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


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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    • "Significantly, engineering aromatic interactions into the M4-M1/M3 interface in ELIC abrogates the potentiating response. Finally, the strength of M4-M1/M3 interactions has no effect on the inhibitory effects of the drug propofol, which acts at a TMD site that does not directly involve M4 (LeBard et al., 2012; Nury et al., 2011). Our proposed model of M4 action is supported by biophysical studies, which have shown that the orientation of nAChR M4, and thus presumably the interactions between M4 and M1/ M3, is sensitive to its surrounding lipid environment (Antollini et al., 2005; Xu et al., 2005). "
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    ABSTRACT: The gating of pentameric ligand-gated ion channels is sensitive to a variety of allosteric modulators that act on structures peripheral to those involved in the allosteric pathway leading from the agonist site to the channel gate. One such structure, the lipid-exposed transmembrane α helix, M4, is the target of lipids, neurosteroids, and disease-causing mutations. Here we show that M4 interactions with the adjacent transmembrane α helices, M1 and M3, modulate pLGIC function. Enhanced M4 interactions promote channel function while ineffective interactions reduce channel function. The interface chemistry governs the intrinsic strength of M4-M1/M3 inter-helical interactions, both influencing channel gating and imparting distinct susceptibilities to the potentiating effects of a lipid-facing M4 congenital myasthenic syndrome mutation. Through aromatic substitutions, functional studies, and molecular dynamics simulations, we elucidate a mechanism by which M4 modulates channel function. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Structure 07/2015; 23(9). DOI:10.1016/j.str.2015.06.020 · 5.62 Impact Factor
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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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