The loss of an electrostatic contact unique to AMPA receptor ligand binding domain 2 slows channel activation

Ligand-gated ion channels undergo conformational changes that transfer the energy of agonist binding to channel opening. Within ionotropic glutamate receptor (iGluR) subunits, this process is initiated in their bilobate ligand binding domain (LBD) where agonist binding to lobe 1 favors closure of lobe 2 around the agonist and allows formation of interlobe hydrogen bonds. AMPA receptors (GluAs) differ from other iGluRs because glutamate binding causes an aspartate-serine peptide bond in a flexible part of lobe 2 to rotate 180° (flipped conformation), allowing these residues to form cross-cleft H-bonds with tyrosine and glycine in lobe 1. This aspartate also contacts the side chain of a lysine residue in the hydrophobic core of lobe 2 by a salt bridge. We investigated how the peptide flip and electrostatic contact (D655-K660) in GluA3 contribute to receptor function by examining pharmacological and structural properties with an antagonist (CNQX), a partial agonist (kainate), and two full agonists (glutamate and quisqualate) in the wildtype and two mutant receptors. Alanine substitution decreased the agonist potency of GluA3(i)-D655A and GluA3(i)-K660A receptor channels expressed in HEK293 cells and differentially affected agonist binding affinity for isolated LBDs without changing CNQX affinity. Correlations observed in the crystal structures of the mutant LBDs included the loss of the D655-K660 electrostatic contact, agonist-dependent differences in lobe 1 and lobe 2 closure, and unflipped D(A)655-S656 bonds. Glutamate-stimulated activation was slower for both mutants, suggesting that efficient energy transfer of agonist binding within the LBD of AMPA receptors requires an intact tether between the flexible peptide flip domain and the rigid hydrophobic core of lobe 2.     

The relationship between agonist potency and AMPA receptor kinetics

AMPA-type glutamate receptors are tetrameric ion channels that mediate fast excitatory synaptic transmission in the mammalian brain. When agonists occupy the binding domain of individual receptor subunits, this domain closes, triggering rearrangements that couple agonist binding to channel opening. Here we compare the kinetic behavior of GluR2 channels activated by four different ligands, glutamate, AMPA, quisqualate, and 2-Me-Tet-AMPA, full agonists that vary in potency by up to two orders of magnitude. After reduction of desensitization with cyclothiazide, deactivation decays were strongly agonist dependent. The time constants of decay increased with potency, and slow components in the multiexponential decays became more prominent. The desensitization decays of agonist-activated currents also contained multiple exponential components, but they were similar for the four agonists. The time course of recovery from desensitization produced by each agonist was described by two sigmoid components, and the speed of recovery varied substantially. Recovery was fastest for glutamate and slowest for 2-Me-Tet-AMPA, and the amplitude of the slow component of recovery increased with agonist potency. The multiple kinetic components appear to arise from closed-state transitions that precede channel gating. Stargazin increases the slow kinetic components, and they likely contribute to the biexponential decay of excitatory postsynaptic currents.     

Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core

Crystal structures of the GluR2 ligand binding core (S1S2) have been determined in the apo state and in the presence of the antagonist DNQX, the partial agonist kainate, and the full agonists AMPA and glutamate. The domains of the S1S2 ligand binding core are expanded in the apo state and contract upon ligand binding with the extent of domain separation decreasing in the order of apo > DNQX > kainate > glutamate approximately equal to AMPA. These results suggest that agonist-induced domain closure gates the transmembrane channel and the extent of receptor activation depends upon the degree of domain closure. AMPA and glutamate also promote a 180 degrees flip of a trans peptide bond in the ligand binding site. The crystal packing of the ligand binding cores suggests modes for subunit-subunit contact in the intact receptor and mechanisms by which allosteric effectors modulate receptor activity.     

Chemical interplay in the mechanism of partial agonist activation in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, one subtype in the family of ionotropic glutamate receptors, are the main receptors responsible for excitatory signaling in the mammalian central nervous system. Previous studies utilitizing the isolated ligand binding domain of these receptors have provided insight into the role of specific ligand-protein interactions in mediating receptor activation. However, these studies relied heavily on the partial agonist kainate, in which the alpha-amine group is constrained in a pyrrolidine ring. Here we have studied a series of substituted and unsubstituted willardiines with primary alpha-amine groups similar to that of the full agonist glutamate whose activation can be varied depending on the size of the substituent. The specific ligand-protein interactions in the mechanism of partial agonism in this subtype were investigated using vibrational spectroscopy, and the large-scale conformational changes in the ligand binding domain were studied with fluorescence resonance energy transfer (FRET). These investigations show that the strength of the interaction at the alpha-amine group correlates with the extent of cleft closure and extent of activation, with the agonist of higher efficacy showing larger cleft closure and stronger interactions at this group, suggesting that this is one of the mechanisms by which the agonist controls receptor activation.     

