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• W2159540841 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. 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 2The abbreviations used are: AMPA2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acidATPA2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acidthio-ATPA2-amino-3-(3-hydroxy-5-tert-butyl-4-isothiazolyl)propionic acidGluRionotropic glutamate receptorGluR2i/GluR2oGluR of the flip or flop splice variantNFRnormal frog RingerQ/R sitesite where a crucial amino acid residue in the channel pore region can be either glutamine or arginine. 2The abbreviations used are: AMPA2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acidATPA2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acidthio-ATPA2-amino-3-(3-hydroxy-5-tert-butyl-4-isothiazolyl)propionic acidGluRionotropic glutamate receptorGluR2i/GluR2oGluR of the flip or flop splice variantNFRnormal frog RingerQ/R sitesite where a crucial amino acid residue in the channel pore region can be either glutamine or arginine. receptors (for review, see Refs. 1Hollmann M. Heinemann S. Annu. Rev. Neurosci. 1994; 17: 31-108Crossref PubMed Scopus (3638) Google Scholar, 2Dingledine R. Borges K. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar, 3Brauner-Osborne H. Egebjerg J. Nielsen E.O. Madsen U. Krogsgaard-Larsen P. J. Med. Chem. 2000; 43: 2609-2645Crossref PubMed Scopus (526) Google Scholar, 4Gouaux E. J. Physiol. (Lond.). 2004; 554: 249-253Crossref Scopus (124) Google Scholar, 5Mayer M.L. Armstrong N. Annu. Rev. Physiol. 2004; 66: 161-181Crossref PubMed Scopus (322) Google Scholar). The AMPA receptor class consists of four members, GluR1 through GluR4, where the subunits associate as two dimers (6Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (785) Google Scholar, 7Ayalon G. Stern-Bach Y. Neuron. 2001; 31: 103-113Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 8Mansour M. Nagarajan N. Nehring R.B. Clements J.D. Rosenmund C. Neuron. 2001; 32: 841-853Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 9Robert A. Irizarry S.N. Hughes T.E. Howe J.R. J. Neurosci. 2001; 21: 5574-5586Crossref PubMed Google Scholar, 10Bowie D. Lange G.D. J. Neurosci. 2002; 22: 3392-3403Crossref PubMed Google Scholar) in a homomeric or heteromeric tetrameric receptor complex (11Rosenmund C. Stern-Bach Y. Stevens C.F. Science. 1998; 280: 1596-1599Crossref PubMed Scopus (644) Google Scholar, 12Chen G.Q. Cui C. Mayer M.L. Gouaux E. Nature. 1999; 402: 817-821Crossref PubMed Scopus (138) Google Scholar) forming a central ion-permeable pore. 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid 2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid 2-amino-3-(3-hydroxy-5-tert-butyl-4-isothiazolyl)propionic acid ionotropic glutamate receptor GluR of the flip or flop splice variant normal frog Ringer site where a crucial amino acid residue in the channel pore region can be either glutamine or arginine. 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid 2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid 2-amino-3-(3-hydroxy-5-tert-butyl-4-isothiazolyl)propionic acid ionotropic glutamate receptor GluR of the flip or flop splice variant normal frog Ringer site where a crucial amino acid residue in the channel pore region can be either glutamine or arginine. 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 (13Koike M. Tsukada S. Tsuzuki K. Kijima H. Ozawa S. J. Neurosci. 2000; 20: 2166-2174Crossref PubMed Google Scholar). High resolution x-ray structures of isolated ligand binding cores from GluR2 (Fig. 1A) in complex with different agonists and antagonists (e.g. Refs. 6Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (785) Google Scholar and 14Hogner A. Kastrup J.S. Jin R. Liljefors T. Mayer M.L. Egebjerg J. Larsen I.K. Gouaux E. J. Mol. Biol. 2002; 322: 93-109Crossref PubMed Scopus (147) Google Scholar, 15Hogner A. Greenwood J.R. Liljefors T. Lunn M.L. Egebjerg J. Larsen I.K. Gouaux E. Kastrup J.S. J. Med. Chem. 2003; 46: 214-221Crossref PubMed Scopus (94) Google Scholar, 16Furukawa H. Gouaux E. EMBO J. 2003; 22: 2873-2885Crossref PubMed Scopus (391) Google Scholar) 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 domains, thereby resulting in the largest separation of the linker connecting the transmembrane region and the binding core. Partial agonists like kainate but also antagonists induce lower domain closure and a subsequent smaller separation of the linker region (6Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (785) Google Scholar, 14Hogner A. Kastrup J.S. Jin R. Liljefors T. Mayer M.L. Egebjerg J. Larsen I.K. Gouaux E. J. Mol. Biol. 2002; 322: 93-109Crossref PubMed Scopus (147) Google Scholar, 15Hogner A. Greenwood J.R. Liljefors T. Lunn M.L. Egebjerg J. Larsen I.K. Gouaux E. Kastrup J.S. J. Med. Chem. 2003; 46: 214-221Crossref PubMed Scopus (94) Google Scholar, 17Jin R. Banke T.G. Mayer M.L. Traynelis S.F. Gouaux E. Nat. Neurosci. 2003; 6: 803-810Crossref PubMed Scopus (335) Google Scholar). Both (S)-glutamate and kainate interact with the receptor through a number of direct hydrogen bonds together with a number of water-mediated hydrogen-bond interactions (6Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (785) Google Scholar). Most notably are the highly conserved interactions between the α-carboxyl group and Arg-485 together with those between the α-amino group and Pro-478, Thr-480, and Glu-705 in GluR2 (6Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (785) Google Scholar). Examination of the crystal structures of co-complexes with different agonists and the ligand binding core of GluR2 suggests that two residues, Tyr-450 and Leu-650, without direct hydrogen bonding to the agonist are located at critical positions for the domain closure. Both Tyr-450 and Leu-650 appear in the crystal structure as wedges between the agonist and domain 1 and domain 2, respectively (Fig. 1). The importance of Leu-650 on the receptor kinetics has been studied in a number of different systems. 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 (18Armstrong N. Mayer M. Gouaux E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5736-5741Crossref PubMed Scopus (131) Google Scholar). Madden et al. (19Madden D.R. Cheng Q. Thiran S. Rajan S. Rigo F. Keinanen K. Reinelt S. Zimmermann H. Jayaraman V. Biochemistry. 2004; 43: 15838-15844Crossref PubMed Scopus (22) Google Scholar) 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)) (19Madden D.R. Cheng Q. Thiran S. Rajan S. Rigo F. Keinanen K. Reinelt S. Zimmermann H. Jayaraman V. Biochemistry. 2004; 43: 15838-15844Crossref PubMed Scopus (22) Google Scholar). 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 (19Madden D.R. Cheng Q. Thiran S. Rajan S. Rigo F. Keinanen K. Reinelt S. Zimmermann H. Jayaraman V. Biochemistry. 2004; 43: 15838-15844Crossref PubMed Scopus (22) Google Scholar). Tyr-450 is conserved among all AMPA and kainate receptors (20Pentikainen O.T. Settimo L. Keinanen K. Johnson M.S. Biochem. Pharmacol. 2003; 66: 2413-2425Crossref PubMed Scopus (20) Google Scholar), and even the prokaryotic GluR0 harbors a tyrosine at the equivalent position (12Chen G.Q. Cui C. Mayer M.L. Gouaux E. Nature. 1999; 402: 817-821Crossref PubMed Scopus (138) Google Scholar). Furthermore, it has been suggested that kainate only induces partial domain closure, primarily because its isopropyl group acts as a “foot in the door,” colliding with Tyr-450 and Leu-650 (18Armstrong N. Mayer M. Gouaux E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5736-5741Crossref PubMed Scopus (131) Google Scholar). We hypothesized that changing the corresponding tyrosine in the full-length GluR2 would affect the properties of the receptor significantly. By combining detailed electrophysiological characterization with high resolution structural information, we examined this hypothesis for GluR2, representing the AMPA family of the ionotropic glutamate receptors. Glutamate Receptor Ligands and Reagents—(S)-Glutamate, kainate, and all other reagents were purchased from regular commercial sources. Mutagenesis—Mutations in the full-length GluR2 receptor were introduced by the standard overlap polymerase chain reaction method using mutagenic primers designed with the desired mutations. We used GluR2Qo as template for the reactions, and the mutants were later subcloned into GluR2Qo(L483Y), GluR2Qi, and GluR2Qi(L483Y) using the restriction sites for ApaI and MunI. All recordings were performed on receptors un-edited in the Q/R site (21Sommer B. Kohler M. Sprengel R. Seeburg P.H. Cell. 1991; 67: 11-19Abstract Full Text PDF PubMed Scopus (1163) Google Scholar). Numbering of GluR1 and GluR2 is according to the mature protein and corresponds to numbering previously reported (22Hollmann M. O'Shea-Greenfield A. Rogers S.W. Heinemann S. Nature. 