A Binding Site Tyrosine Shapes Desensitization Kinetics and Agonist Potency at GluR2

Abstract

Binding of an agonist to the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic acid (AMPA) receptor family of the glutamate receptors (GluRs) results in rapid activation of an ion channel. Continuous application results in a non-desensitizing response for agonists like kainate, whereas most other agonists, such as the endogenous agonist (S)-glutamate, induce desensitization. We demonstrate that a highly conserved tyrosine, forming a wedge between the agonist and the N-terminal part of the bi-lobed ligand-binding site, plays a key role in the receptor kinetics as well as agonist potency and selectivity. The AMPA receptor GluR2, with mutations in Tyr-450, were expressed in Xenopus laevis oocytes and characterized in a two-electrode voltage clamp setup. The mutation GluR2(Y450A) renders the receptor highly kainate selective, and rapid application of kainate to outside-out patches induced strongly desensitizing currents. When Tyr-450 was substituted with the larger tryptophan, the (S)-glutamate desensitization is attenuated with a 10-fold increase in steady-state/peak currents (19% compared with 1.9% at the wild type). Furthermore, the tryptophan mutant was introduced into the GluR2-S1S2J ligand binding core construct and co-crystallized with kainate, and the 2.1-Å x-ray structure revealed a slightly more closed ligand binding core as compared with the wild-type complex. Through genetic manipulations combined with structural and electrophysiological analysis, we report that mutations in position 450 invert the potency of two central agonists while concurrently strongly shaping the agonist efficacy and the desensitization kinetics of the AMPA receptor GluR2.The ionotropic glutamate receptors mediate most of the fast excitatory neurotransmission in the central nervous system. These receptors consist of three structural but also pharmacological and physiological distinct receptors classes; N-methyl-D-aspartic acid receptors, kainate receptors, and AMPA 2 receptors (for review, see Refs. 1-5). The AMPA receptor class consists of four members, GluR1 through GluR4, where the subunits associate as two dimers (6 -10) in a homomeric or heteromeric tetrameric receptor complex (11, 12) forming a central ion-permeable pore.The kinetic properties of the receptor activity are highly dependent on the agonist. The full agonist (S)-glutamate and the partial agonist kainate induce markedly distinct responses on the AMPA receptors. (S)-Glutamate elicits large and almost completely desensitizing currents on GluR2 flip and flop, although kainate elicits non-desensitizing currents, also on both splice variants (13).High resolution x-ray structures of isolated ligand binding cores from GluR2 (Fig. 1A) in complex with different agonists and antagonists (e.g. Refs. 6 and 14 -16) have provided a detailed picture of the network of interactions between the receptor and the ligand. Comparisons of the structural and functional information suggest that full agonists like (S)-glutamate induce a tight (20°) closure of the two ligand binding dom...

The Structure of a Mixed GluR2 Ligand-binding Core Dimer in Complex with (S)-Glutamate and the Antagonist (S)-NS1209

“…6,[8][9][10][11][12][13][14][15][16][17][18] This was observed also for the antagonist (S)-ATPO. 19 Similarly, these residues are of major importance for the binding of (S)-NS1209 (Table 3 and Figure 3(c)), even though this antagonist has neither an a-carboxylate nor an a-ammonium group.…”

“…The D1-D1 dimer interface is very similar to that observed in all other GluR2-S1S2J complex structures, both for antagonists and agonists. 6,[8][9][10][11][12][13][14][15][16][17][18][19] Also, no significant differences are observed for residues known to be involved in desensitisation: e.g. Leu483, Ser497 and Asn754.…”

“…Three-dimensional structures have revealed that ligands bind in a crevice between two domains, D1 and D2, composed mainly of residues from segments S1 and S2, respectively. Several complexes of the GluR2 ligand-binding core with various agonists have been published, 6,[8][9][10][11][12][13][14][15][16][17][18] whereas only two antagonists have been reported in complex with GluR2 to date: the 6,7-dinitro-2,3-quinoxalinedione 3,8 (DNQX; PDB codes 1FTL and 1LB9) and the phosphonate derivative of AMPA, Figure 1). …”

Mutations to the Kainate Receptor Subunit GluR6 Binding Pocket That Selectively Affect Domoate Binding

“…This could be occurring through disruption of domain closure, consistent with the proposed role of the homologous site in GluR2 (Leu650). This has been described as a "wedge" in the binding pocket, preventing complete domain closure in the presence of the relatively bulky agonist KA (Holm et al, 2005). Reducing the size of Leu650 and its GluR4 homolog increases the efficacy of the partial agonist KA relative to Glu (Armstrong et al, 2003;Madden et al, 2004).…”

NMR Spectroscopy of the Ligand-Binding Core of Ionotropic Glutamate Receptor 2 Bound to 5-Substituted Willardiine Partial Agonists

“…22 Crystal structures of GluR2 S1S2 were subsequently determined in uncomplexed and several complexed states as well as in different dimeric configurations. 21,[23][24][25][26][27]20,[28][29][30][31] These structures, when viewed alongside electrophysiological measurements of related receptors, have revealed the structural basis of various aspects of AMPA receptor function.…”