Structural and Single-Channel Results Indicate That the Rates of Ligand Binding Domain Closing and Opening Directly Impact AMPA Receptor Gating

Abstract

At most excitatory central synapses, glutamate is released from presynaptic terminals and binds to postsynaptic AMPA receptors, initiating a series of conformational changes that result in ion channel opening. Efficient transmission at these synapses requires that glutamate binding to AMPA receptors results in rapid and near-synchronous opening of postsynaptic receptor channels. In addition, if the information encoded in the frequency of action potential discharge is to be transmitted faithfully, glutamate must dissociate from the receptor quickly, enabling the synapse to discriminate presynaptic action potentials that are spaced closely in time.The current view is that the efficacy of agonists is directly related to the extent to which ligand binding results in closure of the binding domain. For glutamate to dissociate from the receptor, however, the binding domain must open. Previously, we showed that mutations in glutamate receptor subunit 2 that should destabilize the closed conformation not only sped deactivation but also altered the relative efficacy of glutamate and quisqualate. Here we present x-ray crystallographic and single-channel data that support the conclusions that binding domain closing necessarily precedes channel opening and that the kinetics of conformational changes at the level of the binding domain importantly influence ion channel gating. Our findings suggest that the stability of the closed-cleft conformation has been tuned during evolution so that glutamate dissociates from the receptor as rapidly as possible but remains an efficacious agonist.

Role of Conformational Dynamics in α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) Receptor Partial Agonism

“…Taken together, these data provide strong support for recent proposals that agonist efficacy at AMPA receptors (27), and perhaps kainate receptors (52), is governed not by the extent of closure in a single state of the binding domain but by the relative stability of a range of variously productive closed cleft conformations.…”

“…Based on the x-ray structures, it was initially thought that the extent of cleft closure is the primary mechanism by which agonists mediate receptor activation, i.e., increased cleft closure leads to increased activation (23). However, there were several structures such as those of the glutamate-bound form of the T686A mutant and the structures of the AMPA bound form of the L650T mutant that do not follow this trend (22,(25)(26)(27). Ensemble FRET investigations with the wild type and L650T mutant were consistent with the x-ray structures, further validating these deviations from the cleft closure hypothesis (28,29).…”

“…However, the T686S mutant protein also probed a wider range of cleft closure states that covered more open states than the glutamate bound wild type receptor. Thus, the probability that the T686S mutant was in the closed state was lower than that of the wild type protein when bound to glutamate, and this decrease in probability could lead to lower activation (27,30). These smFRET investigations emphasized the need to study the overall cleft closure landscape and dynamics of the agonistbinding domain to obtain a better insight into the role of cleft closure in activation.…”

Mechanism of AMPA Receptor Activation by Partial Agonists

“…The important structural details of glutamate receptors were determined first by the solution of the structures of the ligand-binding domain (3)(4)(5)(6)(7) and the N-terminal domain (8,9) followed by the structure of the full-length tetrameric GluA2 subtype of AMPA receptor (10). The structures in the presence of various agonists, partial agonists, and antagonists in combination with spectroscopic measurements, electrophysiological measurements, and site-directed mutagenesis have provided a wealth of information on the link between structure and function (11)(12)(13). The binding domain is a bilobed structure to which agonist binds in the cleft between the two lobes.…”

“…Most strikingly, crystal structures of partial agonists of the NMDA receptor show a fully closed lobe (19), leading to the suggestion that partial agonism is fundamentally different for NMDA versus AMPA receptors. An alternative view of partial agonism is that the stability of full lobe closure dictates efficacy (13,15). That is, partial agonists could potentially exist in a dynamic equilibrium among two or more conformations.…”