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Structural Determinants of Agonist-Specific Kinetics at the Ionotropic Glutamate Receptor 2

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Structural Determinants of Agonist-Specific Kinetics at the Ionotropic Glutamate Receptor 2 Structural determinants of agonist-specific kinetics at the ionotropic glutamate receptor 2 Mai Marie Holm*†, Marie-Louise Lunn*‡, Stephen F. Traynelis†, Jette S. Kastrup‡, and Jan Egebjerg*§¶ *Department of Molecular Biology, C. F. Møllers Alle´Building 130, University of Aarhus, DK-8000 Aarhus, Denmark; †Department of Pharmacology, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322; ‡Department of Medicinal Chemistry, Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen, Denmark; and §Department of Molecular Neurobiology, Lundbeck, Ottiliavej 9, DK-2500 Valby, Denmark Communicated by Stephen F. Heinemann, The Salk Institute for Biological Studies, San Diego, CA, June 30, 2005 (received for review February 7, 2005) Glutamate receptors (GluRs) are the most abundant mediators of evoked current in the standard Xenopus oocyte two-electrode the fast excitatory neurotransmission in the human brain. Agonists voltage clamp setup. will, after activation of the receptors, induce different degrees of A variety of AMPA analogues with distinct functional profiles desensitization. The efficacy of agonists strongly correlates with have been synthesized since AMPA itself was originally described the agonist-induced closure of the ligand-binding domain. How- (13, 14). Thio-ATPA (Fig. 1B) was designed as a 3-isothiazolol ever, the differences in desensitization properties are less well analogue of 2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propi- understood. By using high-resolution x-ray structure of the GluR2 onic acid (ATPA) and is a potent agonist at the kainate receptor flop (GluR2o) ligand-binding core protein in complex with the GluR5 (EC50 at 0.10 ␮M) only maintaining moderate activity on partial glutamate receptor agonist (S)-2-amino-3-(3-hydroxy-5- the AMPA receptors GluR1–4 (EC50 at 5.2–32 ␮M) (15). Inter- tert-butyl-4-isothiazolyl)propionic acid [(S)-thio-ATPA], we show estingly, the current characteristics of (S)-thio-ATPA showed max- that (S)-thio-ATPA induces an 18° closure of the binding core similar imal current levels at 78–170% of those evoked by kainate and to another partial agonist, (S)-2-amino-3-(4-bromo-3-hydroxy-5- 120-1600% of those evoked by ATPA. isoxazolyl)propionic acid [(S)-Br-HIBO]. Despite the similar closure In this work, we explore the novel kinetic behaviors of (S)-thio- of the ligand-binding domain, we find in electrophysiological ATPA and a series of structurally distinct AMPA receptor agonists studies that (S)-thio-ATPA induced a 6.4-fold larger steady-state (Fig. 1B) by comparing the activities at the GluR2 flip (GluR2i) and BIOPHYSICS current than (RS)-Br-HIBO, and rapid agonist applications show flop (GluR2o) splice variants in a rapid agonist application setup that (S)-thio-ATPA induces a 3.6-fold higher steady-state͞peak and under two-electrode voltage clamp of Xenopus laevis oocytes. ratio and a 2.2-fold slower desensitization time constant than These results, combined with analysis of a high-resolution x-ray (RS)-Br-HIBO. Structural comparisons reveal that (S)-Br-HIBO, but structure of the ligand-binding core construct of GluR2 (GluR2– not (S)-thio-ATPA, induces a twist of the ligand-binding core S1S2J) complexed with (S)-thio-ATPA, form the basis of further compared with the apostructure, and the agonist-specific confor- understanding of the kinetic behavior of AMPA receptor agonists. mation of Leu-650 correlates with the different kinetic profiles pointing at a key role in defining the desensitization kinetics. We Materials and Methods conclude that, especially for intermediate efficacious agonists, the GluR Ligands and Reagents. (S)-AMPA, (RS)-2-amino-3-(4-bromo- desensitization properties are influenced by additional ligand- 3-hydroxy-5-isoxazolyl)propionic acid [(RS)-Br-HIBO], (S)-thio- induced factors beyond domain closure. ATPA, and (RS)-ATPA were kindly provided by P. Krogsgaard- Larsen and U. Madsen (both of Danish University of 2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid Pharmaceutical Sciences, Copenhagen). Other reagents were pur- (thio-ATPA) ͉ desensitization ͉ domain closure ͉ crystallization chased from Sigma. he fast excitatory synaptic transmission between nerve cells Mutagenesis. The mutations were introduced by the overlap PCR Tis carried out primarily by ionotropic glutamate receptors method using Pfu polymerase, and all of the constructs were (GluRs), mainly localized in the postsynaptic membrane. The subsequently sequenced and inserted into the pGEMHE (16) GluRs are classified into three functionally and pharmacologi- oocyte expression vector. All recordings were performed on re- cally separate groups based on the potencies of their respective ceptors unedited in the Q͞R site (17). agonists, namely the 2-amino-3-(3-hydroxy-5-methyl-4- isoxazolyl)propionic acid (AMPA), kainate, and N-methyl-D- Two-Electrode Voltage Clamp. Oocytes were isolated and prepared aspartic acid (NMDA) receptors (1, 2). The receptor is activated as described in ref. 18. cRNA was transcribed by using the mMES- to an ion-conducting state upon agonist binding, and this process SAGE mMACHINE T7 kit from Ambion (Austin, TX), according can be followed by desensitization. to the supplier’s instructions. To increase survival, some of the The topological and structural characteristics of the GluRs have oocytes were kept in normal frog Ringer (NFR) (10 mM been intensively studied, and currently the favored structural mod- Hepes⅐NaOH, pH 7.4͞115 mM NaCl͞1.8 mM CaCl2͞2.5 mM els predict the AMPA receptors to form tetramers (3, 4) assembled KCl͞0.1 mM MgCl2) supplemented with 1% serum, 10 ␮g͞ml of a pair of dimers (Fig. 1A) (5–10). The mechanism by which penicillin, and 10 ␮g͞ml streptomycin. Data under two-electrode binding of the agonist to the bilobed ligand-binding core is trans- voltage clamp were acquired and analyzed as described in ref. 18. mitted to the pore region is not understood in detail. However, recent studies of 5-substituted willardiines nicely illustrate that a tight closure of the ligand-binding core of GluR2 more efficiently Abbreviations: AMPA, 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid; ATPA, induces a gating conformation of the receptor complex as com- 2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid; Br-HIBO, 2-amino-3-(4- bromo-3-hydroxy-5-isoxazolyl)propionic acid; GluR, ionotropic glutamate receptor; pared with partial agonists inducing more moderate closure of the GluR2i, GluR2 flip; GluR2o, GluR2 flop; GluR2-S1S2J, ligand-binding core construct of binding core (11). A nondesensitizing receptor, harboring the GluR2; NFR, normal frog Ringer. L483Y mutation in the dimer interface (3, 10), has been used to Data deposition: The atomic coordinates and structure factors have been deposited in the characterize agonists of varying chemical structure on the AMPA Protein Data Bank, www.pdb.org (PDB ID code 2AIX). receptor (11, 12). These studies indicate an apparent correlation ¶To whom correspondence should be addressed. E-mail: [email protected]. between the degree of ligand-binding core closure and maximal © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505522102 PNAS ͉ August 23, 2005 ͉ vol. 102 ͉ no. 34 ͉ 12053–12058 Downloaded by guest on September 30, 2021 time constant. All traces displayed in the figures are individual responses. Expression and Purification of GluR2-S1S2J. The GluR2-S1S2J con- struct was kindly provided by E. Gouaux (Columbia University, New York), and the protein was expressed, refolded, and purified essentially as described in refs. 6, 19, and 20. [3H]AMPA Binding. Ligand binding was performed as described in ref. 19. Briefly, for saturation binding refolded GluR2–S1S2J protein (0.08 mg͞ml) was incubated for1honicewith1–200 nM [3H]AMPA (11.1 Ci͞mmol; 1 Ci ϭ 37 GBq) (NEN) in 500 ␮lof binding buffer (100 mM thiocyanate͞2.5 mM CaCl2͞30 mM Tris- HCl pH 7.2). Competition experiments were performed under similar conditions, by using 20 nM [3H]AMPA and 0.2 nM to 1 mM (S)-thio-ATPA. Nonspecific binding was determined in the pres- ence of 1 mM glutamate. The binding experiments were performed in triplicate. Ki values were calculated by using the Cheng–Prusoff equation (21). Crystallization. GluR2–S1S2J in complex with (S)-thio-ATPA was crystallized by the hanging drop vapor diffusion method at 6°C. The protein complex solution contained 5.6 mg͞ml GluR2–S1S2J and 1.6 mg͞ml (S)-thio-ATPA (molar ratio 1:32, in 10 mM Hepes, pH 7.4͞20 mM NaCl͞1 mM EDTA). Crystals were obtained in drops Fig. 1. GluRs and agonists. (A) Diagram of a single GluR subunit highlighted consisting of 1 ␮l of complex solution and 1 ␮l of reservoir solution from the tetrameric assembly. X-ray structures revealed that the ligand- of 0.1 M NaCl, 0.1 M cacodylate buffer (pH 6.5), and 20% binding core is comprised of the two lobes denoted D1 and D2. D1 is formed ͞ polyethylene glycol 8000. The reservoir volume was 0.49 ml. The by S1 plus the C-terminal alternatively spliced flip flop region of S2, and D2 is crystals grew within 1 week to a maximum size of 0.1 mm. primarily composed of S2 and a small C-terminal segment of S1. Agonists bind at the interface between the lobes. Scissors indicate the boundary of the GluR2-S1S2J construct. (B) Structures of agonists used in the study. Data Collection. The data of GluR2–S1S2J in complex with (S)- thio-ATPA were collected on beamline I711 at MAX Lab (Lund, Sweden) at 100K by using a MAR345 image plate detector and Patch-Clamp Recording. Oocytes expressing GluR2 receptors were wavelength of 1.0835 Å. The crystal was transferred to a cryoso- pretested in the standard two-electrode voltage clamp setup. As test lution containing (S)-thio-ATPA and 0.1 M NaCl, 0.1 M cacodylate solution, we used 30 ␮M kainate, while the membrane-potential buffer (pH 6.5), 20% polyethylene glycol 8000, and 15% glycerol for was held at Ϫ60 mV.
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