Binding of ArgTX-636 in the NMDA receptor ion channel

Mette H Poulsen; Jacob Andersen; Rune Christensen; Kasper Bø Hansen; Stephen F Traynelis; Kristian Strømgaard; Anders Skov Kristensen

doi:10.1016/j.jmb.2014.05.017

Binding of ArgTX-636 in the NMDA receptor ion channel

Mette H Poulsen, Jacob Andersen, Rune Christensen, Kasper Bø Hansen, Stephen F Traynelis, Kristian Strømgaard, Anders Skov Kristensen

11 Citations (Scopus)

Abstract

The N-methyl-d-aspartate receptors (NMDARs) constitute an important class of ligand-gated cation channels that are involved in the majority of excitatory neurotransmission in the human brain. Compounds that bind in the NMDAR ion channel and act as blockers are use- and voltage-dependent inhibitors of NMDAR activity and have therapeutic potential for treatment of a variety of brain diseases or as pharmacological tools for studies of the neurobiological role of NMDARs. We have performed a kinetic analysis of the blocking mechanism of the prototypical polyamine toxin NMDAR ion channel blocker argiotoxin-636 (ArgTX-636) at recombinant GluN1/2A receptors to provide detailed information on the mechanism of block. The predicted binding site of ArgTX-636 is in the pore region of the NMDAR ion channel formed by residues in the transmembrane M3 and the M2 pore-loop segments of the GluN1 and GluN2A subunits. To assess the predicted binding mode in further detail, we performed an alanine- and glycine-scanning mutational analysis of this pore-loop segment to systematically probe the role of pore-lining M2 residues in GluN1 and GluN2A in the channel block by ArgTX-636. Comparison of M2 positions in GluN1 and GluN2A where mutation influences ArgTX-636 potency suggests differential contribution of the M2-loops of GluN1 and GluN2A to binding of ArgTX-636. The results of the mutational analysis are highly relevant for the future structure-based development of argiotoxin-derived NMDAR channel blockers.

Original language	English
Journal	Journal of Molecular Biology
Volume	427
Issue number	1
Pages (from-to)	176-89
Number of pages	14
ISSN	0022-2836
DOIs	https://doi.org/10.1016/j.jmb.2014.05.017
Publication status	Published - 16 Jan 2015

Keywords

Animals
Binding Sites
Electrophysiology
Glutamic Acid
HEK293 Cells
Humans
Indoleacetic Acids
Ion Channels
Kinetics
Models, Molecular
Mutagenesis
Mutation
Patch-Clamp Techniques
Polyamines
Protein Conformation
Protein Subunits
Rats
Receptors, N-Methyl-D-Aspartate
Recombinant Fusion Proteins
Spider Venoms

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.jmb.2014.05.017

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@article{c1b2fc19d16946d998c35681597b7522,

title = "Binding of ArgTX-636 in the NMDA receptor ion channel",

abstract = "The N-methyl-d-aspartate receptors (NMDARs) constitute an important class of ligand-gated cation channels that are involved in the majority of excitatory neurotransmission in the human brain. Compounds that bind in the NMDAR ion channel and act as blockers are use- and voltage-dependent inhibitors of NMDAR activity and have therapeutic potential for treatment of a variety of brain diseases or as pharmacological tools for studies of the neurobiological role of NMDARs. We have performed a kinetic analysis of the blocking mechanism of the prototypical polyamine toxin NMDAR ion channel blocker argiotoxin-636 (ArgTX-636) at recombinant GluN1/2A receptors to provide detailed information on the mechanism of block. The predicted binding site of ArgTX-636 is in the pore region of the NMDAR ion channel formed by residues in the transmembrane M3 and the M2 pore-loop segments of the GluN1 and GluN2A subunits. To assess the predicted binding mode in further detail, we performed an alanine- and glycine-scanning mutational analysis of this pore-loop segment to systematically probe the role of pore-lining M2 residues in GluN1 and GluN2A in the channel block by ArgTX-636. Comparison of M2 positions in GluN1 and GluN2A where mutation influences ArgTX-636 potency suggests differential contribution of the M2-loops of GluN1 and GluN2A to binding of ArgTX-636. The results of the mutational analysis are highly relevant for the future structure-based development of argiotoxin-derived NMDAR channel blockers.",

keywords = "Animals, Binding Sites, Electrophysiology, Glutamic Acid, HEK293 Cells, Humans, Indoleacetic Acids, Ion Channels, Kinetics, Models, Molecular, Mutagenesis, Mutation, Patch-Clamp Techniques, Polyamines, Protein Conformation, Protein Subunits, Rats, Receptors, N-Methyl-D-Aspartate, Recombinant Fusion Proteins, Spider Venoms",

