Mark L. Mayer, PhD, Head, Section on Neurophysiology and Biophysics
Andrew Plested, PhD, Postdoctoral Fellow
Yongneng Yao, PhD, Postdoctoral Fellow
Yadong Yu, PhD, Postdoctoral Fellow
Carla Glasser, BS, Technical Specialist
Alison Heffer, BS, Postbaccalaureate Fellow

Ionotropic glutamate receptors (iGluRs) are membrane proteins that act as molecular pores and mediate signal transmission at the majority of excitatory synapses in the mammalian nervous system. The seven gene families of ionotropic glutamate receptors (iGluRs) in humans encode 18 subunits, which assemble to form three major functional families named after the ligands. In the late 1970s, researchers first used the ligands to identify the iGluR subtypes AMPA, kainate, and NMDA. In view of the receptors' essential role in normal brain function and development and a growing body of evidence that dysfunction of iGluR activity mediates several neurological and psychiatric diseases as well as damage during stroke, our laboratory directs substantial effort to the analysis of GluR function at the molecular level. Atomic resolution structures solved by protein crystallization and X-ray diffraction provide a framework in which to design electrophysiological and biochemical experiments to define the allosteric mechanisms underlying ligand recognition and gating of ion channel activity. The resultant information will allow development of subtype-selective antagonists and allosteric modulators with novel therapeutic applications and reveal the inner workings of a complicated protein machine that plays a pivotal role in brain function.

Crystallographic and functional analysis of glutamate receptor ligand complexes

Mayer; in collaboration with Rosenmund

Ionotropic glutamate receptors perform diverse functions in the nervous system. Accordingly, several receptor subtypes have evolved with different kinetics, ion permeability, expression patterns, and regulation by second messengers. Kainate receptors show slower recovery from desensitization than do AMPA receptors, and their affinities for agonists differ. Based on analysis of ligand-binding domain crystal structures, we identified interdomain interactions in the agonist-bound state that are conserved in kainate receptors but are absent from AMPA receptors. Mutations in GluR6 designed to disrupt these contacts reduced apparent agonist affinity, accelerated receptor deactivation, and increased the rate of recovery from desensitization. Conversely, the introduction of mutations in GluR2 that enabled additional interdomain interactions in the agonist-bound state increased apparent agonist affinity 15-fold and slowed both deactivation and recovery from desensitization. We conclude that interdomain interactions have evolved as a distinct mechanism that contributes to the unique kinetic properties of AMPA and kainate receptors.

Biochemical analysis of NR3 ligand-binding selectivity

Yao, Mayer

The NMDA receptor NR3A subunit is expressed widely in the developing central nervous system of mammals. Co-assembly of NR3A with NR1 and NR2 modifies NMDA receptor-mediated responses, reducing calcium permeability. The ligand binding properties of NR3A were unknown but shape the role played by NR3A when incorporated into native NMDA receptors. Based on amino acid sequence alignments and homology with other glutamate receptor ion channels, we constructed a soluble NR3A ligand-binding domain (NR3A S1S2) expressed in E. coli. After affinity, gel filtration, and ion exchange chromatography, purified NR3A S1S2 behaved as a monomer even at a concentration of 20 mg/ml, as determined by size-exclusion chromatography and dynamic light scattering. NR3A S1S2 has a very high affinity for glycine, with an apparent dissociation constant of 40 nM, 650-fold less than the Kd for NR1. Glutamate, which binds to NR2 subunits, also binds to NR3A but with very low affinity at Kd 9.6 mM; in contrast, binding of glutamate to NR1 was not detectable even at a 300 mM concentration. The antagonist-binding profiles of NR3A and NR1 also show striking differences. CNQX and its analogue CGP78608 both bind to NR3A S1S2 with low µM affinity while the affinity of CGP78608 for NR1 is 1,000-fold higher than for CNQX. Other high-affinity NR1 antagonists also show weak binding to NR3A. Proteolysis protection experiments revealed that CNQX and CGP78608 bind to and stabilize domain 1 of NR3A S1S2 but increase proteolysis of domain 2, indicating that the ligands produce conformational changes distinct from those induced by glycine and D-serine. In ongoing work, we are using X-ray diffraction to determine the structure of the ligand binding cores of NR3A and its partner NR3B for a range of ligand complexes.

COLLABORATORS

David Jane, PhD, University of Bristol, Bristol, UK
Christian Rosenmund, PhD, Baylor College of Medicine, Houston, TX
Peter Schuck, PhD, Protein Biophysics Resource, Division of Bioengineering and Physical Science, ORS, NIH, Bethesda, MD

For further information, contact mlm@helix.nih.gov.