1989; 342: 643-648Crossref PubMed Scopus (774) Google Scholar, 23Armstrong N. Sun Y. Chen G.Q. Gouaux E. Nature. 1998; 395: 913-917Crossref PubMed Scopus (602) Google Scholar). GluR2-S1S2J(Y450A) and GluR2-S1S2J(Y450W) were also constructed by the standard overlap polymerase chain reaction method using the wild-type GluR2-S1S2J construct as template (6Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (785) Google Scholar). After PCR, the resulting fragments were digested with PstI and Asp718 and inserted into the wild-type construct, which had previously been digested with the same restriction enzymes. All mutations were verified by sequencing. Expression, refolding, and purification were done essentially as previously described (23Armstrong N. Sun Y. Chen G.Q. Gouaux E. Nature. 1998; 395: 913-917Crossref PubMed Scopus (602) Google Scholar, 24Chen G.Q. Gouaux E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13431-13436Crossref PubMed Scopus (145) Google Scholar) except that a higher concentration of (S)-glutamate (10 mm) was used in the refolding buffer and in all subsequent steps. Two-electrode Voltage Clamp—A female Xenopus laevis frog was anesthetized, and oocytes were isolated, processed, and injected the following day with 50 nl of in vitro transcribed cRNA. In vitro transcription was done using the mMESSAGE mMACHINE T7 kit from Ambion Diagnostics according to the supplied instructions. Oocytes were kept at 18 °C in Barth's solution, and to increase survival some of the oocytes were kept in normal frog Ringer (NFR; 10 mm HEPES-NaOH, pH 7.4, 115 mm NaCl, 1.8 mm CaCl2, 2.5 mm KCl, 0.1 mm MgCl2) supplemented with 1% serum and 10 μg/ml penicillin, 10 μg/ml streptomycin. Recordings were done 2–12 days after injection in a low calcium Ringer solution (10 mm HEPES-NaOH, pH 7.4, 115 mm NaCl, 0.1 mm CaCl2, 2.5 mm KCl, 1.8 mm MgCl2). For further details, see Nielsen et al. 25. The pH in all solutions was routinely checked. We compensated for sodium ions in the (S)-glutamate solutions, and since it has been shown that external ions influence kainate receptor but not AMPA receptor kinetics (26Bowie D. J. Physiol. (Lond.). 2002; 539: 725-733Crossref Scopus (60) Google Scholar), we argue that any alternations in ionic strength would not influence our results. At the wild-type receptors we used 0.9 mm (S)-glutamate, 1 mm kainate, 2.5 mm (RS)-AMPA, and 1 mm (RS)-ATPA as saturating agonist concentrations. When characterizing the Tyr-450 mutants, we used 30 mm (S)-glutamate, 1 mm kainate, 1 mm (RS)-AMPA, and 1 mm (RS)-ATPA (see TABLE THREE and see Fig. 6). Data under two-electrode voltage clamp were acquired and analyzed as previously described (25Nielsen M.M. Liljefors T. Krogsgaard-Larsen P. Egebjerg J. Mol. Pharmacol. 2003; 63: 19-25Crossref PubMed Scopus (20) Google Scholar, 27Holm M.M. Lunn M.-L. Traynelis S.F. Kastrup J.S. Egebjerg J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12053-12058Crossref PubMed Scopus (29) Google Scholar).TABLE THREEAgonist efficacy on wild-type and mutant forms of GluR2o Agonists were applied in saturating concentrations (see “Experimental Procedures”) to two-electrode voltage-clamped oocytes expressing the different receptor types. The (S)-glutamate-elicited response was set at 100%. Values in parentheses represent S.E. from 4–9 oocytes.Receptor/agonistAgonist efficacy (%)(S)-GlutamateKainateAMPAATPAGluR2o100390 (28)aSelected values for kainate and ATPA have previously been published (27)86 (4.3)180 (14)aSelected values for kainate and ATPA have previously been published (27)GluR2o(L483Y)10031 (3.0)aSelected values for kainate and ATPA have previously been published (27)150 (22)98 (1.0)aSelected values for kainate and ATPA have previously been published (27)GluR2o(Y450A)100440 (24)5.2 (0.72)12 (4.5)GluR2o(Y450A,L483Y)100320 (33)1.0 (0.23)1.8 (0.40)GluR2o(Y450W)100190 (5.8)19 (1.3)33 (1.3)GluR2o(Y450W,L483Y)10047 (2.5)68 (3.7)34 (2.5)a Selected values for kainate and ATPA have previously been published (27Holm M.M. Lunn M.