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 construct 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 presence 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. Only oocytes giving responses of Ͼ1 ␮A were a few seconds before flash-cooling. selected for further experiments. Before recording, the vitelline Data were autoindexed and processed with the programs DENZO membrane was removed with forceps after 10-min incubation in and SCALEPACK (HKL Research, Charlottesville, VA) (22). For hyperosmotic medium (200 mM potassium aspartate͞20 mM crystal data and data collection statistics, see Table 4, which is KCl͞1 mM MgCl2͞5 mM EGTA⅐KOH͞10 mM Hepes⅐KOH, pH published as supporting information on the PNAS web site. After 7.4) or 2ϫ NFR. data processing in SCALEPACK, data were transformed to CNS As external recording solution, we used NFR, and the internal format by using the CCP4 suite of programs (23). 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 Structure Determination and Refinements. The structure was solved excised from the oocyte membrane by using fire-polished borosili- by molecular replacement using the program AMORE (24) from cate glass capillaries (outer diameter, 1.5 mm; inner diameter, 1.17 CCP4. The structure of GluR2–S1S2J in complex with the ligand mm; with inner filament) supplied by Harvard Apparatus. The (S)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)-4- patch-pipettes displayed resistances of 2–4 M⍀. Fast application of isoxazolyl]propionic acid [(S)-2-Me-Tet-AMPA] (12) was used as the agonists was obtained by using a piezo-driven (Burleigh Instru- search model for phasing the data of the (S)-thio-ATPA complex, ments, Fishers, NY) double-barreled theta-glass tube (length, 100 including protein atoms only. mm; outer diameter, 2.0 mm; wall thickness, 0.3 mm; septum Initially, the amino acid residues of GluR2–S1S2J were traced by thickness, 0.117 mm) from Hilgenberg (Malsfeld, Germany). Ago- using ARP͞WARP (25), except for 17 amino acids, which were nists were applied with 3-s intervals. To visualize the interface manually built by using program O ((26)). In addition, the ligand between the two solutions, 10 mM sucrose was added to the agonist molecule (S)-thio-ATPA was fitted to the electron density, and solution of all of the agonists, except for (S)-glutamate. Twenty water molecules were gradually added to the structure. Optimal percent to 80% solution exchange was routinely measured by geometry for the isothiazolol ring of (S)-thio-ATPA was obtained stepping from 100% into 60% NFR. Values ranged from 100 to 300 by using ab initio methods. Parameter and topology files for ␮s. The holding potential for all patches was Ϫ120 mV and the programs O and CNS (Version 1.0) (27) were generated by the currents were recorded with a HEKA EPC9 patch-clamp amplifier, HIC-UP server (28). The structure was refined by using CNS, each sampled at 100 kHz and filtered at 2.9 kHz by using the step comprising positional and B-factor refinements. A summary of acquisition program PULSE (both from HEKA Electronics, Lam- the structure refinements is presented in Table 4. Coordinates have brecht, Germany). been deposited in the Protein Data Bank (PDB ID code 2AIX). The responses were analyzed in PULSE-FIT (HEKA Electronics) The HINGEFIND script (29) implemented in VMD (30) was used for Ϫt/␶ by fitting to the exponential equation At ϭ Amaxe ϩ ISS, where an analysis of ligand-induced D1͞D2 domain closure. The interface At is the measured current at time t, Amax is the maximal current accessible surface area was generated by the Protein–Protein amplitude, ISS is the steady-state current, and ␶ is the desensitization Interaction Server (www.biochem.ucl.ac.uk͞bsm͞PP͞server) (31).

12054 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505522102 Holm et al. Downloaded by guest on September 30, 2021 sponses induced by thio-ATPA under two-electrode voltage clamp (15), we crystallized the soluble form of the ligand-binding core of GluR2o (GluR2–S1S2J) in complex with (S)-thio-ATPA. The correct folding of the GluR2–S1S2J protein was determined phar- macologically by saturation binding of [3H]AMPA and by compet- itive displacement of the [3H]AMPA binding by using (S)-thio- 3 ATPA. The protein was saturable to [ H]AMPA binding with a Kd ϭ 22 nM (data not shown), and (S)-thio-ATPA could complete 3 [ H]AMPA binding with a Ki at 0.89 ␮M (Fig. 2A). The structure of the complex of GluR2–S1S2J:(S)-thio-ATPA was determined at 2.17 Å resolution (Rwork ϭ 19.8%; Rfree ϭ 22.9%), and it contained one molecule in the asymmetric unit of the crystal (Fig. 2B). The complex displays an intermediate degree of domain closure at 18° relative to the apostate (6). (S)-thio-ATPA binds with an extensive number of direct and indirect interactions to both D1 and D2 (Fig. 2C; see also Table 5, which is published as supporting information on the PNAS web site). The ␣-amino acid part of (S)-thio-ATPA binds to the receptor as observed for other agonists (6, 12). The hydroxyl group of the isothiazolol ring, which equals one of the oxygen atoms of the ␥-carboxylate moiety of glutamate, makes direct contacts to D2 via three potential hydrogen bonds as well as indirect contacts through a water molecule (W1, Fig. 2C). (S)-thio-ATPA therefore resembles the glutamate- binding mode in GluR2–S1S2J rather than the (S)-AMPA binding mode (6). The interaction with D2 is further stabilized by two hydrogen bonds to the isothiazole nitrogen and two hydrogen bonds

to the sulfur atom in (S)-thio-ATPA. The tert-butyl moiety at the BIOPHYSICS 5-position of the isothiazolol ring points toward a partly hydropho- bic pocket (12, 34), and is stabilized by van der Waals interactions to the side chains of Glu-402, Tyr-450, Pro-478, and Tyr-732 in D1 Fig. 2. Binding characteristics and structure of the ligand-binding core of and to Leu-650, Thr-686, Glu-705, and Met-708 in D2. In addition 3 GluR2 in complex with (S)-thio-ATPA. (A) Diagram of the [ H]AMPA displace- to making contacts to the tert-butyl moiety, Glu-402 and Thr-686 ment from GluR2–S1S2J by (S)-thio-ATPA. (B) Schematic drawing of the struc- also create the lock between D1 and D2 (6, 35). ture of GluR2–S1S2J in complex with (S)-thio-ATPA. Amino acid residues from segment S1 are red, and residues from S2 are orange. The Gly–Thr linker is The trans peptide bond between Asp-651 and Ser-652 has been displayed in yellow. (S)-thio-ATPA and the disulﬁde bridge are shown in reported to exist in two distinct ligand-dependent conformations ball-and-stick representation. (C) Binding mode of (S)-thio-ATPA, including (6). (S)-thio-ATPA stabilizes the peptide conformation similar to potential hydrogen bonds within 3.3 Å (stippled lines). Nitrogen atoms are the apoform, kainate, and the (S)-Br-HIBO complexes but differ- blue, oxygen atoms are red, and sulfur atoms are yellow. Water molecules are ent from the 180° flip observed for the (S)-AMPA and (S)-ATPA shown as red spheres. complexes (6, 12, 36) (Table 1). The two-peptide bond conformations exhibit different hydrogen-bonding pattern to W1. In the MOLSCRIPT (32) and RASTER 3D (33) were used to prepare Fig. 2 (S)-thio-ATPA complex, W1 is further hydrogen bonded to the and Fig. 4. backbone oxygen atom of Ser-652 (3.3 Å) and to the backbone nitrogen atoms of Thr-655 (3.2 Å) and Lys-656 (2.7 Å). In the Results (S)-ATPA and (S)-AMPA complexes, however, W1 makes hydro- X-Ray Structure of GluR2–S1S2J Complexed with (S)-Thio-ATPA. To gen bonds to the oxygen atom of Leu-650, while still binding to the explore the molecular basis of the intriguing large maximal re- backbone nitrogen of Lys-656.

Table 1. Agonist efficacy, binding domain closure, and Asp-651–Ser-652 peptide bond conformation Efficacy,* % Peptide flip,‡ Leu-650 GluR2i GluR2o Domain closure,† GluR2o, conformation,§ Agonist Wild type L483Y Wild type L483Y GluR2o, S1S2J S1S2J GluR2o, S1S2J

Thio-ATPA 290 Ϯ 678Ϯ 3 631 Ϯ 14 95 Ϯ 318 ϪϪ115͞Ϫ166 Glutamate 100 100 100 100 20¶ ϩ͞Ϫ¶ 172͞68 Br-HIBO 134 Ϯ 690Ϯ 198Ϯ 292Ϯ 118࿣ Ϫ࿣ Ϫ170͞64 ATPA 102 Ϯ 0.06 82 Ϯ 1 180 Ϯ 14 98 Ϯ 1 21** ϩ** 178͞66 Kainate 69 Ϯ 1 4.4 Ϯ 1.0 386 Ϯ 28 31 Ϯ 312¶ Ϫ¶ Ϫ94͞166

