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

N1 - Copyright © 2014 Elsevier Ltd. All rights reserved.

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

VL - 427

SP - 176

EP - 189

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 1

ER -