-L. Traynelis S.F. Kastrup J.S. Egebjerg J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12053-12058Crossref PubMed Scopus (29) Google Scholar) Open table in a new tab Rapid Application—Oocytes expressing GluR2 receptors were pretested in the standard two-electrode voltage clamp setup. Only oocytes giving responses >700 nA to 30 μm kainate at -60 mV were selected for further experiments. Before recording the vitelline membrane was removed by incubating the oocyte in hyperosmotic medium (200 mm potassium aspartate, 20 mm KCl, 1 mm MgCl2, 5 mm EGTA-KOH, 10 mm HEPES-KOH, pH 7.4) or, alternatively, 2× NFR. After 10 min of incubation, the vitelline membrane was separated from the oocyte using a pair of fine forceps. As external recording solution we used NFR, and the internal solution was 100 mm KCl, 10 mm EGTA, and 10 mm HEPES, pH 7.4. All agonists were dissolved in NFR. Outside-out patches were excised from the oocyte-membrane using fire-polished borosilicate glass capillaries (outer diameter, 1.5 mm; inner diameter, 1.17 mm; with inner filament) supplied by Harvard apparatus LTD (Kent, UK). The patch pipettes displayed resistances of 2–4 megaohms. Fast application of the agonists was obtained using a piezo-driven (Burleigh Instruments, Fishers, NY) double-barreled theta-glass tube (length, 100 mm; outer diameter, 2.0 mm; wall thickness, 0.3 mm; septum thickness, 0.117 mm) from Hilgenberg GmbH (Malsfeld, Germany). Agonist pulses were applied with a 3-s break between the individual sweeps. To visualize the interface between the two solutions, 10 mm sucrose was added to the agonist solutions except for (S)-glutamate. A 20–80% solution exchange was routinely measured by stepping from 100% into 60% NFR. Values ranged from 100 to 300 μs, which were faster than the rise time of the agonist-activated currents. All patches were clamped at -120 mV, and the currents were recorded with a HEKA EPC9 patch clamp amplifier, sampled at 100 kHz, and filtered at 2.9 kHz using the acquisition program Pulse (both from HEKA electronic GmbH, Lambrecht, Germany). Fast application data were analyzed in Pulse-fit (HEKA) by fitting to the exponential equation At = Amaxe-t/τ + ISS, where At is the measured current at time t, Amax is the maximal current amplitude, ISS is the steady-state current, and τ is the desensitization time constant. The traces were exported from Pulse-fit and drawn in SigmaPlot 3.0 (Jandel Scientific, SPSS Science Inc., Chicago, IL). All traces displayed in the figures are individual raw sweeps. Crystallization—Protein solutions of 8 mg/ml GluR2-S1S2J(Y450W) in 10 mm Hepes pH 7.0, 20 mm NaCl, 1 mm EDTA supplemented with 10 mm (S)-glutamate and kainate, respectively, were used for crystallization. Crystals were grown at 6 °C in 10–20% polyethylene glycol 8000, 0.1 m cacodylate, pH 6.5, 0.1 m Li2SO4 using the hanging-drop method with a 1:1 (v/v) ratio of protein and precipitant solution. Small crystals appeared after a few days and were used for streak seeding in clear drops. Seeded crystals grew to a size of up to 0.1 × 0.1 × 0.05 mm in drops containing kainate, whereas only very small needle-like crystals unsuitable for data collection appeared when the protein was co-crystallized with (S)-glutamate. X-ray Data Collection and Structure Determination—Before data collection crystals of the GluR2-S1S2J(Y450W)-kainate complex were transferred through reservoir solution containing additional 15% glycerol and cryo-cooled using liquid nitrogen. Synchrotron data to 2.1 Å resolution were collected on the beamline I711 at MAX-Lab, Lund, Sweden. The HKL package (Denzo, XdisplayF, and Scalepack) (28Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38252) Google