| The (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole) propionic acid (AMPA) - subtype of glutamate receptor, a ligand-gated ion channel, mediates most of the fast excitatory synaptic transmission in the mammalian central nervous system. In Chapter 2, I present electrophysiological, biochemical and crystallographic studies on the interactions between quisqualate, a high affinity, full agonist, and the GluR2 receptor. Quisqualate induces a fully closed domain conformation in the ligand binding core, similar to what AMPA and glutamate do, reinforcing the concept that full agonists produce similar conformational changes. A detailed comparison among full agonist complexes reveals distinct binding mechanisms, particularly in the region of a hydrophobic pocket that is proximal to the anionic γ-substituents, and demonstrates that water molecules play important roles in mediating agonist-receptor interactions. In Chapters 3 and 4, I report crystallographic and electrophysiological studies on the mechanism of partial agonism on the GluR2 receptor using 5-substituted willardiines, a series of partial agonists that differ by only a single atom. Our studies demonstrate that the GluR2 ligand-binding core can adopt a range of ligand-dependent conformational states that in turn control the open probability of discrete subconductance states of the ion channel. In addition, a novel crystal titration experiment shows that the crystallized ligand-binding core has an affinity for agonists similar to what is observed in solution. In Chapter 5, I report biochemical and crystallographic studies on the interactions between two positive allosteric modulators, CX614 and aniracetam, and the GluR2 ligand-binding core. The co-crystal structures reveal a new binding site for positive modulators of AMPA receptors, which is located in the dimer interface and near the inter-domain hinge regions. We suggest that CX614 and aniracetam stabilize the ligand-binding core in the agonist-bound activated state and slow channel deactivation. At the same time, they stabilize the dimer interface and block channel desensitization. In Chapter 6, I report an efficient over-expression and purification system for the aminoterminal domain (ATD) of GluR2. Large quantities of homogenous GluR2-ATD have provided tremendous help for biochemical and biophysical characterization of ATD. Single crystals of GluR2-ATD have been obtained, which has overcome the major obstacle toward the first crystal structure of an AMPA-ATD. |