Role of the chemical interactions of the agonist in controlling alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor activation

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the main excitatory neurotransmitter receptors in the mammalian central nervous system. Structures of the isolated ligand binding domain of this receptor have provided significant insight into the large-scale conformational changes, which when propagated to the channel segments leads to receptor activation. However, to establish the role of specific molecular interactions in controlling fine details such as the magnitude of the functional response, we have used a multiscale approach, where changes at specific moieties of the agonists have been studied by vibrational spectroscopy, while large-scale conformational changes have been studied using fluorescence resonance energy transfer (FRET) investigations. By exploiting the wide range of activations by the agonists, glutamate, kainate, and AMPA, for the wild type and Y450F and L650T mutants of the GluR2 subtype, and by using the multiscale investigation, we show that the strength of the interactions at the alpha-amine group of the agonist with the protein in all but one case tracks the extent of activation. Since the alpha-amine group forms bridging interactions at the cusp of the ligand binding cleft, this appears to be a critical interaction through which the agonist controls the extent of activation of the receptor.     

Negative cooperativity of glutamate binding in the dimeric metabotropic glutamate receptor subtype 1

Metabotropic glutamate receptor (mGluR) subtype 1 is a Class III G-protein-coupled receptor that is mainly expressed on the post-synaptic membrane of neuronal cells. The receptor has a large N-terminal extracellular ligand binding domain that forms a homodimer, however, the intersubunit communication of ligand binding in the dimer remains unknown. Here, using the intrinsic tryptophan fluorescence change as a probe for ligand binding events, we examined whether allosteric properties exist in the dimeric ligand binding domain of the receptor. The indole ring of the tryptophan 110, which resides on the upper surface of the ligand binding pocket, sensed the ligand binding events. From saturation binding curves, we have determined the apparent dissociation constants (K(0.5)) of representative agonists and antagonists for this receptor (3.8, 0.46, 40, and 0.89 microm for glutamate, quisqualate, (S)-alpha-methyl-4-carboxyphenylglycine ((S)-MCPG), and (+)-2-methyl-4-carboxyphenylglycine (LY367385), respectively). Calcium ions functioned as a positive modulator for agonist but not for antagonist binding (K(0.5) values were 1.3, 0.21, 59, and 1.2 microm for glutamate, quisqualate, (S)-MCPG, and LY367385, respectively, in the presence of 2.0 mm calcium ion). Moreover, a Hill analysis of the saturation binding curves revealed the strong negative cooperativity of glutamate binding between each subunit in the dimeric ligand binding domain. As far as we know, this is the first direct evidence that the dimeric ligand binding domain of mGluR exhibits intersubunit cooperativity of ligand binding.     

On the binding determinants of the glutamate agonist with the glutamate receptor ligand binding domain

Ionotropic glutamate receptors (GluRs) are ligand-gated membrane channel proteins found in the central neural system that mediate a fast excitatory response of neurons. In this paper, we report theoretical analysis of the ligand-protein interactions in the binding pocket of the S1S2 (ligand binding) domain of the GluR2 receptor in the closed conformation. By utilizing several theoretical methods ranging from continuum electrostatics to all-atom molecular dynamics simulations and quantum chemical calculations, we were able to characterize in detail glutamate agonist binding to the wild-type and E705D mutant proteins. A theoretical model of the protein-ligand interactions is validated via direct comparison of theoretical and Fourier transform infrared spectroscopy (FTIR) measured frequency shifts of the ligand's carboxylate group vibrations [Jayaraman et al. (2000) Biochemistry 39, 8693-8697; Cheng et al. (2002) Biochemistry 41, 1602-1608]. A detailed picture of the interactions in the binding site is inferred by analyzing contributions to vibrational frequencies produced by protein residues forming the ligand-binding pocket. The role of mobility and hydrogen-bonding network of water in the ligand-binding pocket and the contribution of protein residues exposed in the binding pocket to the binding and selectivity of the ligand are discussed. It is demonstrated that the molecular surface of the protein in the ligand-free state has mainly positive electrostatic potential attractive to the negatively charged ligand, and the potential produced by the protein in the ligand-binding pocket in the closed state is complementary to the distribution of the electrostatic potential produced by the ligand itself. Such charge complementarity ensures specificity to the unique charge distribution of the ligand.     