Scholar) was used for autoindexing, integration, and scaling of the data (for statistics on data collection, see supplemental Table I). The structure was solved by molecular replacement using the program CNS (29Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16918) Google Scholar) and the wild-type GluR2-S1S2J-kainate structure (PDB code 1FTK without water molecules and ligand) as template followed by simulated annealing. Further refinement was done by repeated cycles of model building in O (30Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar) and maximum-likelihood refinement and individual B-factor refinement using CNS until Rfree converged. Residues 390–506 from S1, a short linker consisting of the amino acids Gly and Thr, followed by residues 632–774 from S2 of the GluR2-S1S2J(Y450W) construct as well as the ligand kainate could be fitted reliably into the electron density. The program Procheck (31Laskowski R.A. MacArthur M.N. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) estimated 91.3% of the residues to be in the allowed region of a Ramachandran plot and 8.7% to be in the generously allowed region. 254 water molecules were located in the electron density in hydrogen-bonding distance from the protein molecule. For statistics on refinement, see supplemental Table I. The importance of the proposed steric interaction between agonists and the Tyr-450 in GluR2 was investigated by mutating the residue to the smaller alanine or the larger tryptophan. Mutations at Tyr-450 Affect Agonist Potencies Making GluR2(Y450A) Kainate-selective—Homomeric receptors composed of GluR2o and GluR2i wild-type or mutant subunits were expressed in X. laevis oocytes, and dose-response relationships were established under a two-electrode voltage clamp (TABLE ONE, Figs. 2 and 3). These experiments revealed a 30- and 60-fold increase in EC50 for (S)-glutamate at GluR2i(Y450W) and GluR2o(Y450W), respectively, as compared with the wild-type receptors. On the other hand, the potency of kainate increased modestly, 2-fold or less for GluR2o(Y450W) and GluR2i(Y450W).TABLE ONEPotencies of (S)-glutamate and kainate on GluR2i/GluR2o and mutants Relative potencies of (S)-glutamate and kainate when characterized in the two-electrode voltage clamp setup on X. laevis oocytes expressing the respective receptors are shown. At least three individual oocytes were tested for each value.Receptor(S)-GlutamateKainateEC50 (min/max)nHEC50 (min/max)nHGluR2iaEC50 values for (S)-glutamate and kainate on GluR2i/o and GluR2i/o(L483Y) have been published previously (27)30 μm (29/31)1.5170 μm (160/190)1.0GluR2i(L483Y)aEC50 values for (S)-glutamate and kainate on GluR2i/o and GluR2i/o(L483Y) have been published previously (27)10 μm (10/10)1.7170 μm (160/190)1.1GluR2oaEC50 values for (S)-glutamate and kainate on GluR2i/o and GluR2i/o(L483Y) have been published previously (27)5.7 μm (5.2/6.4)1.1130 μm (120/140)1.1GluR2o(L483Y)aEC50 values for (S)-glutamate and kainate on GluR2i/o and GluR2i/o(L483Y) have been published previously (27)9.0 μm (7.6/11)1.6110 μm (98/120)1.1GluR2i(Y450A)12 mm (11/13)2.88.1 μm (7.0/9.4)1.2GluR2i(Y450A,L483Y)9.0 mm (7.6/11)1.82.5 μm (2.1/2.8)1.4GluR2o(Y450A)17 mm (16/19)2.03.1 μm (2.9/3.3)1.3GluR2o(Y450A,L483Y)8.3 mm (8.1/8.6)2.21.9 μm (1.7/2.0)1.4GluR2i(Y450W)880 μm (830/930)1.2130 μm (120/140)1.3GluR2i(Y450W,L483Y)190 μm (180/190)1.586 μm (73/100)1.1GluR2o(Y450W)340 μm (300/380)1.162 μm (61/63)1.3GluR2o(Y450W,L483Y)67 μm (61/73)1.538 μm (32/46)1.3a EC50 values for (S)-glutamate and kainate on GluR2i/o and GluR2i/o(L483Y) have been published previously (27Holm M.M. Lunn M.