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Raimund Dutzler - One of the best experts on this subject based on the ideXlab platform.

  • inhibitIon of the prokaryotic pentameric ligand gated Ion Channel elic by divalent catIons
    PLOS Biology, 2012
    Co-Authors: Iwan Zimmermann, Alessandro Marabelli, Carlo Bertozzi, Lucia G Sivilotti, Raimund Dutzler
    Abstract:

    The modulatIon of pentameric Ligand-Gated Ion Channels (pLGICs) by divalent catIons is believed to play an important role in their regulatIon in a physiological context. Ions such as calcium or zinc influence the activity of pLGIC neurotransmitter receptors by binding to their extracellular domain and either potentiate or inhibit Channel activatIon. Here we have investigated by electrophysiology and X-ray crystallography the effect of divalent Ions on ELIC, a close prokaryotic pLGIC homologue of known structure. We found that divalent catIons inhibit the activatIon of ELIC by the agonist cysteamine, reducing both its potency and, at higher concentratIons, its maximum response. Crystal structures of the Channel in complex with barium reveal the presence of several distinct binding sites. By mutagenesis we confirmed that the site responsible for divalent inhibitIon is located at the outer rim of the extracellular domain, at the interface between adjacent subunits but at some distance from the agonist binding regIon. Here, divalent catIons interact with the protein via carboxylate side-chains, and the site is similar in structure to calcium binding sites described in other proteins. There is evidence that other pLGICs may be regulated by divalent Ions binding to a similar regIon, even though the interacting residues are not conserved within the family. Our study provides structural and functIonal insight into the allosteric regulatIon of ELIC and is of potential relevance for the entire family.

  • structural basis of open Channel block in a prokaryotic pentameric ligand gated Ion Channel
    Nature Structural & Molecular Biology, 2010
    Co-Authors: Dirk Trauner, Ricarda J C Hilf, Iwan Zimmermann, Carlo Bertozzi, Alwin Reiter, Raimund Dutzler
    Abstract:

    Open Channel blockers are very different in their chemistry and structure. Now GLIC, a bacterial model for pentameric Ligand-Gated Channels, is used to investigate the structural basis of open Channel block. The work identifies two extended interactIon sites, with large blockers binding to the center of the membrane and small blockers binding to the narrow intracellular regIon.

  • structure of a potentially open state of a proton activated pentameric ligand gated Ion Channel
    Nature, 2009
    Co-Authors: Ricarda J C Hilf, Raimund Dutzler
    Abstract:

    The X-ray structure of a pentameric Ligand-Gated Ion Channel from Erwinia chrysanthemi (ELIC) has recently provided structural insight into this family of Ion Channels at high resolutIon. The structure shows a homo-pentameric protein with a barrel-stave architecture that defines an Ion-conductIon pore located on the fivefold axis of symmetry. In this structure, the wide aqueous vestibule that is encircled by the extracellular ligand-binding domains of the five subunits narrows to a discontinuous pore that spans the lipid bilayer. The pore is constricted by bulky hydrophobic residues towards the extracellular side, which probably serve as barriers that prevent the diffusIon of Ions. This interrupted pore architecture in ELIC thus depicts a non-conducting conformatIon of a pentameric Ligand-Gated Ion Channel, the thermodynamically stable state in the absence of bound ligand. As ligand binding promotes pore opening in these Ion Channels and the specific ligand for ELIC has not yet been identified, we have turned our attentIon towards a homologous protein from the cyanobacterium Gloebacter violaceus (GLIC). GLIC was shown to form proton-gated Channels that are activated by a pH decrease on the extracellular side and that do not desensitize after activatIon. Both prokaryotic proteins, ELIC and GLIC form Ion Channels that are selective for catIons over anIons with poor discriminatIon among monovalent catIons, characteristics that resemble the conductIon properties of the catIon-selective branch of the family that includes acetylcholine and serotonin receptors. Here we present the X-ray structure of GLIC at 3.1 A resolutIon. The structure reveals a conformatIon of the Channel that is distinct from ELIC and that probably resembles the open state. In combinatIon, both structures suggest a novel gating mechanism for pentameric Ligand-Gated Ion Channels where Channel opening proceeds by a change in the tilt of the pore-forming helices.

  • structure of a potentially open state of a proton activated pentameric ligand gated Ion Channel
    Nature, 2009
    Co-Authors: Ricarda J C Hilf, Raimund Dutzler
    Abstract:

    Recently, the first crystal structure of a pentameric Ligand-Gated Ion Channel known as GLIC was published, which represented a closed state of the Channel. Two papers in this issue report the crystal structures of the presumptive open states of a related Channel — ELIC —and show significant tilting of the M2 and M3 α-helices from the closed state. Recently, the first crystal structure of a pentameric Ligand-Gated Ion Channel known as GLIC was published, which represented a closed state of the Channel. In two papers in this issue, presumptive open states of a related Channel — ELIC — have been crystallized and show significant tilting of the M2 and M3 α-helices from the closed state. The X-ray structure of a pentameric Ligand-Gated Ion Channel from Erwinia chrysanthemi (ELIC) has recently provided structural insight into this family of Ion Channels at high resolutIon1. The structure shows a homo-pentameric protein with a barrel-stave architecture that defines an Ion-conductIon pore located on the fivefold axis of symmetry. In this structure, the wide aqueous vestibule that is encircled by the extracellular ligand-binding domains of the five subunits narrows to a discontinuous pore that spans the lipid bilayer. The pore is constricted by bulky hydrophobic residues towards the extracellular side, which probably serve as barriers that prevent the diffusIon of Ions. This interrupted pore architecture in ELIC thus depicts a non-conducting conformatIon of a pentameric Ligand-Gated Ion Channel, the thermodynamically stable state in the absence of bound ligand. As ligand binding promotes pore opening in these Ion Channels and the specific ligand for ELIC has not yet been identified, we have turned our attentIon towards a homologous protein from the cyanobacterium Gloebacter violaceus (GLIC). GLIC was shown to form proton-gated Channels that are activated by a pH decrease on the extracellular side and that do not desensitize after activatIon2. Both prokaryotic proteins, ELIC and GLIC form Ion Channels that are selective for catIons over anIons with poor discriminatIon among monovalent catIons1,2, characteristics that resemble the conductIon properties of the catIon-selective branch of the family that includes acetylcholine and serotonin receptors3,4. Here we present the X-ray structure of GLIC at 3.1 A resolutIon. The structure reveals a conformatIon of the Channel that is distinct from ELIC and that probably resembles the open state. In combinatIon, both structures suggest a novel gating mechanism for pentameric Ligand-Gated Ion Channels where Channel opening proceeds by a change in the tilt of the pore-forming helices.

  • x ray structure of a prokaryotic pentameric ligand gated Ion Channel
    Nature, 2008
    Co-Authors: Ricarda J C Hilf, Raimund Dutzler
    Abstract:

    Pentameric Ligand-Gated Ion Channels (pLGICs) are key players in the early events of electrical signal transductIon at chemical synapses. The family codes for a structurally conserved scaffold of Channel proteins that open in response to the binding of neurotransmitter molecules. All proteins share a pentameric organizatIon of identical or related subunits that consist of an extracellular ligand-binding domain followed by a transmembrane Channel domain. The nicotinic acetylcholine receptor (nAChR) is the most thoroughly studied member of the pLGIC family (for recent reviews see refs 1-3). Two sources of structural informatIon provided an architectural framework for the family. The structure of the soluble acetylcholine-binding protein (AChBP) defined the organizatIon of the extracellular domain and revealed the chemical basis of ligand interactIon. Electron microscopy studies of the nAChR from Torpedo electric ray have yielded a picture of the full-length protein and have recently led to the interpretatIon of an electron density map at 4.0 A resolutIon. Despite the wealth of experimental informatIon, high-resolutIon structures of any family member have so far not been available. Until recently, the pLGICs were believed to be only expressed in multicellular eukaryotic organisms. The abundance of prokaryotic genome sequences, however, allowed the identificatIon of several homologous proteins in bacterial sources. Here we present the X-ray structure of a prokaryotic pLGIC from the bacterium Erwinia chrysanthemi (ELIC) at 3.3 A resolutIon. Our study reveals the first structure of a pLGIC at high resolutIon and provides an important model system for the investigatIon of the general mechanisms of Ion permeatIon and gating within the family.

