| 1. |
- Goni, Maria Fernanda Sanchez, et al.
(författare)
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The ACER pollen and charcoal database : a global resource to document vegetation and fire response to abrupt climate changes during the last glacial period
- 2017
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Ingår i: Earth System Science Data. - : Copernicus GmbH. - 1866-3508 .- 1866-3516. ; 9:2, s. 679-695
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Tidskriftsartikel (refereegranskat)abstract
- Quaternary records provide an opportunity to examine the nature of the vegetation and fire responses to rapid past climate changes comparable in velocity and magnitude to those expected in the 21st-century. The best documented examples of rapid climate change in the past are the warming events associated with the Dansgaard-Oeschger (D-O) cycles during the last glacial period, which were sufficiently large to have had a potential feedback through changes in albedo and greenhouse gas emissions on climate. Previous reconstructions of vegetation and fire changes during the D-O cycles used independently constructed age models, making it difficult to compare the changes between different sites and regions. Here, we present the ACER (Abrupt Climate Changes and Environmental Responses) global database, which includes 93 pollen records from the last glacial period (73-15 ka) with a temporal resolution better than 1000 years, 32 of which also provide charcoal records. A harmonized and consistent chronology based on radiometric dating (C-14, U-234/Th-230, optically stimulated luminescence (OSL), Ar-40/Ar-39-dated tephra layers) has been constructed for 86 of these records, although in some cases additional information was derived using common control points based on event stratigraphy. The ACER database compiles metadata including geospatial and dating information, pollen and charcoal counts, and pollen percentages of the characteristic biomes and is archived in Microsoft Access (TM) at https://doi. org/10.1594/PANGAEA. 870867.
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| 2. |
- Heusser, Stephanie A., et al.
(författare)
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Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel
- 2016
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Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 138:2, s. 243-253
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Tidskriftsartikel (refereegranskat)abstract
- The superfamily of pentameric ligand-gated ion channels includes neurotransmitter receptors that mediate fast synaptic transmission in vertebrates, and are targets for drugs including alcohols, anesthetics, benzodiazepines, and anticonvulsants. However, the mechanisms of ion channel opening, gating, and modulation in these receptors leave many open questions, despite their pharmacological importance. Subtle conformational changes in both the extracellular and transmembrane domains are likely to influence channel opening, but have been difficult to characterize given the limited structural data available for human membrane proteins. Recent crystal structures of a modified Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in multiple states offer an appealing model system for structure-function studies. However, the pharmacology of the crystallographic GluCl construct is not well established. To establish the functional relevance of this system, we used two-electrode voltage-clamp electrophysiology in Xenopus oocytes to characterize activation of crystallographic and native-like GluCl constructs by L-glutamate and ivermectin. We also tested modulation by ethanol and other anesthetic agents, and used site-directed mutagenesis to explore the role of a region of Loop F which was implicated in ligand gating by molecular dynamics simulations. Our findings indicate that the crystallographic construct functionally models concentration-dependent agonism and allosteric modulation of pharmacologically relevant receptors. Specific substitutions at residue Leu174 in loop F altered direct L-glutamate activation, consistent with computational evidence for this region's role in ligand binding. These insights demonstrate conservation of activation and modulation properties in this receptor family, and establish a framework for GluCl as a model system, including new possibilities for drug discovery. In this study, we elucidate the validity of a modified glutamate-gated chloride channel (GluCl(cryst)) as a structurally accessible model for GABA(A) receptors. In contrast to native-like controls, GluCl(cryst) exhibits classical activation by its neurotransmitter ligand L-glutamate. The modified channel is also sensitive to allosteric modulators associated with human GABA(A) receptors, and to site-directed mutations predicted to alter channel opening.
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| 3. |
- Heusser, Stephanie A., et al.
(författare)
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Functional Validation of Virtual Screening for Novel Agents with General Anesthetic Action at Ligand-Gated Ion Channelss
- 2013
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Ingår i: Molecular Pharmacology. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0026-895X .- 1521-0111. ; 84:5, s. 670-678
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Tidskriftsartikel (refereegranskat)abstract
- GABA(A) receptors play a crucial role in the actions of general anesthetics. The recently published crystal structure of the general anesthetic propofol bound to Gloeobacter violaceus ligand-gated ion channel (GLIC), a bacterial homolog of GABA(A) receptors, provided an opportunity to explore structure-based ligand discovery for pentameric ligand-gated ion channels (pLGICs). We used molecular docking of 153,000 commercially available compounds to identify molecules that interact with the propofol binding site in GLIC. In total, 29 compounds were selected for functional testing on recombinant GLIC, and 16 of these compounds modulated GLIC function. Active compounds were also tested on recombinant GABA(A) receptors, and point mutations around the presumed binding pocket were introduced into GLIC and GABA(A) receptors to test for binding specificity. The potency of active compounds was only weakly correlated with properties such as lipophilicity or molecular weight. One compound was found to mimic the actions of propofol on GLIC and GABA(A), and to be sensitive to mutations that reduce the action of propofol in both receptors. Mutant receptors also provided insight about the position of the binding sites and the relevance of the receptor's conformation for anesthetic actions. Overall, the findings support the feasibility of the use of virtual screening to discover allosteric modulators of pLGICs, and suggest that GLIC is a valid model system to identify novel GABA(A) receptor ligands.
