A structural rationale for N-methylbicuculline acting as a promiscuous competitive antagonist of inhibitory pentameric ligand gated ion channels

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Abstract

Bicuculline, a valued chemical tool in neurosciences research, is a competitive antagonist of specific GABA A receptors and affects other pentameric ligand gated ion channels including the glycine, nicotinic acetylcholine and 5-hydroxytryptamine type 3 receptors. We used a fluorescence quenching assay and isothermal titration calorimetry to record low micromolar dissociation constants for N-methylbicuculline interacting with acetylcholine binding protein and an engineered version called glycine-binding protein (GBP), which provides a surrogate for the heteromeric interface of the extracellular domain of the glycine receptor (GlyR). The 2.4 Å resolution crystal structure of the GBP:N-methylbicuculline complex, sequence and structural alignments reveal similarities and differences between GlyR and the GABA A receptor bicuculline interactions. N-methylbicuculline displays a similar conformation in different structures but adopts distinct orientations enforced by interactions and steric blocks with key residues and plasticity in the binding sites. These features explain the promiscuous activity of bicuculline against the principal inhibitory pentameric ligand gated ion channels in the CNS.

Original languageEnglish
Number of pages19
JournalChemBioChem
Early online date20 Dec 2019
DOIs
Publication statusE-pub ahead of print - 20 Dec 2019

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Ligand-Gated Ion Channels
Bicuculline
Glycine
Glycine Receptors
Carrier Proteins
GABA-A Receptors
Acetylcholine
GABA-A Receptor Antagonists
Receptors, Serotonin, 5-HT3
Calorimetry
Sequence Alignment
Neurosciences
Titration
Crystal orientation
Plasticity
Conformations
Quenching
Assays
Crystal structure
Fluorescence

Keywords

  • acetylcholine binding protein
  • alkaloid
  • competitive antagonist
  • GABAA-receptor
  • glycine receptor

Cite this

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title = "A structural rationale for N-methylbicuculline acting as a promiscuous competitive antagonist of inhibitory pentameric ligand gated ion channels",
abstract = "Bicuculline, a valued chemical tool in neurosciences research, is a competitive antagonist of specific GABA A receptors and affects other pentameric ligand gated ion channels including the glycine, nicotinic acetylcholine and 5-hydroxytryptamine type 3 receptors. We used a fluorescence quenching assay and isothermal titration calorimetry to record low micromolar dissociation constants for N-methylbicuculline interacting with acetylcholine binding protein and an engineered version called glycine-binding protein (GBP), which provides a surrogate for the heteromeric interface of the extracellular domain of the glycine receptor (GlyR). The 2.4 {\AA} resolution crystal structure of the GBP:N-methylbicuculline complex, sequence and structural alignments reveal similarities and differences between GlyR and the GABA A receptor bicuculline interactions. N-methylbicuculline displays a similar conformation in different structures but adopts distinct orientations enforced by interactions and steric blocks with key residues and plasticity in the binding sites. These features explain the promiscuous activity of bicuculline against the principal inhibitory pentameric ligand gated ion channels in the CNS.",
keywords = "acetylcholine binding protein, alkaloid, competitive antagonist, GABAA-receptor, glycine receptor",
author = "Jones, {Mathew J.} and Alice Dawson and Hales, {Tim G.} and Hunter, {William N.}",
note = "Funded with support from a Wellcome Trust PhD studentship and equipment grant [094090].",
year = "2019",
month = "12",
day = "20",
doi = "10.1002/cbic.201900680",
language = "English",
journal = "ChemBioChem",
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T1 - A structural rationale for N-methylbicuculline acting as a promiscuous competitive antagonist of inhibitory pentameric ligand gated ion channels

AU - Jones, Mathew J.

AU - Dawson, Alice

AU - Hales, Tim G.

AU - Hunter, William N.

N1 - Funded with support from a Wellcome Trust PhD studentship and equipment grant [094090].

PY - 2019/12/20

Y1 - 2019/12/20

N2 - Bicuculline, a valued chemical tool in neurosciences research, is a competitive antagonist of specific GABA A receptors and affects other pentameric ligand gated ion channels including the glycine, nicotinic acetylcholine and 5-hydroxytryptamine type 3 receptors. We used a fluorescence quenching assay and isothermal titration calorimetry to record low micromolar dissociation constants for N-methylbicuculline interacting with acetylcholine binding protein and an engineered version called glycine-binding protein (GBP), which provides a surrogate for the heteromeric interface of the extracellular domain of the glycine receptor (GlyR). The 2.4 Å resolution crystal structure of the GBP:N-methylbicuculline complex, sequence and structural alignments reveal similarities and differences between GlyR and the GABA A receptor bicuculline interactions. N-methylbicuculline displays a similar conformation in different structures but adopts distinct orientations enforced by interactions and steric blocks with key residues and plasticity in the binding sites. These features explain the promiscuous activity of bicuculline against the principal inhibitory pentameric ligand gated ion channels in the CNS.

AB - Bicuculline, a valued chemical tool in neurosciences research, is a competitive antagonist of specific GABA A receptors and affects other pentameric ligand gated ion channels including the glycine, nicotinic acetylcholine and 5-hydroxytryptamine type 3 receptors. We used a fluorescence quenching assay and isothermal titration calorimetry to record low micromolar dissociation constants for N-methylbicuculline interacting with acetylcholine binding protein and an engineered version called glycine-binding protein (GBP), which provides a surrogate for the heteromeric interface of the extracellular domain of the glycine receptor (GlyR). The 2.4 Å resolution crystal structure of the GBP:N-methylbicuculline complex, sequence and structural alignments reveal similarities and differences between GlyR and the GABA A receptor bicuculline interactions. N-methylbicuculline displays a similar conformation in different structures but adopts distinct orientations enforced by interactions and steric blocks with key residues and plasticity in the binding sites. These features explain the promiscuous activity of bicuculline against the principal inhibitory pentameric ligand gated ion channels in the CNS.

KW - acetylcholine binding protein

KW - alkaloid

KW - competitive antagonist

KW - GABAA-receptor

KW - glycine receptor

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JF - ChemBioChem

SN - 1439-4227

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