*Relative maximal response measured in oocytes and calculated as the ratio of the maximal glutamate response. Maximal response was obtained by 0.5 mM (S)-thio-ATPA, 0.9 mM glutamate, 0.5 mM (S)-Br-HIBO, 1.0 mM (RS)-ATPA, or 1.0 mM kainate. †Domain closure reflects the degree of ligand-induced closure of the ligand-binding core. ‡Peptide flip denotes unflipped (Ϫ) or flipped (ϩ) conformations of the Asp-651–Ser-652 peptide bond (6). §Values (°) given correspond to those of the torsional angles ␹1͞␹2 of the Leu-650 side chain. PDB ID codes: thio-ATPA (2AIX), glutamate (1FTJ, mol B), Br-HIBO (1M5C), ATPA (1NNP, mol A), kainate (1FW0). ¶Values from Armstrong and Gouaux (6). ࿣Values from Hogner et al. (12). **Values from Lunn et al. (36).

Holm et al. PNAS ͉ August 23, 2005 ͉ vol. 102 ͉ no. 34 ͉ 12055 Downloaded by guest on September 30, 2021 Table 2. Agonist potency on GluR2 mutants and splice variants (S)-Glutamate Kainate (RS)-ATPA (S)-Thio-ATPA

Mutant EC50 nH EC50 nH EC50 nH EC50 nH

GluR2i WT 30 (29; 31) 1.5 170 (160; 190) 1.0 140 (120; 160) 1.0 40 (39; 41) 1.1 GluR2i(L483Y) 10 (10; 10) 1.7 170 (160; 190) 1.1 37 (34; 40) 1.4 5.0 (4.9; 5.1) 0.9 GluR2o WT 5.7 (5.2; 6.4) 1.1 130 (120; 140) 1.1 49 (38; 63) 1.0 13 (11; 16) 1.5 GluR2o(L483Y) 9.0 (7.6; 11) 1.6 110 (98; 120) 1.1 56 (49; 65) 1.6 2.9 (2.7; 3.1) 1.5

Potency of (S)-glutamate, kainate, (RS)-ATPA, and (S)-thio-ATPA at the unedited (Q variant) AMPA receptors; GluR2i, GluR2i(L483Y), GluR2o, and GluR2o(L483Y). Recordings were performed under two-electrode voltage clamp. EC50 values are in ␮M. Numbers in parentheses denote (min; max) of at least three individual oocytes.