-L. Traynelis S.F. Kastrup J.S. Egebjerg J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12053-12058Crossref PubMed Scopus (29) Google Scholar) Open table in a new tab FIGURE 3Dose-response relationships for (S)-glutamate and kainate on the GluR2 mutant and wild-type receptors. The agonists were applied to wild-type GluR2 (open circles), GluR2(L483Y) (filled circles), GluR2(Y450A) (open triangles), GluR2(Y450A,L483Y) (filled triangles), GluR2(Y450W) (open squares), and GluR2-(Y450W,L483Y) (filled squares). A, (S)-glutamate tested on the flip variants. As in the other panels responses were normalized to an estimated maximal value and fitted to the Hill equation to estimate the EC50 values and Hill coefficients listed in TABLE ONE. B, (S)-glutamate tested on the flop variants. C and D, kainate tested on the flip and flop variants, respectively. In all four panels at least three oocytes were tested for each combination, and most of the error bars are less than half the size of the symbols used for the respective receptors.View Large Image Figure ViewerDownload Hi-res image Download (PPT) When determining the dose-response relationships for the Y450A mutant, we observed a dramatic reduction in the potency for (S)-glutamate, with an almost 3000-fold shift at GluR2o(Y450A) and a 400-fold shift at GluR2i(Y450A). In contrast, kainate exhibited higher potency at the GluR2(Y450A) mutant, with a 21- and 42-fold decrease in EC50 at GluR2i(Y450A) and GluR2o(Y450A), respectively. Thus, kainate exhibits more than 1400- and 5400-fold higher potencies at GluR2i(Y450A) and GluR2o(Y450A) than (S)-glutamate. Mutation of Leu-507 in GluR3 (corresponding to Leu-483 in GluR2) to any aromatic residue has been shown to block or reduce the conformational changes required for the receptor complex to enter the desensitized state (31Laskowski R.A. MacArthur M.N. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). To investigate the contribution from desensitization, we also determined the potency of the agonists in the presence of the non-desensitizing conferring mutation (32Stern-Bach Y. Russo S. Neuman M. Rosenmund C. Neuron. 1998; 21: 907-918Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). The largest effect was observed for (S)-glutamate on GluR2o(Y450W,L483Y), showing a 5-fold higher potency on this receptor as compared with GluR2o(Y450W). Introduction of L483Y in either of the GluR2(Y450A) mutants changed the potency of both agonists less than 3-fold, showing that differences in the desensitization contributed less to the large shift in the ligand potency than the mutations in position 450 (TABLE ONE, Fig. 3). Mutations at Tyr-450 Change the Desensitization Properties—To determine the kinetic properties of the mutant GluR2 receptors, we used a piezo-driven rapid application setup. At this setup we applied agonists to outside-out patches excised from X. laevis oocytes with a solution exchange less than 300 μs. Given the reduced potency of (S)-glutamate on GluR2o(Y450W) (TABLE ONE), this mutant was activated with high concentrations of (S)-glutamate. 100 mm (S)-glutamate elicited desensitizing currents in response to rapid agonist application but with slower kinetics and a 10-fold higher steady-state/peak ratio compared with the wild-type receptor (Fig. 4). We determined the desensitization time constant,τ,to4.5 ± 0.3 ms and the steady-state/peak ratio to 19 ± 2.3% on GluR2o(Y450W), compared with 2.0 ± 0.2 ms and 1.9 ± 0.4%, respectively, for the wild-type receptor (TABLE TWO). When (S)-glutamate was applied to outside-out patches containing the GluR2i(Y450W) homomeric receptors, the responses were almost non-desensitizing, with a steady-state/peak ratio at 86 ± 0.04% (Fig. 4A).TABLE TWOKinetics of wild-type and mutant GluR2i and GluR2o Desensitization kinetics of wild-type and mutant GluR2 are revealed from outside-out patches isolated from X. laevis oocytes expressing the given homomeric receptors. In this study, at all recordings in the fast application setup (S)-glutamate denotes 100 mm (S)-glutamate, and kainate denotes 1 mm kainate. Different procedures applied in other reports are indicated in table footnotes. ss, steady state; Non-des, completely non-desensitizing currents with ss/peak ∼100%. —, not determined. At least three patches were tested for each value (n ≥ 3).(S)-Glu" @default.
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• W2159540841 date "2005-10-01" @default.
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• W2159540841 title "A Binding Site Tyrosine Shapes Desensitization Kinetics and Agonist Potency at GluR2" @default.
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