α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARsa
) are a subtype of glutamate-gated ion channels that mediate most fast excitatory synaptic transmission by ensuring rapid responses to synaptically released glutamate.1–3
In addition, activity-dependent changes in the number of AMPARs at synapses modulates the strength of synaptic transmission, an essential component of the mechanism underlying various forms of synaptic plasticity including learning and memory.4–8
AMPARs are composed of four modular subunits (GluR1–4 or GluRA–D), each consisting of an amino-terminal domain (NTD) that modulates receptor assembly, a ligand-binding domain (LBD) that gates the pore of the receptor, three transmembrane segments (M1, M3, M4), a reentrant loop (M2) that lines the pore of the channel, and a cytoplasmic C-terminal domain that influences receptor trafficking (Figure S1, Supporting Information
High-resolution crystal structures of an engineered ligand-binding core (S1S2J) with several bound ligands have provided insight into the structure and function of full-length receptors.9,10
Gouaux and co-workers provided the first high-resolution structures of the GluR2 AMPAR ligand-binding core (Figure S1, Supporting Information
These structures revealed that the ligand-binding core, formed from two discontinuous polypeptide segments (Figures S1 and S2, Supporting Information
), adopts a clamshell-like shape that is open in two states, unliganded (apo) and with a competitive antagonist bound. The clamshell is closed with agonist bound. Notably, structurally related ligands within a given class produce distinct degrees of clamshell closure.14–17
Coupled with electrophysiological experiments carried out on full-length receptors, these studies suggested that the degree of closure affects the conductance (ion permeation) of the channel, providing a model for channel gating. In addition to modulating channel biophysics, ligand binding also appears to influence the trafficking of AMPARs. For example, both agonists and antagonists have been shown to induce the internalization of AMPARs from neuronal plasma membranes.18
Although the mechanistic basis for this effect is not understood, it is likely that conformational changes within the ligand-binding domain are translated to the intracellular C-terminal domains, which play a critical role in receptor trafficking.
Quinoxaline-2,3-diones are a major class of competitive AMPAR antagonists, frequently used in studies focused on characterizing the activity of AMPARs.19
Key members of this family of antagonists are 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Recently we reported the development of ANQX, a new member of the family of quinoxaline-2,3-diones containing an ortho
-nitrophenylazide, which irreversibly inhibits AMPARs in the presence of ultraviolet light and provides a means of rapidly inactivating receptors expressed on the surfaces of neurons.20–22
In the absence of ultraviolet light, ANQX reversibly inhibits AMPARs. The mechanism by which ANQX irreversibly antagonizes AMPARs has not been previously described. Here we report that ANQX forms two products upon photolysis in the presence of the GluR2 ligand-binding core (S1S2J). Following irradiation with ultraviolet light, ANQX loses dinitrogen to form a highly reactive nitrene that either reacts intramolecularly to form [1,2,5]oxadiazolo[3,4-G
)-dione 1-oxide (FQX) or intermolecularly to form a covalent adduct with Glu705 located in the binding pocket. The high-resolution crystal structure of FQX bound to the S1S2J revealed that FQX binds in the same orientation and promotes the same degree of closure of the S1S2J around the ligand as the previously reported structures complexed with DNQX and CNQX.12,23
Together, these data indicate a common orientation of quinoxaline-2,3-diones within the AMPAR LBD and reveal the mechanism of receptor photoinactivation with ANQX.