Jean-pierre Changeux - One of the best experts on this subject based on the ideXlab platform.

  • crystal structures of a pentameric ligand gated Ion Channel provide a mechanism for activatIon
    Proceedings of the National Academy of Sciences of the United States of America, 2014
    Co-Authors: L Sauguet, Jean-pierre Changeux, Pierre-jean Corringer, Christa Le Huon, Azadeh Shahsavar, Frederic Poitevin, Anais Menny, Akos Nemecz, Ahmed Haouz, Marc Delarue
    Abstract:

    Pentameric Ligand-Gated Ion Channels mediate fast chemical transmissIon of nerve signals. The structure of a bacterial proton-gated homolog has been established in its open and locally closed conformatIons at acidic pH. Here we report its crystal structure at neutral pH, thereby providing the X-ray structures of the two end-points of the gating mechanism in the same pentameric Ligand-Gated Ion Channel. The large structural variability in the neutral pH structure observed in the four copies of the pentamer present in the asymmetric unit has been used to analyze the intrinsic fluctuatIons in this state, which are found to prefigure the transitIon to the open state. In the extracellular domain (ECD), a marked quaternary change is observed, involving both a twist and a blooming motIon, and the pore in the transmembrane domain (TMD) is closed by an upper bend of helix M2 (as in locally closed form) and a kink of helix M1, both helices no longer interacting across adjacent subunits. On the tertiary level, detachment of inner and outer β sheets in the ECD reshapes two essential cavities at the ECD–ECD and ECD–TMD interfaces. The first one is the ligand-binding cavity; the other is close to a known divalent catIon binding site in other pentameric Ligand-Gated Ion Channels. In additIon, a different crystal form reveals that the locally closed and open conformatIons coexist as discrete ones at acidic pH. These structural results, together with site-directed mutagenesis, physiological recordings, and coarse-grained modeling, have been integrated to propose a model of the gating transitIon pathway.

  • the nicotinic acetylcholine receptor the founding father of the pentameric ligand gated Ion Channel superfamily
    Journal of Biological Chemistry, 2012
    Co-Authors: Jean-pierre Changeux
    Abstract:

    A critical event in the history of biological chemistry was the chemical identificatIon of the first neurotransmitter receptor, the nicotinic acetylcholine receptor. Disciplines as diverse as electrophysiology, pharmacology, and biochemistry joined together in a unified and ratIonal manner with the common goal of successfully identifying the molecular device that converts a chemical signal into an electrical one in the nervous system. The nicotinic receptor has become the founding father of a broad family of pentameric membrane receptors, paving the way for their identificatIon, including that of the GABAA receptors.

  • x ray structures of general anaesthetics bound to a pentameric ligand gated Ion Channel
    Nature, 2011
    Co-Authors: Hugues Nury, Jean-pierre Changeux, Catherine Van Renterghem, Yun Weng, Marc Baaden, Alphonso Tran, Virginie Dufresne
    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.

  • X-ray structure of a pentameric Ligand-Gated Ion Channel in an apparently open conformatIon
    Nature, 2009
    Co-Authors: Nicolas Bocquet, Jean-pierre Changeux, Hugues Nury, Marc Delarue, Marc Baaden, Chantal Le Poupon, Pierre-jean Corringer
    Abstract:

    Pentameric Ligand-Gated Ion Channels from the Cys-loop family mediate fast chemo-electrical transductIon, but the mechanisms of Ion permeatIon and gating of these membrane proteins remain elusive. Here we present the X-ray structure at 2.9 A resolutIon of the bacterial Gloeobacter violaceus pentameric Ligand-Gated Ion Channel homologue (GLIC) at pH 4.6 in an apparently open conformatIon. This catIonic Channel is known to be permanently activated by protons. The structure is arranged as a funnel-shaped transmembrane pore widely open on the outer side and lined by hydrophobic residues. On the inner side, a 5 A constrictIon matches with rings of hydrophilic residues that are likely to contribute to the Ionic selectivity. Structural comparison with ELIC, a bacterial homologue from Erwinia chrysanthemi solved in a presumed closed conformatIon, shows a wider pore where the narrow hydrophobic constrictIon found in ELIC is removed. Comparative analysis of GLIC and ELIC reveals, in concert, a rotatIon of each extracellular beta-sandwich domain as a rigid body, interface rearrangements, and a reorganizatIon of the transmembrane domain, involving a tilt of the M2 and M3 alpha-helices away from the pore axis. These data are consistent with a model of pore opening based on both quaternary twist and tertiary deformatIon.