A binding site tyrosine shapes desensitization kinetics and agonist potency at GluR2. A mutagenic, kinetic, and crystallographic study

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-A 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.     

Chemistry and conformation of the ligand-binding domain of GluR2 subtype of glutamate receptors

In the present report, using vibrational spectroscopy we have probed the ligand-protein interactions for full agonists (glutamate and alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA)) and a partial agonist (kainate) in the isolated ligand-binding domain of the GluR2 subunit of the glutamate receptor. These studies indicate differences in the strength of the interactions of the alpha-carboxylates for the various agonists, with kainate having the strongest interactions and glutamate having the weakest. Additionally, the interactions at the alpha-amine group of the agonists have also been probed by studying the environment of the non-disulfide-bonded Cys-425, which is in close proximity to the alpha-amine group. These investigations suggest that the interactions at the alpha-amine group are stronger for full agonists such as glutamate and AMPA as evidenced by the increase in the hydrogen bond strength at Cys-425. Partial agonists such as kainate do not change the environment of Cys-425 relative to the apo form, suggesting weak interactions at the alpha-amine group of kainate. In addition to probing the ligand environment, we have also investigated the changes in the secondary structure of the protein. Results clearly indicate that full agonists such as glutamate and AMPA induce similar secondary structural changes that are different from those of the partial agonist kainate; thus, a spectroscopic signature is provided for identifying the functional consequences of a specific ligand binding to this protein.     

Interactions Between N-Acetylaspartylglutamate and AMPA, Kainate, and NMDA Binding Sites

Abstract: The structure of N -acetylaspartylglutamate (NAAG) suggests this neuronal dipeptide as a candidate for interaction with discrete subclasses of ionotropic and metabotropic acidic amino acid receptors. A substantial difficulty in the assay of these interactions is posed by membrane-bound peptidase activity that converts the dipeptide to glutamate and N -acetylaspartate, molecules that will interfere with receptor assays. We have developed two sets of unique receptor assay conditions and applied one standard assay to measure the interactions, under equilibrium binding conditions, of [³H]kainate, [³H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([³H]AMPA), and [³H]CGS-19755 with the three classes (kainate, quisqualate, and N -methyl- d -aspartate) of ionotropic glutamate receptors, while inhibiting peptidase activity against NAAG. Under these conditions, NAAG exhibits apparent inhibition constants (IC₅₀) of 500, 790, and 8.8 µ M in the kainate, AMPA, and CGS-19755 receptor binding assays, respectively. Glutamate was substantially more effective and less specific in these competition assays, with inhibition constants of 0.36, 1.1, and 0.37 µ M . These data support the hypothesis that, relative to glutamate, NAAG functions as a specific, low potency agonist at N -methyl- d -aspartate subclass of ionotropic acidic amino acid receptors, but the peptide is not likely to activate directly the kainate or quisqualate subclasses of excitatory ionotropic receptors under physiologic conditions.     

Mechanisms for ligand binding to GluR0 ion channels: crystal structures of the glutamate and serine complexes and a closed apo state

High-resolution structures of the ligand binding core of GluR0, a glutamate receptor ion channel from Synechocystis PCC 6803, have been solved by X-ray diffraction. The GluR0 structures reveal homology with bacterial periplasmic binding proteins and the rat GluR2 AMPA subtype neurotransmitter receptor. The ligand binding site is formed by a cleft between two globular alpha/beta domains. L-Glutamate binds in an extended conformation, similar to that observed for glutamine binding protein (GlnBP). However, the L-glutamate gamma-carboxyl group interacts exclusively with Asn51 in domain 1, different from the interactions of ligand with domain 2 residues observed for GluR2 and GlnBP. To address how neutral amino acids activate GluR0 gating we solved the structure of the binding site complex with L-serine. This revealed solvent molecules acting as surrogate ligand atoms, such that the serine OH group makes solvent-mediated hydrogen bonds with Asn51. The structure of a ligand-free, closed-cleft conformation revealed an extensive hydrogen bond network mediated by solvent molecules. Equilibrium centrifugation analysis revealed dimerization of the GluR0 ligand binding core with a dissociation constant of 0.8 microM. In the crystal, a symmetrical dimer involving residues in domain 1 occurs along a crystallographic 2-fold axis and suggests that tetrameric glutamate receptor ion channels are assembled from dimers of dimers. We propose that ligand-induced conformational changes cause the ion channel to open as a result of an increase in domain 2 separation relative to the dimer interface.     

