X-Ray Structures of General Anaesthetics Bound to a Pentameric Ligand-Gated Ion Channel

Institut Pasteur, Groupe Récepteurs-Canaux, F-75015 Paris, France.
Nature (Impact Factor: 41.46). 01/2011; 469(7330):428-31. DOI: 10.1038/nature09647
Source: PubMed

ABSTRACT General anaesthetics have enjoyed long and widespread use but their molecular mechanism of action remains poorly understood. There is good evidence that their principal targets are pentameric ligand-gated ion channels (pLGICs) such as inhibitory GABA(A) (γ-aminobutyric acid) receptors and excitatory nicotinic acetylcholine receptors, which are respectively potentiated and inhibited by general anaesthetics. The bacterial homologue from Gloeobacter violaceus (GLIC), whose X-ray structure was recently solved, is also sensitive to clinical concentrations of general anaesthetics. Here we describe the crystal structures of the complexes propofol/GLIC and desflurane/GLIC. These reveal a common general-anaesthetic binding site, which pre-exists in the apo-structure in the upper part of the transmembrane domain of each protomer. Both molecules establish van der Waals interactions with the protein; propofol binds at the entrance of the cavity whereas the smaller, more flexible, desflurane binds deeper inside. Mutations of some amino acids lining the binding site profoundly alter the ionic response of GLIC to protons, and affect its general-anaesthetic pharmacology. Molecular dynamics simulations, performed on the wild type (WT) and two GLIC mutants, highlight differences in mobility of propofol in its binding site and help to explain these effects. These data provide a novel structural framework for the design of general anaesthetics and of allosteric modulators of brain pLGICs.

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Available from: Jean-Pierre Changeux, Sep 27, 2015
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    • "Current evidence suggests that some of these anesthetics act, in part, via binding at different regions within the TM domain or the central pore region of the receptors (Cummins, 2007). For example, ethanol and volatile anesthetics (isoflurane, and its structural isomer, enflurane) are reported to share overlapping sites of action in the TM2 and TM3 domain that are distinctly different from the site of action of the intravenous anesthetic, propofol (Mascia et al., 1996; Mihic et al., 1997; Krasowski et al., 1998, 2001; Nury et al., 2011; Sauguet et al., 2013). Mutations in the TM domain of GABA A Rs at positions 270 or 291 in a2, or at positions 265 or 286 in b2 rendered these receptors insensitive to isoflurane, while preserving receptor sensitivity to propofol (Krasowski et al., 1998). "
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    ABSTRACT: We recently developed Ultra-Sensitive Ethanol Receptors (USERs) as a novel tool for investigation of single receptor subunit populations sensitized to extremely low ethanol concentrations that do not affect other receptors in the nervous system. To this end, we found that mutations within the extracellular Loop 2 region of glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABAARs) can significantly increase receptor sensitivity to micro-molar concentrations of ethanol resulting in up to a 100-fold increase in ethanol sensitivity relative to wild type (WT) receptors. The current study investigated: 1) Whether structural manipulations of Loop 2 in α1 GlyRs could similarly increase receptor sensitivity to other anesthetics; and 2) If mutations exclusive to the C-terminal end of Loop 2 are sufficient to impart these changes. We expressed α1 GlyR USERs in Xenopus oocytes and tested the effects of three classes of anesthetics, isoflurane (volatile), propofol (intravenous), and lidocaine (local), known to enhance glycine-induced chloride currents using two-electrode voltage clamp electrophysiology. Loop 2 mutations produced a significant 10-fold increase in isoflurane and lidocaine sensitivity, but no increase in propofol sensitivity compared to WT α1 GlyRs. Interestingly, we also found that structural manipulations in the C-terminal end of Loop 2 were sufficient and selective for α1 GlyR modulation by ethanol, isoflurane, and lidocaine. These studies are the first to report the extracellular region of α1 GlyRs as a site of lidocaine action. Overall, the findings suggest that Loop 2 of α1 GlyRs is a key region that mediates isoflurane and lidocaine modulation. Moreover, the results identify important amino acids in Loop 2 that regulate isoflurane, lidocaine, and ethanol action. Collectively, these data indicate the commonality of the sites for isoflurane, lidocaine, and ethanol action, and the structural requirements for allosteric modulation on α1 GlyRs within the extracellular Loop 2 region. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
    Neuroscience 03/2015; 297. DOI:10.1016/j.neuroscience.2015.03.034 · 3.36 Impact Factor
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    • "Similarly, a transmembrane binding site for propofol has been identified in the bacterial pentameric Gloeobacter violaceus ligand-gated ion channel by means of X-ray crystallography (Nury et al., 2011). Electrophysiological studies with a variety of pentameric ligand-gated ion channels provide additional evidence that propofol interacts via a transmembrane site (Ghosh et al., 2013; Lynagh and Laube, 2014). "
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    ABSTRACT: In common with other members of the Cys-loop family of pentameric ligand-gated ion channels, 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are activated by the binding of a neurotransmitter to an extracellular orthosteric site, located at the interface of two adjacent receptor subunits. In addition, a variety of compounds have been identified that modulate agonistevoked responses of 5-HT3Rs, and other Cys-loop receptors, by binding to distinct allosteric sites. In this study, we examined the pharmacological effects of a group of monoterpene compounds on recombinant 5-HT3Rs expressed in Xenopus oocytes. Two phenolic monoterpenes (carvacrol and thymol) display allosteric agonist activity on human homomeric 5-HT3ARs (64 6 7% and 80 6 4% of the maximum response evoked by the endogenous orthosteric agonist 5-HT, respectively). In addition, at lower concentrations, where agonist effects are less apparent, carvacrol and thymol act as potentiators of responses evoked by submaximal concentrations of 5-HT. By contrast, carvacrol and thymol have no agonist or potentiating activity on the closely related mouse 5-HT3ARs. Using subunit chimeras containing regions of the human and mouse 5-HT3A subunits, and by use of site-directed mutagenesis, we have identified transmembrane amino acids that either abolish the agonist activity of carvacrol and thymol on human 5-HT3ARs or are able to confer this property on mouse 5-HT3ARs. By contrast, these mutations have no significant effect on orthosteric activation of 5-HT3ARs by 5-HT. We conclude that 5-HT3ARs can be activated by the binding of ligands to an allosteric transmembrane site, a conclusion that is supported by computer docking studies.
    Molecular pharmacology 01/2015; 87:87 - 95. DOI:10.1124/mol.114.094540 · 4.13 Impact Factor
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    • "A definitive proof of an allosteric mechanism of action of a ligand is to show by protein crystallography or a similar high-resolution approach that its binding site is spatially distinct and nonoverlapping from that of the orthosteric site(s), ideally by solving the ternary complex or, more likely, as two separate costructure determinations. Such evidence has been provided in the field of enzymology, where allosteric ligand binding sites have been identified on HIV-1 reverse transcriptase , p38 MAP kinase, and glucokinase by crystallography (Hardy and Wells, 2004) and, more recently, for LGICs (Nury et al., 2011; Sauguet et al., 2013) and for GPCRs (Kruse et al., 2013), as described above (sections IV.A, IV.B, and IV.D). "
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    ABSTRACT: Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties.
    Pharmacological reviews 10/2014; 66(4):918-47. DOI:10.1124/pr.114.008862 · 17.10 Impact Factor
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