TY - JOUR

T1 - Tweaking agonist efficacy at N-methyl-D-aspartate receptors by site-directed mutagenesis

AU - Hansen, Kasper B

AU - Clausen, Rasmus P

AU - Bjerrum, Esben J

AU - Bechmann, Christian

AU - Greenwood, Jeremy R

AU - Christensen, Caspar

AU - Kristensen, Jesper L

AU - Egebjerg, Jan

AU - Bräuner-Osborne, Hans

PY - 2005/12

Y1 - 2005/12

N2 - The structural basis for partial agonism at N-methyl-D-aspartate (NMDA) receptors is currently unresolved. We have characterized several partial agonists at the NR1/NR2B receptor and investigated the mechanisms underlying their reduced efficacy by introducing mutations in the glutamate binding site. Key residues were selected for mutation based on ligand-protein docking studies using a homology model of NR2B-S1S2 built from the X-ray structure of NR1-S1S2 in complex with glycine. Wild-type and mutant forms of NR2B were coexpressed with NR1 in Xenopus laevis oocytes and characterized by two-electrode voltage-clamp electrophysiology. By combining mutagenesis of residues His486 or Val686 with activation by differently substituted partial agonists, we introduce varying degrees of steric clash between the ligand and the two binding domains S1 and S2. In cases where ligand-protein docking predicts increased steric clashes between agonists and the residues forming the S1-S2 interface, the agonists clearly show decreased relative efficacy. Furthermore, we demonstrate that the mutation S690A affects both potency and efficacy in an agonist-specific manner. The results indicate that essential residues in the ligand binding pocket of NR2B may adopt different conformations depending on the agonist bound. Together, these data indicate that agonist efficacy at the NR2B subunit can be controlled by the extent of steric clashes between the agonist and the ligand binding domains and by ligand-dependent arrangements of residues within the binding pocket.

AB - The structural basis for partial agonism at N-methyl-D-aspartate (NMDA) receptors is currently unresolved. We have characterized several partial agonists at the NR1/NR2B receptor and investigated the mechanisms underlying their reduced efficacy by introducing mutations in the glutamate binding site. Key residues were selected for mutation based on ligand-protein docking studies using a homology model of NR2B-S1S2 built from the X-ray structure of NR1-S1S2 in complex with glycine. Wild-type and mutant forms of NR2B were coexpressed with NR1 in Xenopus laevis oocytes and characterized by two-electrode voltage-clamp electrophysiology. By combining mutagenesis of residues His486 or Val686 with activation by differently substituted partial agonists, we introduce varying degrees of steric clash between the ligand and the two binding domains S1 and S2. In cases where ligand-protein docking predicts increased steric clashes between agonists and the residues forming the S1-S2 interface, the agonists clearly show decreased relative efficacy. Furthermore, we demonstrate that the mutation S690A affects both potency and efficacy in an agonist-specific manner. The results indicate that essential residues in the ligand binding pocket of NR2B may adopt different conformations depending on the agonist bound. Together, these data indicate that agonist efficacy at the NR2B subunit can be controlled by the extent of steric clashes between the agonist and the ligand binding domains and by ligand-dependent arrangements of residues within the binding pocket.

KW - Amino Acid Substitution

KW - Animals

KW - Binding Sites

KW - Electrophysiology

KW - Glutamic Acid

KW - Models, Molecular

KW - Mutagenesis, Site-Directed

KW - Oocytes

KW - Rats

KW - Receptors, N-Methyl-D-Aspartate

KW - Transduction, Genetic

KW - Xenopus laevis

U2 - 10.1124/mol.105.014795

DO - 10.1124/mol.105.014795

M3 - Journal article

C2 - 16131614

VL - 68

SP - 1510

EP - 1523

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 0026-895X

IS - 6

ER -