Discrimination between agonists and antagonists by the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-selective glutamate receptor. A mutation analysis of the ligand-binding domain of GluR-D subunit

The crystal structures of the ligand-binding core of the agonist complexes of the glutamate receptor-B (GluR-B) subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor indicate that the distal anionic group of agonist molecules are stabilized by interactions with an N-terminal region of an alpha-helix (helix F) in the lobe 2 ("domain 2," Armstrong, N., and Gouaux, E. (2000) Neuron 28, 165-181) of the two-lobed ligand-binding domain. We used site-directed mutagenesis to further analyze the role of this region in the recognition of both agonists and antagonists by the AMPA receptor. Wild-type and mutated versions of the ligand-binding domain of GluR-D were expressed in insect cells as secreted soluble polypeptides and subjected to binding assays using [(3)H]AMPA, an agonist, and [(3)H]Ro 48-8587 (9-imidazol-1-yl-8-nitro-2,3,5,6-tetrahydro[1,2,4]triazolo[1,5-c] quinazoline-2,5-dione), a high affinity AMPA receptor antagonist, as radioligands. Single alanine substitutions at residues Leu-672 and Thr-677 severely affected the affinities for all agonists, as seen in ligand competition assays, whereas similar mutations at residues Asp-673, Ser-674, Gly-675, Ser-676, and Lys-678 selectively affected the binding affinities of one or two of the agonists. In striking contrast, the binding affinities of [(3)H]Ro 48-8587 and of another competitive antagonist, 6,7-dinitroquinoxaline-2,3-dione, were not affected by any of these alanine mutations, suggesting the absence of critical side-chain interactions. Together with ligand docking experiments, our results indicate a selective engagement of the side chains of the helix F region in agonist binding, and suggest that conformational changes involving this region may play a critical role in receptor activation.     

Crystal structures of the GluR5 and GluR6 ligand binding cores: molecular mechanisms underlying kainate receptor selectivity

Little is known about the molecular mechanisms underlying differences in the ligand binding properties of AMPA, kainate, and NMDA subtype glutamate receptors. Crystal structures of the GluR5 and GluR6 kainate receptor ligand binding cores in complexes with glutamate, 2S,4R-4-methylglutamate, kainate, and quisqualate have now been solved. The structures reveal that the ligand binding cavities are 40% (GluR5) and 16% (GluR6) larger than for GluR2. The binding of AMPA- and GluR5-selective agonists to GluR6 is prevented by steric occlusion, which also interferes with the high-affinity binding of 2S,4R-4-methylglutamate to AMPA receptors. Strikingly, the extent of domain closure produced by the GluR6 partial agonist kainate is only 3 degrees less than for glutamate and 11 degrees greater than for the GluR2 kainate complex. This, together with extensive interdomain contacts between domains 1 and 2 of GluR5 and GluR6, absent from AMPA receptors, likely contributes to the high stability of GluR5 and GluR6 kainate complexes.     

Regulation of AMPA receptor gating by ligand binding core dimers

Ionotropic glutamate receptors are tetramers, the isolated ligand binding cores of which assemble as dimers. Previous work on nondesensitizing AMPA receptor mutants, which combined crystallography, ultracentrifugation, and patch-clamp recording, showed that dimer formation by the ligand binding cores is required for activation of ion channel gating by agonists. To define the mechanisms responsible for stabilization of dimer assembly in native AMPA receptors, contacts between the adjacent ligand binding cores were individually targeted by amino acid substitutions, using the GluR2 crystal structure as a guide to design mutants. We show that disruption of a salt bridge, hydrogen bond network, and intermolecular van der Waals contacts between helices D and J in adjacent ligand binding cores greatly accelerates desensitization. Conservation of these contacts in AMPA and kainate receptors indicates that they are important determinants of dimer stability and that the dimer interface is a key structural element in the gating mechanism of these glutamate receptor families.     

Coupled control of desensitization and gating by the ligand binding domain of glutamate receptors

The kinetics of ligand gated ion channels are tuned to permit diverse roles in cellular signaling. To follow high-frequency excitatory synaptic input, postsynaptic AMPA-type glutamate receptors must recover rapidly from desensitization. Chimeras between AMPA and the related kainate receptors demonstrate that the ligand binding domains alone control the lifetime of the desensitized state. Mutation of nonconserved amino acids in the lower lobe (domain 2) of the ligand binding domain conferred slow recovery from desensitization on AMPA receptors, and fast recovery on kainate receptors. Single-channel recordings and a correlation between the rate of deactivation and the rate of recovery across panels of mutant receptors revealed that domain 2 also controls ion channel gating. Our results demonstrate that the same mechanism that ensures fast recovery also sharpens the response of AMPA channels to synaptically released glutamate.     