Dose-Response Relationships on GluR2 Splice Variants. Homomeric Br-HIBO-induced current on GluR2 resembles the (S)-glutamate- unedited forms of GluR2i͞o were expressed in Xenopus oocytes induced currents more than those elicited by (S)-thio-ATPA (Fig. and the potencies of selected agonists were determined under 3 and Table 3). To explain the kinetic differences between (S)- two-electrode voltage clamp (Fig. 1B and Table 2). Dose–response thio-ATPA and (S)-Br-HIBO, we performed a more detailed relationships are shown in Fig. 5, which is published as supporting comparison of the crystal structures. We found four noticeable information on the PNAS web site. The flop splice form responded differences between the two complexes. First, within the binding to (S)-glutamate, (RS)-ATPA, and (S)-thio-ATPA with 3- to 5-fold pocket, the same 17 amino acid residues are observed to be in lower EC50 values than the flip splice forms. The apparent differ- contact with (S)-thio-ATPA and (S)-Br-HIBO. However, only 9 ence in potency seems to reflect different desensitization kinetics of potential direct hydrogen bonds (distance Ͻ 3.3 Å) are formed to the splice forms, because recording from the nondesensitizing (S)-Br-HIBO as compared with 14 to (S)-thio-ATPA (Table 5). GluR2(L483Y) mutant almost entirely eliminates these differences Secondly, a twist of D1 relative to D2 (projection deviation angle in potency. The potency of (S)-Br-HIBO has previously been of Ϸ6°) occurs in the (S)-Br-HIBO structure as compared with the determined to 4.4 Ϯ 0.6 ␮M at GluR2i(L483Y) (12). apo-GluR2–S1S2J. This twist is not observed in the structure of (S)-thio-ATPA (Fig. 4A). The twist might be induced to accom- Agonist Efficacy on Wild-Type (WT) and Nondesensitizing Receptor modate the ring system of (S)-Br-HIBO, which is positioned with Mutants. Saturating agonist concentrations were used to determine an angle of Ϸ30° to the ring system of (S)-thio-ATPA (Fig. 4B). the maximal currents under a two-electrode voltage clamp. The (S)-Br-HIBO will not be able to bind in the nontwisted protein values were normalized to the maximal current activated by (S)- conformation seen in the (S)-thio-ATPA complex because of steric glutamate (Table 1). A remarkable difference between the splice clashes with the D2 residues Leu-650, Thr-686, and Leu-704. forms was observed for the kainate-induced current relative to the The last two differences between the (S)-Br-HIBO and (S)-thio- steady-state glutamate-induced current, whereas (S)-thio-ATPA ATPA structures are observed in the conformation of the side was the most efficacious agonist on both of the splice forms. Despite chains of Leu-650 and Met-708. In the two structures, the Leu-650 the fact that (S)-Br-HIBO induced a domain closure similar to side-chain ␹1 angle adopts a gauche and an anti arrangement in the (S)-thio-ATPA, (RS)-Br-HIBO activated maximal currents that (S)-Br-HIBO and (S)-thio-ATPA structures, respectively (Table 1). were 2.2- and 6.4-fold smaller than (S)-thio-ATPA on GluR2i and The side chain of Met-708 is pushed toward Pro-403 due to an GluR2o, respectively (Table 1). otherwise steric clash with the tert-butyl group of (S)-thio-ATPA, Because the amplitude of the maximal current recorded in whereas the position of the S␦-C␧ part of the Met-708 side chain is oocytes is influenced by a number of parameters, including desen- optimized for favorable contacts to the bromide atom of (S)-Br- sitization, we performed additional experiments on the nondesen- HIBO (Fig. 4B). sitizing mutant GluR2(L483Y) (10, 37). At this mutant, (S)- glutamate becomes the most efficacious agonist, and because Discussion kainate already induces nondesensitizing currents on the WT Domain Closure Correlates with Efficacy at GluR2o but Not GluR2i. receptors (38), the ratio to the glutamate-activated currents be- The 3D structure of the GluR2o ligand-binding core has been comes smaller on this receptor (Table 1). Analyzing (RS)-Br-HIBO determined for the apoform as well as a large number of complexes on this mutant showed a maximal current similar to (S)-thio-ATPA with antagonists and agonists that induce varied electrophysiolog- on GluR2o(L483Y), while it was significantly higher (Imax, 90% Ϯ ical responses. Functional studies, primarily performed on GluR2i, 1%; P ϭ 0.001, t test) on GluR2i(L483Y), adding support to the have revealed a striking correlation between the degree of domain model that the relative domain closure correlates with the steady- closure and the maximal response recorded as the peak current, or state conductance on GluR2o but surprisingly not for GluR2i the relative maximal current in the presence of cyclothiazide or the (Table 1). nondesensitizing mutant GluR2(L483Y) (11, 12, 40). We also observe a correlation between the degree of domain closure and the Receptor Kinetics on GluR2i͞o Splice Variants. The desensitization relative maximal current (Table 1) by using the nondesensitizing time course was determined from outside-out patches isolated from form of GluR2o, where glutamate and ATPA as full agonists Xenopus oocytes expressing high receptor levels (Table 3). By using induce the largest relative maximal current (98–100%) and a rapid agonist application with saturating concentrations, (S)-thio- closure of 21° (6, 41). Br-HIBO and thio-ATPA induce an inter- ATPA evoked a fast desensitizing current on GluR2o (␶ ϭ 6.6 Ϯ mediate maximal current (92–95%) and a less tight domain closure 0.7 ms), whereas an almost nondesensitizing current was activated of 18° (12), whereas the low domain closure (12°) induced by on GluR2i (Fig. 3A and Table 3), in strong contrast to the fast and kainate (6) also induces the lowest maximal current (31%). Inter- almost complete desensitization induced by (S)-glutamate on both estingly, when the relative maximal currents were determined on splice forms (Fig. 3B and Table 3). the nondesensitizing GluR2i mutant, there were concordance between the closure and the maximal current for many of the Comparison of (S)-Thio-ATPA and (S)-Br-HIBO in GluR2. (RS)-Br- agonists except for the tert-butyl substituted compounds, (RS)- HIBO induces currents on GluR1o and GluR3o, similar to those ATPA and (S)-thio-ATPA. They became less efficacious (the elicited by (S)-glutamate (39). Likewise, the kinetics of the (RS)- relative maximal current changed from 98% to 82% and 95% to