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| 4. |
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| 5. |
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| 6. |
- Bergh, Cathrine, et al.
(författare)
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Markov state models of proton- and pore-dependent activation in a pentameric ligand-gated ion channel
- 2021
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Ingår i: eLIFE. - : eLife Sciences Publications, Ltd. - 2050-084X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Ligand-gated ion channels conduct currents in response to chemical stimuli, mediating electrochemical signaling in neurons and other excitable cells. For many channels, the details of gating remain unclear, partly due to limited structural data and simulation timescales. Here, we used enhanced sampling to simulate the pH-gated channel GLIC, and construct Markov state models (MSMs) of gating. Consistent with new functional recordings, we report in oocytes, our analysis revealed differential effects of protonation and mutation on free-energy wells. Clustering of closed- versus open-like states enabled estimation of open probabilities and transition rates, while higher-order clustering affirmed conformational trends in gating. Furthermore, our models uncovered state- and protonation-dependent symmetrization. This demonstrates the applicability of MSMs to map energetic and conformational transitions between ion-channel functional states, and how they reproduce shifts upon activation or mutation, with implications for modeling neuronal function and developing state-selective drugs.
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| 7. |
- Fourati, Zaineb, et al.
(författare)
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Structural Basis for a Bimodal Allosteric Mechanism of General Anesthetic Modulation in Pentameric Ligand-Gated Ion Channels
- 2018
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 23:4, s. 993-1004
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Tidskriftsartikel (refereegranskat)abstract
- Ion channel modulation by general anesthetics is a vital pharmacological process with implications for receptor biophysics and drug development. Functional studies have implicated conserved sites of both potentiation and inhibition in pentameric ligand-gated ion channels, but a detailed structural mechanism for these bimodal effects is lacking[1] . The prokaryotic model protein GLIC recapitulates anesthetic modulation of human ion channels, and is accessible to structure determination in both apparent open and closed states. Here, we report ten X-ray structures and electrophysiological characterization of GLIC variants in the presence and absence of general anesthetics, including the surgical agent propofol. We show that general anesthetics can allosterically favor closed channels by binding in the pore, or favor open channels via various subsites in the transmembrane domain. Our results support an integrated, multi-site mechanism for allosteric modulation, and provide atomic details of both potentiation and inhibition by one of the most common general anesthetics.
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| 8. |
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| 9. |
- Heusser, Stephanie A., 1987-
(författare)
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Allosteric modulation of pentameric ligand-gated ion channels by general anesthetics
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pentameric ligand-gated ion channels (pLGICs) are key components of fast synaptic transmission and are targets of neuroactive drugs such as benzodiazepines, alcohol and muscle relaxants. Although early theories of general anesthesia suggested non-specific lipid interaction as the mechanism of anesthetic action, it has now become evident that they too bind to pLGICs. While general anesthetics act as positive allosteric modulators on most anion-conducting pLGICs, they inhibit cation-conducting channels. A detailed structural mechanism of how such opposite allosteric effects emerge has yet to be presented.This thesis investigates the structure-function relationship underlying the dynamics of channel activation and explores the mechanisms behind allosteric modulation by general anesthetics. Key model systems include the glutamate-gated chloride channel of C. elegans (GluCl) and the G. violaceus ligand-gated ion channel (GLIC), that show considerable structural homology to mammalian channel but with the added simplicity of homomeric assembly and accessibility to crystallization. Functional assessment is performed through recombinant expression of the channels in Xenopus oocytes, which are then used for two-electrode voltage clamp electrophysiology. These measurements are combined with recent advances in structure determination and computational simulations to propose structural mechanisms behind the functional effects.In this thesis I present the exploration and validation of the crystallographic construct GluCl as a model system to explore fundamental questions of mammalian pLGIC function. Further studies contribute to the understanding of the basis of allosteric modulation by identifying responsible binding sites for both potentiation and inhibition by general anesthetics in GLIC and substantiate a structural mechanism for these effects. The studies also offer a link between receptor- and lipid-based theories of anesthesia, and demonstrate successful discovery of new lead compounds with general anesthetic properties using virtual screening. The thesis therefore makes a contribution to the fundamental understanding of allosteric modulation in pLGICs and builds on the basis for rational drug discovery.
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| 10. |
- Heusser, Stephanie A., et al.
(författare)
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Allosteric potentiation of a ligand-gated ion channel is mediated by access to a deep membrane-facing cavity
- 2018
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : NATL ACAD SCIENCES. - 0027-8424 .- 1091-6490. ; 115:42, s. 10672-10677
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Tidskriftsartikel (refereegranskat)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 similar 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.
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