The nicotinic receptor ligand binding domain

The ligand binding domain (LBD) of the nicotinic acetylcholine receptor has served as a prototype for understanding molecular recognition in the family of neurotransmitter-gated ion channels. During the past fifty years, studies progressed from fundamental electrophysiological analyses of ACh-evoked ion flow, to biochemical purification of the receptor protein, pharmacological measurements of ligand binding, molecular cloning of receptor subunits, site-directed mutagenesis combined with functional analysis and recently, atomic structural determination. The emerging picture of the nicotinic receptor LBD is a specialized pocket of aromatic and hydrophobic residues formed at interfaces between protein subunits that changes conformation to convert agonist binding into gating of an intrinsic ion channel.     

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitro-quinoxaline-2,3-dione ([3H]-CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [3H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD = 67 +/- 9.0 nM and Bmax = 3.56 +/- 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of [3H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [3H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KD1 = 9.0 +/- 3.5 nM, Bmax = 0.15 +/- 0.05 pmol/mg protein, KD2 = 278 +/- 50 nM, and Bmax = 1.54 +/- 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of L-[3H]glutamate to quisqualate receptors and to sites labeled with [3H]AMPA. These results suggest that the non-NMDA glutamate receptor antagonist [3H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.     

A Charge-inverting Mutation in the “Linker” Region of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Alters Agonist Binding and Gating Kinetics Independently of Allosteric Modulators

AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728–4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.     

Dynamics of Cleft Closure of the GluA2 Ligand-binding Domain in the Presence of Full and Partial Agonists Revealed by Hydrogen-Deuterium Exchange

The majority of excitatory neurotransmission in the CNS is mediated by tetrameric AMPA receptors. Channel activation begins with a series of interactions with an agonist that binds to the cleft between the two lobes of the ligand-binding domain of each subunit. Binding leads to a series of conformational transitions, including the closure of the two lobes of the binding domain around the ligand, culminating in ion channel opening. Although a great deal has been learned from crystal structures, determining the molecular details of channel activation, deactivation, and desensitization requires measures of dynamics and stabilities of hydrogen bonds that stabilize cleft closure. The use of hydrogen-deuterium exchange at low pH provides a measure of the variation of stability of specific hydrogen bonds among agonists of different efficacy. Here, we used NMR measurements of hydrogen-deuterium exchange to determine the stability of hydrogen bonds in the GluA2 (AMPA receptor) ligand-binding domain in the presence of several full and partial agonists. The results suggest that the stabilization of hydrogen bonds between the two lobes of the binding domain is weaker for partial than for full agonists, and efficacy is correlated with the stability of these hydrogen bonds. The closure of the lobes around the agonists leads to a destabilization of the hydrogen bonding in another portion of the lobe interface, and removing an electrostatic interaction in Lobe 2 can relieve the strain. These results provide new details of transitions in the binding domain that are associated with channel activation and desensitization.     

Stereochemistry of glutamate receptor agonist efficacy: engineering a dual-specificity AMPA/kainate receptor

Upon agonist binding, the bilobate ligand-binding domains of the ionotropic glutamate receptors (iGluR) undergo a cleft closure whose magnitude correlates broadly with the efficacy of the agonist. AMPA (alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid) and kainate are nonphysiological agonists that distinguish between subsets of iGluR. Kainate acts with low efficacy at AMPA receptors. Here we report that the structure-based mutation L651V converts the GluR4 AMPA receptor into a dual-specificity AMPA/kainate receptor fully activated by both agonists. To probe the stereochemical basis of partial agonism, we have also investigated the correlation between agonist efficacy and a series of vibrational and fluorescence spectroscopic signals of agonist binding to the corresponding wild-type and mutant GluR4 ligand-binding domains. Two signals track the extent of channel activation: the maximal change in intrinsic tryptophan fluorescence and the environment of the single non-disulfide bonded C426, which appears to probe the strength of interactions with the ligand alpha-amino group. Both of these signals arise from functional groups that are poised to detect changes in the extent of channel cleft closure and thus provide additional information about the coupling between conformational changes in the ligand-binding domain and activation of the intact receptor.