David B Sattelle - One of the best experts on this subject based on the ideXlab platform.

  • the cys loop ligand gated Ion Channel gene superfamily of the parasitoid wasp nasonia vitripennis
    Heredity, 2010
    Co-Authors: Andrew K Jones, A N Bera, Kristin Lees, David B Sattelle
    Abstract:

    Members of the cys-loop Ligand-Gated Ion Channel (cysLGIC) superfamily mediate chemical neurotransmissIon and are studied extensively as potential targets of drugs used to treat neurological disorders, such as Alzheimer's disease. Insect cys-loop LGICs also have central roles in the nervous system and are targets of highly successful insecticides. Here, we describe the cysLGIC superfamily of the parasitoid wasp, Nasonia vitripennis, which is emerging as a highly useful model organism and is deployed as a biological control of insect pests. The wasp superfamily consists of 26 genes, which is the largest insect cysLGIC superfamily characterized, whereas Drosophila melanogaster, Apis mellifera and Tribolium castaneum have 23, 21 and 24, respectively. As with Apis, Drosophila and Tribolium, Nasonia possesses Ion Channels predicted to be gated by acetylcholine, gamma-amino butyric acid, glutamate and histamine, as well as orthologues of the Drosophila pH-sensitive chloride Channel (pHCl), CG8916 and CG12344. Similar to other insects, wasp cysLGIC diversity is broadened by alternative splicing and RNA A-to-I editing, which may also serve to generate species-specific receptor isoforms. These findings on N. vitripennis enhance our understanding of cysLGIC functIonal genomics and provide a useful basis for the study of their functIon in the wasp model, as well as for the development of improved insecticides that spare a major beneficial insect species.

  • the cys loop ligand gated Ion Channel gene superfamily of the red flour beetle tribolium castaneum
    BMC Genomics, 2007
    Co-Authors: Andrew K Jones, David B Sattelle
    Abstract:

    Members of the cys-loop Ligand-Gated Ion Channel (cys-loop LGIC) superfamily mediate chemical neurotransmissIon and are studied extensively as potential targets of drugs used to treat neurological disorders such as Alzheimer's disease. Insect cys-loop LGICs are also of interest as they are targets of highly successful insecticides. The red flour beetle, Tribolium castaneum, is a major pest of stored agricultural products and is also an important model organism for studying development. As part of the T. castaneum genome sequencing effort, we have characterized the beetle cys-loop LGIC superfamily which is the third insect superfamily to be described after those of Drosophila melanogaster and Apis mellifera, and also the largest consisting of 24 genes. As with Drosophila and Apis, Tribolium possesses Ion Channels gated by acetylcholine, γ-amino butyric acid (GABA), glutamate and histamine as well as orthologs of the Drosophila pH-sensitive chloride Channel subunit (pHCl), CG8916 and CG12344. Similar to Drosophila and Apis, Tribolium cys-loop LGIC diversity is broadened by alternative splicing although the beetle orthologs of RDL and GluCl possess more variants of exon 3. Also, RNA A-to-I editing was observed in two Tribolium nicotinic acetylcholine receptor subunits, Tcasα6 and Tcasβ1. Editing in Tcasα6 is evolutIonarily conserved with D. melanogaster, A. mellifera and Heliothis virescens, whereas Tcasβ1 is edited at a site so far only observed in the beetle. Our findings reveal that in diverse insect species the cys-loop LGIC superfamily has remained compact with only minor changes in gene numbers. However, alternative splicing, RNA editing and the presence of divergent subunits broadens the cys-loop LGIC proteome and generates species-specific receptor isoforms. These findings on Tribolium castaneum enhance our understanding of cys-loop LGIC functIonal genomics and provide a useful basis for the development of improved insecticides that target an important agricultural pest.