author = "Poulsen, {Mette H} and Jacob Andersen and Rune Christensen and Hansen, {Kasper B{\o}} and Traynelis, {Stephen F} and Kristian Str{\o}mgaard and Kristensen, {Anders Skov}",

year = "2015",

month = jan,

day = "16",

doi = "10.1016/j.jmb.2014.05.017",

language = "English",

volume = "427",

pages = "176--89",

journal = "Journal of Molecular Biology",

issn = "0022-2836",

publisher = "Academic Press",

number = "1",

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TY - JOUR

T1 - Binding of ArgTX-636 in the NMDA receptor ion channel

AU - Poulsen, Mette H

AU - Andersen, Jacob

AU - Christensen, Rune

AU - Hansen, Kasper Bø

AU - Traynelis, Stephen F

AU - Strømgaard, Kristian

AU - Kristensen, Anders Skov

PY - 2015/1/16

Y1 - 2015/1/16

N2 - The N-methyl-d-aspartate receptors (NMDARs) constitute an important class of ligand-gated cation channels that are involved in the majority of excitatory neurotransmission in the human brain. Compounds that bind in the NMDAR ion channel and act as blockers are use- and voltage-dependent inhibitors of NMDAR activity and have therapeutic potential for treatment of a variety of brain diseases or as pharmacological tools for studies of the neurobiological role of NMDARs. We have performed a kinetic analysis of the blocking mechanism of the prototypical polyamine toxin NMDAR ion channel blocker argiotoxin-636 (ArgTX-636) at recombinant GluN1/2A receptors to provide detailed information on the mechanism of block. The predicted binding site of ArgTX-636 is in the pore region of the NMDAR ion channel formed by residues in the transmembrane M3 and the M2 pore-loop segments of the GluN1 and GluN2A subunits. To assess the predicted binding mode in further detail, we performed an alanine- and glycine-scanning mutational analysis of this pore-loop segment to systematically probe the role of pore-lining M2 residues in GluN1 and GluN2A in the channel block by ArgTX-636. Comparison of M2 positions in GluN1 and GluN2A where mutation influences ArgTX-636 potency suggests differential contribution of the M2-loops of GluN1 and GluN2A to binding of ArgTX-636. The results of the mutational analysis are highly relevant for the future structure-based development of argiotoxin-derived NMDAR channel blockers.

AB - The N-methyl-d-aspartate receptors (NMDARs) constitute an important class of ligand-gated cation channels that are involved in the majority of excitatory neurotransmission in the human brain. Compounds that bind in the NMDAR ion channel and act as blockers are use- and voltage-dependent inhibitors of NMDAR activity and have therapeutic potential for treatment of a variety of brain diseases or as pharmacological tools for studies of the neurobiological role of NMDARs. We have performed a kinetic analysis of the blocking mechanism of the prototypical polyamine toxin NMDAR ion channel blocker argiotoxin-636 (ArgTX-636) at recombinant GluN1/2A receptors to provide detailed information on the mechanism of block. The predicted binding site of ArgTX-636 is in the pore region of the NMDAR ion channel formed by residues in the transmembrane M3 and the M2 pore-loop segments of the GluN1 and GluN2A subunits. To assess the predicted binding mode in further detail, we performed an alanine- and glycine-scanning mutational analysis of this pore-loop segment to systematically probe the role of pore-lining M2 residues in GluN1 and GluN2A in the channel block by ArgTX-636. Comparison of M2 positions in GluN1 and GluN2A where mutation influences ArgTX-636 potency suggests differential contribution of the M2-loops of GluN1 and GluN2A to binding of ArgTX-636. The results of the mutational analysis are highly relevant for the future structure-based development of argiotoxin-derived NMDAR channel blockers.

KW - Animals

KW - Binding Sites

KW - Electrophysiology

KW - Glutamic Acid

KW - HEK293 Cells

KW - Humans

KW - Indoleacetic Acids

KW - Ion Channels

KW - Kinetics

KW - Models, Molecular

KW - Mutagenesis

KW - Mutation

KW - Patch-Clamp Techniques

KW - Polyamines

KW - Protein Conformation

KW - Protein Subunits

KW - Rats

KW - Receptors, N-Methyl-D-Aspartate

KW - Recombinant Fusion Proteins

KW - Spider Venoms

U2 - 10.1016/j.jmb.2014.05.017

DO - 10.1016/j.jmb.2014.05.017

M3 - Journal article

C2 - 24862283

SN - 0022-2836

VL - 427

SP - 176

EP - 189

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 1

ER -

Binding of ArgTX-636 in the NMDA receptor ion channel

Abstract

Keywords

UN SDGs

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