The loss of an electrostatic contact unique to AMPA receptor ligand binding domain 2 slows channel activation.

Glutamate mediates excitatory synaptic transmission in brain by activation of AMPA (GluA1–4), kainate (GluK1–5) and NMDA (GluN1–3) receptors. The molecular diversity of the ionotropic glutamate receptor (iGluR) subfamilies is important for their divergent functional roles. AMPA receptors mediate fast synaptic transmission in the central nervous system, while NMDA channels, with slower activation, high Ca2+ permeability, and voltage-sensitivity to block by external and internal Mg2+ ions, act as coincidence detectors at the synapse (1) and activate transduction mechanisms for nuclear signaling (2–4). Together, AMPA and NMDA receptors mediate activity-dependent synaptic plasticity underlying learning and memory formation (5–8), and Ca2+-permeable AMPA channels contribute to excitotoxic cell death in epilepsy, hypoxic ischemia and neurodegenerative diseases (8–11). Deeper understanding of AMPA receptor activation will be useful for developing agents to regulate sleep and respiration, and to treat some forms of epilepsy and cognitive disorders whose pathological features are related to dysregulation of GluA receptor function. Knowledge of the conformational changes and molecular rearrangements that contribute to agonist activation of GluA receptors is essential to understanding how drugs might be designed to modify channel function. Detailed structures of the ligand binding domains of iGluRs (12–15) showed that ligands bind in a cleft between the two lobes in the LBD (Figure 1A, GluA3 with quisqualate bound). AMPA receptor LBDs bound to full agonists are closed by 20–22° relative to unliganded apo structures, and there is a 180° rotation around a peptide bond in lobe 2 hypothesized to play a role in receptor activation (12, 16). In this flipped position, there is an additional hydrogen bond between the carbonyl of D655 through a water molecule to the amide of Y452 and another between the carbonyl of S656 and the amide of G453 (Figure 1B). D655 also forms a salt bridge with K660 of GluA3 (Figure 1C). This salt bridge is seen GluA structures, and as suggested by primary sequences and observed in crystal structures of other iGluR LBDs (Figure 1D), it appears to be unique to AMPA-type iGluR receptors (14, 15, 17, 18). Figure 1 Structural elements of iGluRs The activation of ligand-gated receptors has been described as a wave of conformational changes starting with the initial interaction of agonist with the receptor and ending in the opening of the channel gate (19, 20). We hypothesized that the D655–K660 salt bridge may contribute to the transmission of the energy of agonist binding to channel opening by tethering D655 to the F-helix in the hydrophobic core of lobe 2 and investigated this by single alanine substitutions at each residue. Comparing wildtype and mutant LBD structures demonstrated the loss of the D655–K660 salt bridge and changes in binding affinity that were ligand-dependent. Electrophysiological analysis revealed increased agonist EC50 values (decreased potency), and glutamate response activation was slower for both mutations. Together these observations provide evidence that the energy of agonist induced conformational changes in the LBD is transmitted more effectively to the channel when the D655–K660 salt bridge is intact, and further support a role for the peptide flip conformational change in AMPA receptor activation.