Correlating AMPA Receptor Activation and Cleft Closure across Subunits: Crystal Structures of the GluR4 Ligand-Binding Domain in Complex with Full and Partial Agonists

Abstract

AMPA receptors are glutamate-gated ion channels that are essential mediators of synaptic signals in the central nervous system. They form tetramers that are assembled as combinations of the subunits GluR1-4, each of which contains a ligand-binding domain (LBD). Crystal structures of the GluR2 LBD have revealed an agonist-binding cleft, which is located between two lobes and which acts like a Venus flytrap. In general, agonist efficacy is correlated with the extent of cleft closure. However, recent observations show that cleft closure is not the sole determinant of relative efficacy for glutamate receptors. In addition, these studies have focused on the GluR2 subunit, which is the specific target of a physiologically important RNA-editing modification in vivo. We therefore wished to test the generality of the cleft closure:efficacy correlation for other AMPA-R subunits. Here, we present crystal structures of the GluR4 flip LBD in complex with both full and partial agonists. As for GluR2, both agonists stabilize a closed-cleft conformation, and the partial agonist induces a smaller cleft closure than the full agonist. However, a detailed analysis of LBD:kainate interactions reveals the importance of subtle backbone conformational changes in the ligand-binding pocket in determining the magnitude of agonist-associated conformational changes. Furthermore, the GluR4 subunit exhibits a different correlation between receptor activation and LBD cleft closure than does GluR2. Keywordsionotropic glutamate receptor; AMPA receptor ion channel; GluR4 ligand-binding domain; X-ray crystallography; electrophysiology; subunit dimerization; relative agonist efficacy; conformational change 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 † A.G. was supported in part by the John H. Copenhaver, Jr., and William H. Thomas, M.D., 1952 Fellowship Fund and 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...

Mechanism of AMPA Receptor Activation by Partial Agonists

“…In addition, dynamics near the lobe interface suggest that large-scale lobe motions may be present when the LBD is bound to IW (21,23). In contrast, when the wild type LBD is bound to kainate, the lobes are in a relatively open orientation in all crystal structures to date (4,18,24,25), and this is supported by the average structure determined using NMR spectroscopy (15). The idea has been that the lobes remain separated by a steric clash between the isoprenyl group of kainate and the side chain of Leu-650 (3,18).…”

“…Although the lobes are slightly open relative to glutamate, two interlobe H-bonds (Gly-451-Gly-653 and Tyr-450-H 2 O-Ser-652) produced by a 180°rota-tion of the peptide bond are present in all three copies. These H-bonds are absent in most crystal structures of partial agonists and have been absent in all previously determined structures of kainate-bound AMPA receptors (3,18,24,25). Previous crystal structures have suggested that kainate prevents further lobe closure due to the steric clash between the side chain of Leu-650 with the isoprenyl group of kainate (foot-in-the-door mechanism) (18).…”

“…Furthermore, little evidence for microseconds-to-milliseconds timescale dynamics in the presence of kainate have been observed in NMR studies (23). However, structures of GluA3 (24) and GluA4 (25) in the presence of kainate are more closed than GluA2 and the D651A mutation of GluA3 results in even further closure of the lobes due to a rotation of the side chain of Leu-650 (GluA2 numbering). 3 Cysteine-trapping studies (i.e.…”

Glutamate Receptor Ion Channels: Structure, Regulation, and Function

“…2; see also section V.A). Crystallography studies together with homology modeling of the AMPA receptor subunits GluA1 to GluA4 show that residues that directly interact with ␣-carboxyl and ␣-amino groups of glutamate, AMPA, and kainate are conserved (Armstrong et al, 1998;Armstrong and Gouaux, 2000;Bjerrum et al, 2003, Pentikä inen et al, 2003Gill et al, 2008) (Fig. 2).…”

The Structure of (−)-Kaitocephalin Bound to the Ligand Binding Domain of the (S)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA)/Glutamate Receptor, GluA2

“…The lobe opening of the GluA2 LBD bound to various antagonists or the apo state was measured relative to the A protomer of the structure of GluA2 LBD bound to glutamate (3DP6) as described by Gill et al (32). The distance between the ␤-carbons of P632 on each monomer within a dimer is given.…”