Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Allosteric modulation of pentameric ligand-gated ion channels by general anesthetics
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0003-3224-4547
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2018.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-154106ISBN: 978-91-7797-151-1 (print)ISBN: 978-91-7797-152-8 (electronic)OAI: oai:DiVA.org:su-154106DiVA, id: diva2:1190799
Public defence
2018-05-04, Magnéli Hall, Chemical Practice Laboratory, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Accepted. Paper 4: Manuscript.

Available from: 2018-04-11 Created: 2018-03-15 Last updated: 2018-04-09
List of papers
1. Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel
Open this publication in new window or tab >>Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel
Show others...
2016 (English)In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 138, no 2, p. 243-253Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
anesthetic, cys-loop receptor, GluCl, Ion channel, pLGIC
National Category
Biophysics Medicinal Chemistry
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-133228 (URN)10.1111/jnc.13644 (DOI)000380263700004 ()27102368 (PubMedID)
Available from: 2016-09-12 Created: 2016-09-05 Last updated: 2018-03-27Bibliographically approved
2. Functional Validation of Virtual Screening for Novel Agents with General Anesthetic Action at Ligand-Gated Ion Channelss
Open this publication in new window or tab >>Functional Validation of Virtual Screening for Novel Agents with General Anesthetic Action at Ligand-Gated Ion Channelss
Show others...
2013 (English)In: Molecular Pharmacology, ISSN 0026-895X, E-ISSN 1521-0111, Vol. 84, no 5, p. 670-678Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-96087 (URN)10.1124/mol.113.087692 (DOI)000325534800002 ()
Note

AuthorCount:9

Available from: 2013-11-14 Created: 2013-11-11 Last updated: 2018-03-27Bibliographically approved
3. Structural Basis for a Bimodal Allosteric Mechanism of General Anesthetic Modulation in Pentameric Ligand-Gated Ion Channels
Open this publication in new window or tab >>Structural Basis for a Bimodal Allosteric Mechanism of General Anesthetic Modulation in Pentameric Ligand-Gated Ion Channels
Show others...
2018 (English)In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 23, no 4, p. 993-1004Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biochemistry and Molecular Biology Structural Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-154102 (URN)10.1016/j.celrep.2018.03.108 (DOI)000432453100009 ()
Available from: 2018-03-15 Created: 2018-03-15 Last updated: 2018-06-25Bibliographically approved
4. Propofol potentiation in the pentameric ion channel GLIC is mediated by a deep membrane-facing cavity
Open this publication in new window or tab >>Propofol potentiation in the pentameric ion channel GLIC is mediated by a deep membrane-facing cavity
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology Computer and Information Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-154105 (URN)
Available from: 2018-03-15 Created: 2018-03-15 Last updated: 2018-03-27Bibliographically approved

Open Access in DiVA

Allosteric modulation of pentameric ligand-gated ion channels by general anesthetics(3068 kB)54 downloads
File information
File name FULLTEXT01.pdfFile size 3068 kBChecksum SHA-512
c0f6beeb7e229bf3ce75e846fc16825618c2ae462ab3ca06e9176362b8d1f8313310f29df1c5a047502c09c7ac5fa2cbdc03e4a2f02676d412296b4d535acd91
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Heusser, Stephanie A.
By organisation
Department of Biochemistry and Biophysics
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 54 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 588 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf