Comparison of the agonist binding site of homomeric, heteromeric, and chimeric GluR1o and GluR3o AMPA receptors

Banke, Tue G.; Schousboe, Arne; Pickering, Darryl S.

doi:10.1002/(sici)1097-4547(19970715)49:2<176::aid-jnr6>3.0.co;2-6

Cited by 13 publications

(14 citation statements)

References 48 publications

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“…In good agreement with the characteristics of KA in previous studies using fluorescent Ca 2+ indicator assays, KA was a pronounced partial agonist at all four AMPA receptors, whereas it displayed full agonism at GluR5Q and GluR6Q [16,17]. In agreement with previous studies, (S)-Quis was a potent full agonist at AMPA receptors displaying >10-fold lower potencies at GluR5Q and GluR6Q, whereas (RS)-ACPA was less discriminative between the AMPA and KA receptors (Tables 1-3) [16,26,27,29,32,[35][36][37][38]. (RS)-ACPA displayed slightly higher potencies at GluR3i and GluR4i than at GluR1i and GluR2Qi but these differences were not significant ( Table 1).…”

Section: Discussionsupporting

confidence: 89%

“…The rank orders of potencies for the seven standard GluR agonists obtained at the six constructed GluR-HEK293 cell lines in the Fluo-4/AM assay are in excellent agreement with previously reported results from studies of GluRs expressed in Xenopus oocytes or mammalian cells and assayed by means of electrophysiology [17,[26][27][28][29][30][31][32][33], fluorescent Ca 2+ indicator dyes [16,17] or in binding assays [16,27,28,[34][35][36][37][38]. The Hill slopes of the agonists in this study were typically between 1.1 and 1.7, which is similar to the values observed for agonists at stable GluR3i-GluR5Q-and GluR6Q-HEK293 cell lines in Fura-2 and Fluo-3 assays [16,17].…”

Section: Discussionsupporting

confidence: 88%

“…(S)-Glu was essentially non-selective amongst the six non-NMDA receptors and equipotent to (RS)-AMPA at GluR1i-GluR4i (Tables 1-3) [16,17,[26][27][28]. KA was an agonist at all six GluRs displaying >10-fold higher potency at GluR6Q than at the other five receptors (Tables 1 and 2).…”

Section: Discussionmentioning

confidence: 99%

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Functional Characterisation of Homomeric Ionotropic Glutamate Receptors GluR1-GluR6 in a Fluorescence-Based High Throughput Screening Assay

Strange¹,

Bräuner‐Osborne²,

Jensen

2006

CCHTS

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We have constructed stable HEK293 cell lines expressing the rat ionotropic glutamate receptor subtypes GluR1(i), GluR2Q(i), GluR3(i), GluR4(i), GluR5Q and GluR6Q and characterised the pharmacological profiles of the six homomeric receptors in a fluorescence-based high throughput screening assay using Fluo-4/AM as a fluorescent Ca2+ indicator. In this assay, the pharmacological properties of nine standard GluR ligands correlated nicely with those previously observed in electrophysiology studies of GluRs expressed in Xenopus oocytes or mammalian cells. The potencies and efficacies displayed by the agonists (S)-glutamate, (S)-quisqualate, kainate, (RS)-AMPA, (RS)-ATPA, (RS)-ACPA] and (S)-4-AHCP at the six GluRs were in concordance with electrophysiological studies. Furthermore, the Ki values exhibited by the competitive antagonists NBQX and (RS)-ATPO were also in agreement with findings of previous studies. Finally, the effects of various concentrations of Ca2+ in the assay buffer and of the allosteric modulators cyclothiazide and concanavalin A on GluR signalling were examined. This study represents the most elaborate functional characterisation of multiple AMPA and KA receptor subtypes in the same assay reported to date. We propose that high throughput screening of compound libraries at the six GluR-HEK293 cell lines could be helpful in the search for structurally and pharmacologically novel ligands acting at the receptors.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionsupporting

confidence: 88%

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Functional Characterisation of Homomeric Ionotropic Glutamate Receptors GluR1-GluR6 in a Fluorescence-Based High Throughput Screening Assay

Strange¹,

Bräuner‐Osborne²,

Jensen

2006

CCHTS

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“…Desensitization of the responses was blocked by coapplication of a saturating concentration of cyclothiazide (100 M) in the presence of a saturating concentration (Ն 22-fold EC 50 ) of agonist. The EC 50 values of the agonists used have been reported previously (Banke et al, 1997;Coquelle et al, 2000;Kizelsztein et al, 2000;Vogensen et al, 2000;Campiani et al, 2001). The efficacies of KA and (S)-CPW399 relative to (S)-glutamate were measured at GluR2(Q) i :KA ϭ 0.212 Ϯ 0.032 (n ϭ 19), (S)-CPW399 ϭ 0.432 Ϯ 0.036 (n ϭ 21), and GluR3 i :KA ϭ 0.242 Ϯ 0.015 (n ϭ 21), (S)-CPW399 ϭ 0.147 Ϯ 0.017 (n ϭ 12).…”

Section: Resultsmentioning

confidence: 99%

Tyr702 Is an Important Determinant of Agonist Binding and Domain Closure of the Ligand-Binding Core of GluR2

Frandsen

Pickering²,

Vestergaard³

et al. 2004

Mol Pharmacol

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Ionotropic glutamate receptors mediate most rapid excitatory synaptic transmission in the mammalian central nervous system, and their involvement in neurological diseases has stimulated widespread interest in their structure and function. Despite a large number of agonists developed so far, few display selectivity among (S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid (AMPA)-receptor subtypes. The present study provides X-ray structures of the glutamate receptor 2 (GluR2)-selective partial agonist (S)-2-amino-3-(1,3,5,6,7-pentahydro-2,4-dioxocyclopenta[e] pyrimidin-1-yl) propanoic acid [(S)-CPW399] in complex with the ligand-binding core of GluR2 (GluR2-S1S2J) and with a (Y702F)GluR2-S1S2J mutant. In addition, the structure of the nonselective partial agonist kainate in complex with (Y702F)GluR2-S1S2J was determined. The results show that the selectivity of (S)-CPW399 toward full-length GluR2 relative to GluR3 is reflected in the binding data on the two soluble constructs, allowing the use of (Y702F)GluR2-S1S2J as a model system for studying GluR2/GluR3 selectivity. Structural comparisons suggest that selectivity arises from disruption of a watermediated network between ligand and receptor. A D1-D2 domain closure occurs upon agonist binding. (S)-CPW399 and kainate induce greater domain closure in the Y702F mutant, indicating that these partial agonists here act in a manner more reminiscent of full agonists. Both kainate and (S)-CPW399 exhibited higher efficacy at (Y702F)GluR2(Q) i than at wild-type GluR2(Q) i . Whereas an excellent correlation exists between domain closure and efficacy of a range of agonists at full-length GluR2 determined by electrophysiology in Xenopus laevis oocytes, a direct correlation between agonist induced domain closure of (Y702F)GluR2-S1S2J and efficacy at the GluR3 receptor is not observed. Although it clearly controls selectivity, mutation of this residue alone is insufficient to explain agonist-induced conformational rearrangements occurring in this variant.Binding of (S)-glutamate to ionotropic glutamate receptors (iGluRs) is a key step in the predominant mechanism of rapid excitatory synaptic transmission among nerve cells within the mammalian central nervous system. iGluRs are important in the development and function of the central nervous system and are implicated in learning and memory formation. Furthermore, iGluRs also seem to be associated with certain neurological and psychiatric diseases (e.g., Alzheimer-type diseases, Parkinson's disease, epilepsy, stroke, amyotrophic lateral sclerosis, and schizophrenia). Therefore, the iGluRs are considered potential drug targets (Dingledine et al., 1999;Brä uner-Osborne et al., 2000). The iGluRs have been divided into three different classes: the (S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid (AMPA), The structures reported in this article have been deposited within the Protein Databank with accession numbers 1SYH, 1SYI, and 1XHY.Article, publication date, and citation information can be found at ...

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“…With this question in mind, we analyzed the properties of homomeric GFP-tagged GluR3 receptors expressed in Xenopus oocytes. GluR3 f lop was chosen because it is one of the most common AMPA receptors in the central nervous system and because it produces functional homomeric receptors that generate large current responses (19).…”

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confidence: 99%

Properties of GluR3 receptors tagged with GFP at the amino or carboxyl terminus

Limón

Reyes-Ruiz

Eusebi

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Anatomical visualization of neurotransmitter receptor localization is facilitated by tagging receptors, but this process can alter their functional properties. We have evaluated the distribution and properties of WT glutamate receptor 3 (GluR3) ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (WT GluR3) and two receptors in which GFP was tagged to the amino terminus (GFP-GluR3) or to the carboxyl terminus (GluR3-GFP). Although the fluorescence in Xenopus oocytes was stronger in the vegetal hemisphere because of localization of internal structures (probable sites of production, storage or recycling of receptors), the insertion of receptors into the plasma membrane was polarized to the animal hemisphere. The fluorescence intensity of oocytes injected with GluR3-GFP RNA was approximately double that of oocytes injected with GFP-GluR3 RNA. Accordingly, GluR3-GFP oocytes generated larger kainate-induced currents than GFP-GluR3 oocytes, with similar EC 50 values. Currents elicited by glutamate, or AMPA coapplied with cyclothiazide, were also larger in GluR3-GFP oocytes. The glutamate-to kainate-current amplitude ratios differed, with GluR3-GFP being activated more efficiently by glutamate than the WT or GFP-GluR3 receptors. This pattern correlates with the slower decay of glutamate-induced currents generated by GluR3-GFP receptors. These changes were not observed when GFP was tagged to the amino terminus, and these receptors behaved like the WT. The antagonistic effects of 6-nitro-7-sulfamoylbenzo-[f]quinoxaline-2,3-dione (NBQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were not altered in any of the tagged receptors. We conclude that GFP is a useful and convenient tag for visualizing these proteins. However, the effects of different sites of tag insertion on receptor characteristics must be taken into account in assessing the roles played by these receptor proteins.fluorescent tag ͉ receptor expression ͉ Xenopus oocytes ͉ kainate ͉ glutamate G lutamate is the main excitatory neurotransmitter in the mammalian central nervous system. Its receptors can either link to a second messenger receptor channel-coupling system (metabotropic) or can themselves form ligand-gated ion channels (ionotropic) (1, 2). The ionotropic glutamate receptors are divided into three groups, according to their differential affinity for the agonists N-methyl-D-aspartate (NMDA), kainate (Kai), and ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) (2). Functional AMPA receptors are made up of homomeric or heteromeric arrangements of the glutamate receptor (GluR)-1 to -4 subunits; their membrane topology indicates an extracellular amino terminus, three transmembrane segments, one membrane reentrant loop, and an intracellular carboxyl-terminal end (3, 4).

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Comparison of the agonist binding site of homomeric, heteromeric, and chimeric GluR1o and GluR3o AMPA receptors

Cited by 13 publications

References 48 publications

Functional Characterisation of Homomeric Ionotropic Glutamate Receptors GluR1-GluR6 in a Fluorescence-Based High Throughput Screening Assay

Functional Characterisation of Homomeric Ionotropic Glutamate Receptors GluR1-GluR6 in a Fluorescence-Based High Throughput Screening Assay

Tyr702 Is an Important Determinant of Agonist Binding and Domain Closure of the Ligand-Binding Core of GluR2

Properties of GluR3 receptors tagged with GFP at the amino or carboxyl terminus

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