  • the nicotinic acetylcholine receptor gene family of the nematode caenorhabditis elegans an update on nomenclature
    Invertebrate Neuroscience, 2007
    Co-Authors: Andrew K Jones, Paul H Davis, Jonathan Hodgkin, David B Sattelle
    Abstract:

    The simple nematode, Caenorhabditis elegans, possesses the most extensive known gene family of nicotinic acetylcholine receptor (nAChR)-like subunits. Whilst all show greatest similarity with nAChR subunits of both invertebrates and vertebrates, phylogenetic analysis suggests that just over half of these (32) may represent other members of the cys-loop Ligand-Gated Ion Channel superfamily. We have introduced a novel nomenclature system for these "Orphan" subunits, designating them as lgc genes (Ligand-Gated Ion Channels of the cys-loop superfamily), which can also be applied in future to unnamed and uncharacterised members of the cys-loop Ligand-Gated Ion Channel superfamily. We present here the resulting updated versIon of the C. elegans nAChR gene family and related Ligand-Gated Ion Channel genes.

Sarah C. R. Lummis - One of the best experts on this subject based on the ideXlab platform.

  • modulatIon of the erwinia ligand gated Ion Channel elic and the 5 ht3 receptor via a common vestibule site
    eLife, 2020
    Co-Authors: Marijke Brams, Kerry L Price, Radovan Spurny, Cedric Govaerts, Kumiko Kambara, Anant Gharpure, Els Pardon, Genevieve L Evans, D Bertrand, Sarah C. R. Lummis
    Abstract:

    Pentameric Ligand-Gated Ion Channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to Channel opening. In this study, we developed nanobodies (single domain antibodies), which are functIonally active as allosteric modulators, and solved co-crystal structures of the prokaryote (Erwinia) Channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functIonal importance of the vestibule binding site to the human 5-HT3 receptor, suggesting a common mechanism of modulatIon in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities in pLGICs with an accessible vestibule site.

  • ModulatIon of the Erwinia Ligand-Gated Ion Channel (ELIC) and the 5-HT 3 receptor via a common vestibule site
    'Organisation for Economic Co-Operation and Development (OECD)', 2020
    Co-Authors: Brams Marijke, Spurny Radovan, Bertrand Daniel, Govaerts Cedric, Pardon Els, Kambara Kumiko, Gharpure Anant, Evans, Genevieve G.l., Price, Kerry L, Sarah C. R. Lummis
    Abstract:

    Funder: Instruct-ERICPentameric Ligand-Gated Ion Channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to Channel opening. In this study, we developed nanobodies (single domain antibodies), which are functIonally active as allosteric modulators, and solved co-crystal structures of the prokaryote (Erwinia) Channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functIonal importance of the vestibule binding site to the human 5-HT3 receptor, suggesting a common mechanism of modulatIon in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities in pLGICs with an accessible vestibule site

  • Probing Proline Residues in the Prokaryotic Ligand-Gated Ion Channel, ELIC
    2018
    Co-Authors: Richard Mosesso, Dennis A. Dougherty, Sarah C. R. Lummis
    Abstract:

    Erwinia Ligand-Gated Ion Channel (ELIC) is a bacterial homologue of vertebrate pentameric Ligand-Gated Ion Channels (pLGICs) and has proven to be a valuable model for understanding the structure and functIon of this important protein family. There is nevertheless still a questIon about whether molecular details can be accurately extrapolated from this protein to those found in eukaryotes. Here we explore the role of proline residues (Pros) in ELIC by creating mutant receptors, expressing them in Xenopus laevis oocytes, and using whole-cell voltage-clamp electrophysiology to monitor Channel activity. In contrast to eukaryotic pLGICs, proline-to-alanine (Pro-to-Ala) substitutIon in ELIC mostly resulted in gain of functIon, and even altering highly conserved Pro residues in M1 and the M2–M3 loop did not ablate functIon. These substitutIons also mostly resulted in ablatIon of the modulatIon by Ca2+ observed in wild-type receptors. SubstitutIon of the Pro in the “Cys loop”, however, did result in nonfunctIonal receptors. Probing this residue with noncanonical amino acids revealed a requirement for a substituted amine at this positIon, as well as a general preference for Pro analogues with greater intrinsic cis biases. We propose there is likely a cis bond at the apex of the Cys loop in this protein, which is consistent with some, but not all, findings from other pLGICs. Overall, the data show that the roles of proline residues are less critical in ELIC than in other pLGICs, supporting other studies that suggest cautIon must be applied in using data from this prokaryotic receptor to understand molecular details of eukaryotic pLGIC receptor functIon

