On the Mechanisms of α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) Receptor Binding to Glutamate and Kainate

Abstract

The ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of ionotropic glutamate receptors mediates much of the fast excitatory neurotransmission in the central nervous system. The ability of these receptors to shape such responses appears to be due in part to dynamic processes induced by agonists in the ligand-binding domain. Previous studies employing fluorescence spectroscopy and whole cell recording suggest that agonist binding is followed by sequential transitions to one or more distinct conformational states. Here, we used hydrogen-deuterium exchange to determine the mechanisms of binding of glutamate and kainate (full and partial agonists, respectively) to a soluble ligand-binding domain of GluR2. Our results provide a structural basis for sequential state models of agonist binding and the free energy changes of the associated state-to-state transitions. For glutamate, a multi-equilibrium binding reaction was discerned involving distinct ligand docking, domain isomerization, and lobe-locking steps. In contrast, kainate binding involves a simpler dock-isomerization process in which the isomerization equilibrium is shifted dramatically toward open domain conformations. In light of increasing evidence that the stability, in addition to the extent, of domain closure is a critical component of the channel activation mechanism, the differences in domain opening and closing equilibria detected for glutamate and kainate should be useful structural measures for interpreting the markedly different current responses evoked by these agonists.As a consequence of glutamate binding, AMPA 2 receptors produce a rapid flux of cations into post-synaptic neurons that is vital for information transfer in the central nervous system. In addition, these receptors undergo fast deactivation and desensitization along with slower recovery kinetics, which together shape the time course of current decay and restoration (1-6). Structurally, AMPA receptors are tetrameric membranebound proteins composed of modular subunits having two large extracellular domains, a membrane-spanning ion channel, and a C-terminal intracellular region (4, 7-12). One of the extracellular domains, S1S2, is a bilobed structure that binds agonists in a cleft between its lobes (13). Domain closure of S1S2 around the agonist leads to channel opening, and maximum currents have been attributed to complete lobe closure upon complex formation with full agonists (13-17). However, among the full agonists examined, those like glutamate, which are less potent and which bind to S1S2 with lower affinity, produce higher rates of channel deactivation and resensitization (18), despite inducing the same degree of cleft closure in S1S2. This suggests that AMPA receptor function is dependent not only on the extent of lobe closure caused by the agonist but also on the lobe closing and opening dynamics associated with agonist binding and dissociation (19).A prevailing model for the binding reaction is

Mechanisms of Modal Activation of GluA3 Receptors

“…When full agonists are bound, the Asp655/Ser656 peptide bond flip results in the formation of two H-bonds between the carbonyl of Ser656 (lobe 2) and the amide of Gly453 (lobe 1) and between the carbonyl of Asp655and the amide of Tyr452 through a water molecule. The fully closed, H-bonding pattern exists on the time scale of approximately 100 ms (Fenwick and Oswald, 2010), which is the time scale of modal gating. The number and arrangement of subunits bound to agonist and in the H-bond locked state could determine the mode.…”

“…However, NMR studies and crystal structures of mutant GluA2 LBDs have suggested that the stability of the closed lobe may also contribute to agonist efficacy. Agonist binding consists of at least three steps: 1) binding to lobe 1, 2) closure of the lobes and contacts with lobe 2, and 3) interlobe contacts (including H-bonds) to stabilize the closed lobe form (Abele et al, 2000;Armstrong and Gouaux, 2000;Cheng et al, 2005;Robert et al, 2005;Weston et al, 2006;Zhang et al, 2008a;Fenwick and Oswald, 2010). Lobe closure is required for channel gating, but the degree of lobe closure that can trigger gating is not clear (i.e., does gating require a full closure?…”

Dynamics of Cleft Closure of the GluA2 Ligand-binding Domain in the Presence of Full and Partial Agonists Revealed by Hydrogen-Deuterium Exchange

“…After an initial control spectrum was taken in water, the protein was lyophilized and resuspended in D 2 O. A series of 1 H, 15 N HSQC/TROSY experiments (generally 12 sequential experiments) was performed at each of several temperatures, starting at 10°C up to 25°C (16).…”

“…The protein was then concentrated and stored in 20 mM sodium acetate, 25 mM sodium chloride, 1 mM sodium azide, and 10 mM glutamate at pH 5.5. The protein used for the glutamate (10 mM) and kainate experiments was prepared as described previously (16). AMPA (5 mM), kainate (10 mM), IW (2 mM), FW (2 mM), and NW (2 mM) were exchanged for glutamate by successive concentration and dilution.…”

“…We used NMR measurements of hydrogen-deuterium (HD) exchange to probe the equilibrium between the formation and breakage of hydrogen bonds (15) that stabilize GluA2 LBD lobe closure (16) and to illustrate the differences caused by the binding of full and partial agonists. We found that binding of agonist involves not only formation of new H-bonds within the binding site but also changes in the stability of H-bonding outside of the binding site that affect activation, deactivation, and desensitization of the receptor.…”