Investigators in the LCMN are working on the molecular mechanisms that underlie the function of the glutamate receptor gene family, which encodes AMPA, kainate, and NMDA receptors. These ligand-gated ion channels mediate excitatory synaptic transmission throughout the brain and spinal cord. We use the techniques of structural biology, protein chemistry, neurophysiology and biophysics, and molecular biology. The diversity of our approaches requires investigators and trainees to develop an unusually broad range of skills that cross traditional boundaries in the life sciences. Using these approaches, we have characterized many of the key functional properties of glutamate receptors. Work by the laboratory revealed for the first time the mechanisms that allow NMDA receptor channels to function as Hebbian switches, or coincidence detectors. The key properties of NMDA receptors discovered by the laboratory include voltage-dependent channel block by Mg2+and a high permeability to Ca2+. Studies on AMPA and kainate receptors using concentration jump experiments revealed the unusually rapid kinetics of desensitization and the diverse actions of allosteric modulators, including cyclothiazide, lectins, aniracetam, and polyamines. As our work has progressed, we have been able to incorporate increasing detail into our picture of glutamate receptor function and are now poised to ask mechanistic questions within the context of receptor subunit domains of known structure.

Crystal Structure of the GluR0 Ligand-Binding Core
Mayer
GluR0, a glutamate receptor ion channel from the photosynthetic bacterium Synechocystis PCC 6803, is a likely candidate for the precursor that evolved into the ligand-gated ion channels mediating excitatory synaptic transmission in the vertebrate brain. The ligand-binding core of GluR0 was overexpressed as a soluble protein in E. coli and crystallized. The structure of a glutamate-bound complex was solved by x-ray diffraction to 1.6 Å resolution. The GluR0 structure reveals homology with bacterial periplasmic binding proteins and GluR2 AMPA subtype neurotransmitter receptor ligand-binding cores. However, in each protein, the mechanisms by which bind ligands are bound are distinct. The ligand-binding sites are all formed by a cleft between two globular a/b domains. In GluR0, L-glutamate binds in an extended conformation similar to that observed for glutamine-binding protein (GlnBP). However, the L-glutamate g-carboxyl group interacts exclusively with an asparagine residue (51) in domain 1 while, for GluR2 and GlnBP, the ligand g-carboxyl and g-amide groups interact with domain 2 residues. Ion channel gating for GluR0 is activated by both acidic and neutral amino acids. To address how neutral amino acids are recognized, we solved the structure of the binding site complex with L-serine at 1.9 Å resolution. The structure revealed solvent molecules acting as surrogate ligand atoms, such that the serine OH group, makes solvent-mediated hydrogen bonds with Asn51 via a water molecule that replaces one of the L-glutamate g-carboxyl group oxygen atoms. Such atomic resolution studies provide our first glimpse of the structures that underlie the specificity of ligands for individual receptor subtypes. Current experiments are characterizing additional glutamate receptor domains for crystallographic studies, which will define the mechanisms for ligand selectivity at AMPA, kainate, and NMDA receptors.

GluR0 Gating Mechanisms
Cui, Mayer
The laboratory examined mechanisms regulating the activity of GluR0 by using single-channel recording from out-side-out patches and transiently transfected HEK cells. We have discovered that GluR0 can be independently gated by glutamate, by external protons, and by lowering the external Ca2+ concentration. Single-channel responses gated by either protons or by lowering [Ca2+]o to micromolar concentrations exhibited a voltage-dependent block by external Na+, which was similar to that of glutamate-gated responses. The single-channel activities gated by all three conditions typically displayed two types of single-channel kinetics: long open times interspersed with bursts interrupted by brief closures at high frequency. Proton-gated currents, EC50 = 30 mM (pH 4.5), exhibited a pH-dependent block of the single-channel conductance at -60 mV and were cross-desensitized by 1mM glutamate. Similarly, GluR0 responses activated by lowering [Ca2+]o, IC50 = 10 µM but not [Mg2+]o, were desensitized by 1mM glutamate. The independence of these gating mechanisms was suggested by a binding site mutant, GluR0/R117K, which maintained normal pH and Ca2+ sensitivity while virtually abolishing the activation by glutamate. Although the gating of many ion channels can be modulated by pH or Ca2+, GluR responses gated by protons and blocked by Ca2+ in the absence of ligands have not been reported before. Future experiments will attempt to identify the molecular mechanisms underlying gating by Ca2+, protons, and glutamate.

FIG 11

The dimer surface shown here is mediated by hydrophobic contacts (yellow) as well as by the interaction of a lysine side chain (dark blue) with peptide bond carbonyl oxygens (red). In GluR0, the dimer interface occurs along a crystallographic two-fold axis. In GluR2, there are molecules of both crystallographic and noncrystallographic symmetry. To determine whether the ligand-binding cores dimerize in solution, the Gouaux laboratory performed equilibrium centrifugation analysis. The analysis revealed dimerization of the GluR0 ligand-binding core with a dissociation constant of 0.8 µM; for GluR2, the equilibrium Kd was nearly 1,000 times higher. Earlier work has identified an AMPA receptor mutation that exchanges a tyrosine for leucine side chain, which greatly diminishes the extent of desensitization, similar to the action of the allosteric modulator cyclothiazide. This mutation, as well as those that alter the binding of cyclothiazide, map to the dimer interface of the ligand-binding core as revealed in crystallographic studies. In a series of ongoing experiments in collaboration with the Gouaux laboratory, we are examining changes in the kinetics and the extent of desensitization of mutants that alter the environment of the mutant tyrosine side chain. Our experiments suggest that locking the dimer in a stable conformation prevents desensitization while mutants that disrupt the dimer interface enhance desensitization.

The Role of Ligand-Binding Core Dimers in Glutamate Receptor Gating
Horning, Mayer, Gouaux
Crystal structures of the GluR0 and GluR2 ligand-binding cores revealed the formation of dimers with a nearly identical organization involving contacts exclusively between domain 1.