  • Insights into the binding of GABA to the insect RDL receptor from atomistic simulatIons: a comparison of models
    2016
    Co-Authors: Federico Comitani, Sarah C. R. Lummis, Netta Cohen, Jamie Ashby, Dominic Botten, Carla Molteni
    Abstract:

    Abstract The resistance to dieldrin (RDL) receptor is an insect pentameric Ligand-Gated Ion Channel (pLGIC). It is activated by the neurotransmitter c-aminobutyric acid (GABA) binding to its extracellular domain; hence eluci-dating the atomistic details of this interactIon is important for understanding how the RDL receptor functIons. As no high resolutIon structures are currently available, we built homology models of the extracellular domain of the RDL receptor using different templates, including the widely used acetylcholine binding protein and two pLGICs, the Erwinia Chrysanthemi Ligand-Gated Ion Channel (ELIC) and the more recently resolved GluCl. We then docked GABA into the selected three dimensIonal structures, which we used as starting points for classical molecular dynamics simulatIons. This allowed us to analyze in detail the behavior of GABA in the binding sites, including the hydrogen bond and catIon-p interactIon networks it formed, the conformers it visited and the possible role of water molecules in mediating the interactIons; we also estimated the binding free energies. The models were all stable and showed common features, including interactIons consistent with experimental data and similar to other pLGICs; differences could be attributed to the quality of the models, which increases with increasing sequence identity, and the use of a pLGIC template. We supple-mented the molecular dynamics informatIon with meta-dynamics, a rare event method, by exploring the free energy landscape of GABA binding to the RDL receptor. Overall, we show that the GluCl template provided the best models. GABA forming direct salt-bridges with Arg211 and Glu204, and catIon-p interactIons with an aromatic cage including Tyr109, Phe206 and Tyr254, represents a favorable binding arrangement, and the interactIon with Glu204 can also be mediated by a water molecule

  • predictIon of 5 ht3 receptor agonist binding residues using homology modeling
    Biophysical Journal, 2003
    Co-Authors: David C Reeves, Muhammed Sayed, Paklee Chau, Kerry L Price, Sarah C. R. Lummis
    Abstract:

    5-HT3 receptors demonstrate significant structural and functIonal homology to other members of the Cys-loop Ligand-Gated Ion Channel superfamily. The extracellular domains of these receptors share similar sequence homology (∼20%) with Limnaea acetylcholine binding protein, for which an x-ray crystal structure is available. We used this structure as a template for computer-based homology modeling of the 5-HT3 receptor extracellular domain. AutoDock software was used to dock 5-HT into the putative 5-HT3 receptor ligand-binding site, resulting in seven alternative energetically favorable models. Residues located no more than 5 A from the docked 5-HT were identified for each model; of these, 12 were found to be common to all seven models with five others present in only certain models. Some docking models reflected the catIon-π interactIon previously demonstrated for W183, and data from these and other studies were used to define our preferred models.

Erik Lindahl - One of the best experts on this subject based on the ideXlab platform.

  • allosteric potentiatIon of a ligand gated Ion Channel is mediated by access to a deep membrane facing cavity
    Proceedings of the National Academy of Sciences of the United States of America, 2018
    Co-Authors: Stephanie A Heusser, Rebecca J Howard, Erik Lindahl, Marie Lycksell, Xueqing Wang, Sarah Mccomas
    Abstract:

