Correlating AMPA receptor activation and cleft closure across subunits: crystal structures of the GluR4 ligand-binding domain in complex with full and partial agonists

Within the central nervous system, most fast excitatory synaptic signals are mediated by ionotropic glutamate receptors (iGluRs) that are selective for the synthetic agonist AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) (1). In addition to their role in neurotransmission, the AMPA receptors (AMPA-Rs) contribute to synaptic plasticity and are thus thought to play important roles in learning and memory (2). AMPA-Rs have also been implicated in neuropathological conditions such as epilepsy, ischemia, neurodegeneration, glioma, and multiple sclerosis (3–5). AMPA-Rs are assembled as tetramers of subunits known as GluR1-GluR4. The composition of the receptors in a given neuron depends on which subunits are expressed, and on a variety of post-transcriptional modifications, including alternative splicing and RNA editing (reviewed in ref. 1). A particularly important modification involves the selective RNA editing of the mRNA encoding the GluR2 subunit, which converts a genetically encoded Gln codon common to all subunits to an Arg codon at position 586 in the mature sequence of GluR2. This editing is nearly 100% efficient in adults. Heteromeric channels that include edited GluR2-R subunits are the most common form of AMPA-R in vivo. They are non-rectifying and conduct predominantly monovalent cations. In contrast, AMPA-Rs that do not include GluR2-R subunits display inward rectification and are permeable to both monovalent ions and Ca2+. Although less widespread, there is increasing evidence that such Ca2+-permeable AMPA-Rs play important roles in both physiological and pathophysiological processes (reviewed in refs. 6, 7). The GluR2 ligand-binding domain (LBD) forms a bilobate structure that binds ligands in a deep cleft located between the two lobes. High-resolution X-ray structures of multiple LBD:ligand complexes have shown that full agonists stabilize a fully closed cleft, while antagonists stabilize an open cleft, and partial agonists induce intermediate degrees of cleft closure (8). Thus, although the ligand-free form of the core LBD exhibits some conformational flexibility (9), the ligand-bound GluR2 structures generally show a direct correlation between the degree of cleft closure and the extent of channel activation (10). Recent LRET-reporter assays suggest that the conformational changes seen in the isolated LBD are reflective of changes that take place in functional receptors (11). However, the same correlation does not hold in the case of the N-methyl-D-aspartate (NMDA)-selective family of ionotropic glutamate receptors. Instead, it has been observed that both full and partial agonists induce a full cleft closure of the NR1 LBD (12). Furthermore, a GluR2 mutation (T686A) has been identified that differentially affects the efficacies of glutamate and quisqualate without altering the extent of cleft closure seen in the isolated LBD (13). To account for this observation, it has been proposed that the T686A mutation destabilizes the closed versus the open cleft conformation. While the fully edited GluR2 subunit plays an important role in the regulation of the Ca2+ permeability of iGluRs (14), the edited GluR2 subunit is poorly expressed and forms only weakly conducting ion channels by itself (15, 16). As a result, functional studies involving GluR2 homomers have been carried out on the GluR2-Q form, taking advantage of the fact that the electrophysiological behavior of the unedited GluR2 subunit is similar to that of the non-GluR2 subunits. To date, our understanding of the role of cleft closure in AMPA-R activation is based on this work, coupled with crystal structures of the GluR2-LBD. In contrast, structures are available for multiple members of the other major families of iGluRs (selective for NMDA and kainate, respectively) (12, 17, 18). As a result, it is important to assess the extent to which the relationship between cleft closure and relative efficacy is a general characteristic of AMPA-R LBDs. To address this question, we have pursued the crystallization and structure determination of the GluR4flip subunit. GluR4 plays a crucial role during the early postnatal phase in the proper development of synaptic circuitry by forming functional channels in response to weak synaptic activity (19). GluR4 subunits are co-expressed with GluR2 in the rat subthalamic cells (20) and in granule cells of the cerebellum and cochlear nucleus (21), and GluR2 and GluR4 are known to form functional heteromeric channels (6, 22). Furthermore, during the development of cerebellar granule cells, expression of the GluR4flip isoform predominates during early development, but remains relatively constant, whereas expression of the GluR4flop isoform, which has different biophysical characteristics, increases with age (23, 24). Studies carried out in cultured hippocampal neurons subjected to sublethal ischemic conditions have shown a shift in AMPA-R expression patterns, characterized by increased GluR4 expression levels, and a greater permeability to Ca2+ ions which could lead to post-ischemic neuronal damage (25). Ultimately, GluR4 may therefore be an important therapeutic target. Here, we present a structural analysis of the GluR4flip LBD core in complexes with the physiological full agonist glutamate and the partial agonist kainate (KA), together with an electrophysiological analysis of the GluR4 channel responses to these agonists. Our studies reveal a dependence of relative agonist efficacy on cleft closure that is qualitatively similar to, but quantitatively distinct from that seen for GluR2. In addition, these structures allow us to analyze the compatibility of trans-subunit dimerization interactions and to investigate stereochemical differences in LBD:ligand interactions. These results emphasize the three-dimensional nature of the activation process in ligand-gated ion channels and the resulting need for high-resolution structural information on fully assembled receptors.