus. GLIC s exprime à la membrane des cellules eucaryotes et procaryotes sous forme d homo-pentamère, et forme des canaux cationiques activés par les protons. Dans un deuxième temps, suite à l'optimisation de la sur-expression en système hétérologue et de la purification en détergent de GLIC, des cristaux ont été générés à pH acide afin de capter une conformation ouverte du canal. La structure de GLIC à 2,9A révèle un canal ionique ouvert en forme d'entonnoir. En comparant la structure d'ELIC, un autre homologue cristallisé dans la forme fermée, nous avons pu mettre en évidence un mécanisme d'ouverture du canal impliquant des mouvements quaternaires globaux de la protéine ainsi que des déformations tertiaires à l'interface entre domaine agoniste et partie membranaire. Ces travaux ont donc contribué à la découverte d une nouvelle famille de pLGICs d origine bactérienne ainsi qu'à l'étude au niveau atomique de leurs transitions allostériques.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Nicotinic Receptors at the Amino Acid Level

International audiencenAChRs are pentameric transmembrane proteins into the superfamily of ligand-gated ion channels that includes the 5HT3, glycine, GABAA, and GABAC receptors. Electron microscopy, affinity labeling, and mutagenesis experiments, together with secondary structure predictions and measurements, suggest an all-beta folding of the N-terminal extracellular domain, with the connecting loops contributing to the ACh binding pocket and to the subunit interfaces that mediate the allosteric transitions between conformational states. The ion channel consists of two distinct elements symmetrically organized along the fivefold axis of the molecule: a barrel of five M2 helices, and on the cytoplasmic side five loops contributing to the selectivity filter. The allosteric transitions of the protein underlying the physiological ACh-evoked activation and desensitization possibly involve rigid body motion of the extracellular domain of each subunit, linked to a global reorganization of the transmembrane domain responsible for channel gating

A novel mechanism of neural plasticity?

Nicotine enhances intracellular nicotinic receptor maturation

Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system

International audienceNicotinic receptors - a family of ligand-gated ion channels that mediate the effects of the neurotransmitter acetylcholine - are among the most well understood allosteric membrane proteins from a structural and functional perspective. There is also considerable interest in modulating nicotinic receptors to treat nervous-system disorders such as Alzheimer's disease, schizophrenia, depression, attention deficit hyperactivity disorder and tobacco addiction. This article describes both recent advances in our understanding of the assembly, activity and conformational transitions of nicotinic receptors, as well as developments in the therapeutic application of nicotinic receptor ligands, with the aim of aiding novel drug discovery by bridging the gap between these two rapidly developing fields