    Theories of general anesthesia have shifted in focus from bulk lipid effects to specific interactIons with membrane proteins. Target receptors include several subtypes of pentameric Ligand-Gated Ion Channels; however, structures of physiologically relevant proteins in this family have yet to define anesthetic binding at high resolutIon. Recent cocrystal structures of the bacterial protein GLIC provide snapshots of state-dependent binding sites for the common surgical agent propofol (PFL), offering a detailed model system for anesthetic modulatIon. Here, we combine molecular dynamics and oocyte electrophysiology to reveal differential motIon and modulatIon upon modificatIon of a transmembrane binding site within each GLIC subunit. WT Channels exhibited net inhibitIon by PFL, and a contractIon of the cavity away from the pore-lining M2 helix in the absence of drug. Conversely, in GLIC variants exhibiting net PFL potentiatIon, the cavity was persistently expanded and proximal to M2. MutatIons designed to favor this deepened site enabled sensitivity even to subclinical concentratIons of PFL, and a uniquely prolonged mode of potentiatIon evident up to ∼30 min after washout. Dependence of these prolonged effects on exposure time implicated the membrane as a reservoir for a lipid-accessible binding site. However, at the highest measured concentratIons, potentiatIon appeared to be masked by an acute inhibitory effect, consistent with the presence of a discrete, water-accessible site of inhibitIon. These results support a multisite model of transmembrane allosteric modulatIon, including a possible link between lipid- and receptor-based theories that could inform the development of new anesthetics.

  • altering the open vs closed state balance of the glic ligand gated Ion Channel through mutagenesis enables molecular simulatIon of the reversible gating process
    Biophysical Journal, 2014
    Co-Authors: Iman Pouya, Samuel Murail, Goran Klement, Peter M Kasson, Ozge Yoluk, Erik Lindahl
    Abstract:

    The prokaryotic Gloeobacter violaceus pentameric Ligand-Gated Ion Channel (GLIC) is an important template for studies of Cys-loop receptors in the human central nervous system. It is also a key model system to understand the transitIons and stabilizatIon of different conformatIons of membrane proteins through allosteric modulatIon - a ligand that opens the Channel can either stabilize the open state, or destabilize the closed one. Capturing these processes on the molecular level will not only enable us to understand gating, but also make it possible to predict functIonal responses rather than merely binding properties. Over the last years, we have studied mutants of the GLIC Channel that make it more similar to the human Cys-loop receptors both experimentally and in simulatIons, and shown how this provides evidence for a dual allosteric modulatIon mechanism with separate inhibitory and potentiating binding sites. Here, I will present our new results on mutatIons in the central GLIC pore. By modifying the hydrophobicity of one or several residues in the pore it is possible to alter the pore hydratIon level, which in turn has paramount effects on the conformatIon - molecular simulatIons show that it is possible to stabilize the Channel either in the open or closed conformatIon independent of pH. By using ensemble molecular dynamics simulatIons covering several tens of microseconds, we show that it is possible to reversibly simulate transitIons between conformatIons that are shown to actually conduct Ions or block the current in simulatIons. In vivo electrophysiology and single-Channel recordings on the mutants confirm this influence on the kinetics, and provide us with a way to couple molecular-level dynamics and kinetics to the experimentally observed equilibrium and transitIons between conformatIons.

  • stabilizatIon of the glucl ligand gated Ion Channel in the presence and absence of ivermectin
    Biophysical Journal, 2013
    Co-Authors: Ozge Yoluk, Erik Lindahl, James R Trudell, Torben Bromstrup, Edward Bertaccini
    Abstract:

    Improving our understanding of the mechanisms and effects of anesthetics is a critically important part of neuroscience. The currently dominant theory is that anesthetics and similar molecules act by binding to Cys-loop receptors in the postsynaptic terminal of nerve cells and potentiate or inhibit their functIon. Although structures for some of the most important mammalian Channels have still not been determined, a number of important results have been derived from work on homologous catIonic Channels in bacteria. However, partly due to the lack of a nervous system in bacteria, there are a number of questIons about how these results relate to higher organisms. The recent determinatIon of a structure of the eukaryotic chloride Channel, GluCl, is an important step toward accurate modeling of mammalian Channels, because it is more similar in functIon to human Cys-loop receptors such as GABAAR or GlyR. One potential issue with using GluCl to model other receptors is the presence of the large ligand ivermectin (IVM) positIoned between all five subunits. Here, we have performed a series of microsecond molecular simulatIons to study how the dynamics and structure of GluCl change in the presence versus absence of IVM. When the ligand is removed, subunits move at least 2 A closer to each other compared to simulatIons with IVM bound. In additIon, the pore radius shrinks to 1.2 A, all of which appears to support a model where IVM binding between subunits stabilizes an open state, and that the relaxed nonIVM conformatIons might be suitable for modeling other Channels. Interestingly, the presence of IVM also has an effect on the structure of the important loop C located at the neurotransmitter-binding pocket, which might help shed light on its partial agonist behavior.

