Stereochemistry of Glutamate Receptor Agonist Efficacy:  Engineering a Dual-Specificity AMPA/Kainate Receptor

Madden, Dean R.; Cheng, Qing; Thiran, Shalita; Rajan, Shanti; Rigo, Frank; Keinänen, Kari; Reinelt, Stephan; Zimmermann, Herbert; Jayaraman, Vasanthi

doi:10.1021/bi048447y

Cited by 23 publications

(17 citation statements)

References 28 publications

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“…There is a strong correlation between the domain closure induced by an agonist and the efficacy (5,6,14), whereas the correlation to the desensitization kinetics and the steady-state/peak ratios also depend on other properties of the ligand-receptor interaction than the domain closure (19,27). For agonists of similar structural scaffolds, as e.g.…”

Section: Discussionmentioning

confidence: 99%

“…Substituting Leu-650 with a threonine decreases the potency of (S)-glutamate, AMPA, and quisqualate 8 -70-fold, but conversely, the mutation increases the potency of kainate by 3-fold (18). Madden et al (19) recently reported a Fourier transform infrared difference spectroscopy study on the soluble GluR4 ligand binding core with the GluR4(L651V) mutation (equivalent to GluR2(Leu-650)) (19). Notably, they revealed that receptor kinetics could not alone be attributed to a rigid body movement of the two binding domains but that a rearrangement of GluR4(Leu-651) also contributed to the distinct kinetics induced by different agonists (19).…”

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confidence: 99%

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

Holm

Naur

Vestergaard

et al. 2005

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Holm

Naur

Vestergaard

et al. 2005

Journal of Biological Chemistry

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“…For the S1S2-D construct, affinity and ion-exchange purifications in the absence of glutamate were followed by serial dialysis against glutamate-free SAXS buffers (overall volumetric dilution Նϳ1.5 ϫ 10 7 -fold). This protocol has been used in previous spectroscopic studies that would have been sensitive to the presence of significant levels of bound agonist: none was detected (9,25,26). For the S1S2J and S1S2L constructs, 1 mM glutamate was present in the samples following ion-exchange purification, but was removed by serial dialysis against glutamate-free SAXS buffers (overall volumetric dilution Ն10 13 -fold).…”

Section: Methodsmentioning

confidence: 99%

Solution X-ray Scattering Evidence for Agonist- and Antagonist-induced Modulation of Cleft Closure in a Glutamate Receptor Ligand-binding Domain

Madden

Armstrong

Svergun

et al. 2005

Journal of Biological Chemistry

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“…The Y450F point mutation was introduced into the plasmid using the Stratagene QuikChange Site-Directed Mutagenesis kit (Stratagene), and the final construct was verified by sequencing the coding region of the DNA. For the electrophysiological measurements, the wild type and mutant receptors were expressed in human embryonic kidney 293 (HEK 293) cells (ATCC CRL 1573) using the same protocol as outlined in Madden et al (24). Briefly, the cells were cultured in DMEM supplemented with 10% fetal bovine serum (Life Technologies, Bethesda, MD) and transfections were performed using Fugene 6 (Roche, Indianapolis, IN) transfection reagent, with 1-2 μg of glutamate receptor cDNA and 0.5 μg of plasmid containing the cDNA for green fluorescent protein.…”

Section: Electrophysiological Measurementsmentioning

confidence: 99%

Role of the Chemical Interactions of the Agonist in Controlling α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor Activation

Mankiewicz

Rambhadran

et al. 2007

Biochemistry

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Abstractα-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, in order to establish the role of specific molecular interactions in controlling such fine details 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,Y450F, and L650T mutants and by using the multiscale investigation, we show that the strength of the interactions at the α-amine group of the agonist with the protein in all but one case tracks the extent of activation. Since the α-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.α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, a member of the ionotropic glutamate receptor family, are the main mediators of fast excitatory synaptic transmission in the mammalian central nervous system. Signal transmission is initiated by glutamate binding to an extracellular ligand binding domain, which leads to the formation of cation specific transmembrane channels (1-6). Large scale expression of the isolated extracellular ligand binding domain (S1S2) has led the way for detailed structural studies of this domain. X-ray (7-10), nuclear magnetic resonance (NMR) (4,(11)(12)(13), and vibrational spectroscopic (14-16) investigations using this soluble protein have provided significant insight into the relationship between structure and function in this subtype.The structures of S1S2 show a graded cleft closure conformational change upon binding agonists with varying efficacy in the bilobed ligand binding domain (9,10). The extent of cleft closure induced by a given agonist in most cases exhibits a direct correlation to the extent of activation of the receptor, suggesting that this is one of the possible modes of coupling between the ligand binding domain and opening of the ion channel. Vibrational spectroscopic investigations using the S1S2 protein provide a more detailed view of the specific interactions between the agonist and the extracellular ligand binding domain and their role in the functioning of the receptor. The frequency shifts in the asymmetric carboxylate vibrational mode, which is sensitive to the strength of the non-covalent interactions at this moiety, indicate that partial *Address correspondence to: Vasanthi Jayaraman, Department of Integrative Biolo...

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Stereochemistry of Glutamate Receptor Agonist Efficacy: Engineering a Dual-Specificity AMPA/Kainate Receptor

Cited by 23 publications

References 28 publications

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

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

Solution X-ray Scattering Evidence for Agonist- and Antagonist-induced Modulation of Cleft Closure in a Glutamate Receptor Ligand-binding Domain

Role of the Chemical Interactions of the Agonist in Controlling α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor Activation

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