  • structural basis for alcohol modulatIon of a pentameric ligand gated Ion Channel
    Proceedings of the National Academy of Sciences of the United States of America, 2011
    Co-Authors: Rebecca J Howard, Pierre-jean Corringer, Samuel Murail, Kathryn E Ondricek, Erik Lindahl, James R Trudell, Adron R Harris
    Abstract:

    Despite its long history of use and abuse in human culture, the molecular basis for alcohol actIon in the brain is poorly understood. The recent determinatIon of the atomic-scale structure of GLIC, a prokaryotic member of the pentameric Ligand-Gated Ion Channel (pLGIC) family, provides a unique opportunity to characterize the structural basis for modulatIon of these Channels, many of which are alcohol targets in brain. We observed that GLIC recapitulates bimodal modulatIon by n-alcohols, similar to some eukaryotic pLGICs: methanol and ethanol weakly potentiated proton-activated currents in GLIC, whereas n-alcohols larger than ethanol inhibited them. Mapping of residues important to alcohol modulatIon of Ionotropic receptors for glycine, γ-aminobutyric acid, and acetylcholine onto GLIC revealed their proximity to transmembrane cavities that may accommodate one or more alcohol molecules. Site-directed mutatIons in the pore-lining M2 helix allowed the identificatIon of four residues that influence alcohol potentiatIon, with the directIon of their effects reflecting α-helical structure. At one of the potentiatIon-enhancing residues, decreased side chain volume converted GLIC into a highly ethanol-sensitive Channel, comparable to its eukaryotic relatives. Covalent labeling of M2 positIons with an alcohol analog, a methanethiosulfonate reagent, further implicated residues at the extracellular end of the helix in alcohol binding. Molecular dynamics simulatIons elucidated the structural consequences of a potentiatIon-enhancing mutatIon and suggested a structural mechanism for alcohol potentiatIon via interactIon with a transmembrane cavity previously termed the “linking tunnel.” These results provide a unique structural model for independent potentiating and inhibitory interactIons of n-alcohols with a pLGIC family member.

  • normal mode gating motIons of a ligand gated Ion Channel persist in a fully hydrated lipid bilayer model
    ACS Chemical Neuroscience, 2010
    Co-Authors: Edward J Bertaccini, James R Trudell, Erik Lindahl
    Abstract:

    We have previously used molecular modeling and normal-mode analyses combined with experimental data to visualize a plausible model of a transmembrane Ligand-Gated Ion Channel. We also postulated how the gating motIon of the Channel may be affected by the presence of various ligands, especially anesthetics. As is typical for normal-mode analyses, those studies were performed ut vacuo to reduce the computatIonal complexity of the problem. While such calculatIons constitute an efficient way to model the large scale structural flexibility of transmembrane proteins, they can be criticized for neglecting the effects of an explicit phospholipid bilayer or hydrated environment. Here, we show the successful calculatIon of normal-mode motIons for our model of a glycine alpha-1 receptor, now suspended in a fully hydrated lipid bilayer. Despite the almost uniform atomic density, the introductIon of water and lipid does not grossly distort the overall gating motIon. Normal-mode analysis revealed that even a fully immersed glycine alpha-1 receptor continues to demonstrate an iris-like Channel gating motIon as a low-frequency, high-amplitude natural harmonic vibratIon consistent with Channel gating. Furthermore, the introductIon of periodic boundary conditIons allows the examinatIon of simultaneous harmonic vibratIons of lipid in synchrony with the protein gating motIons that are compatible with reasonable lipid bilayer perturbatIons. While these perturbatIons tend to influence the overall protein motIon, this work provides continued support for the iris-like motIon model that characterizes gating within the family of Ligand-Gated Ion Channels.