12056 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505522102 Holm et al. Downloaded by guest on September 30, 2021 Table 3. Desensitization kinetics Steady-state͞peak, % Decay time; ␶,ms

GluR2i GluR2o GluR2i GluR2o

Thio-ATPA 69 Ϯ 714Ϯ 2 Ͼ100 6.6 Ϯ 0.7 Glutamate 6.9 Ϯ 1.7 1.9 Ϯ 0.4 9.8 Ϯ 0.8 2.0 Ϯ 0.2 Br-HIBO 11 Ϯ 1 3.9 Ϯ 0.8 13 Ϯ 1 3.0 Ϯ 0.1

Desensitization time constants and steady-state͞peak ratios from responses evoked by 100-ms applications of 10 mM (S)-glutamate, 2.5 mM (S)-thio-ATPA, and 5 mM (RS)-Br-HIBO, respectively. The agonists were applied to outside-out patches pulled from Xenopus oocytes expressing homomeric GluR2i or GluR2o. Values are presented as mean Ϯ SEM, n Ն 3.

78%, respectively), whereas no significant change was observed for (RS)-Br-HIBO. Similar discrepancies between the GluR2o crystal structure and the GluR2i functional data were observed when comparing kainate, Br-willardiine, and I-willardiine. The latter two induced a 15° and 11° domain closure and relative maximal currents of 37% and 34% (11), respectively, whereas kainate induced a closure of 12° (6) together with a relative maximal current of 4.4% on GluR2i(L483Y) (Table 1). Thus, structurally different compounds might induce different degrees of closure in the GluR2i and GluR2o despite the fact that all residues involved in ligand interactions are conserved between the two splice forms. Alternatively,

similar structures in the binding pocket might affect the subunit BIOPHYSICS interface differently, thereby stabilizing other intersubunit config- urations with different coupling efficiency to the conducting states of the channel (11).

Structural Differences Affecting the Desensitization. Despite the similar degree of domain closure induced by Br-HIBO and thio- Fig. 4. Superimposition of GluR2-S1S2J:(S)-thio-ATPA and GluR2-S1S2J:(S)- ATPA, we observed a Ͼ3.5-fold difference in the steady-state͞peak Br-HIBO dimers. Residues from D1 of protomer 1 (left) were used for the ratio and a Ͼ2.2-fold difference in decay time on GluR2o (corre- superimposition (rms deviation 0.45 Å on C␣ atoms). (A) D1 residues of the sponding to a 6.2- and Ͼ8-fold difference on GluR2i, respectively). GluR2-S1S2J:(S)-thio-ATPA dimer are colored red, and D2 residues are colored A comparison of the structures of the GluR2 ligand-binding core orange. The GluR2-S1S2J:(S)-Br-HIBO dimer is colored cyan. The agonists complexed with (S)-thio-ATPA and (S)-Br-HIBO revealed four (S)-thio-ATPA and (S)-Br-HIBO are shown in ball-and-stick representation. Dotted lines indicate the distance between Ile-633 of the two protomers of the marked differences between the two structures. Two involve the GluR2-S1S2J:(S)-thio-ATPA dimer (orange) and GluR2-S1S2J:(S)-Br-HIBO dimer residues Leu-650 and Met-708. Leu-650 is located in a dynamic part (cyan). The D1͞D2 domain closure is 18° in both structures, and the arrow of the binding pocket adjacent to Asp-651–Ser-652, reported to indicates the different twist of D2 between the two structures. (B) The adopt multiple peptide-bond conformations (6). However, we find ligand-binding site. The (S)-thio-ATPA and (S)-Br-HIBO complexes are super- no correlation between the peptide flip conformation and the imposed and colored as in A. Ligands are shown in ball-and-stick and water agonist-induced currents (Table 1). In addition, Leu-650 forms an molecules as spheres. Nitrogen atoms are blue, oxygen atoms are red, and interdomain bridge by means of nonpolar interactions and is able sulfur atoms are yellow. to adopt at least two markedly distinct conformations (Table 1 and Fig. 4B). (S)-Br-HIBO, (S)-glutamate, (S)-AMPA, and (S)-ATPA all induce similar conformations of Leu-650, whereas e.g., (S)-thio- ATPA and kainate induce the other conformation (6, 12, 36). Mutating the Leu at the equivalent position to a Thr in GluR1 (42) or a Val in GluR4 (43) changes the desensitization properties of the receptors. NMR dynamics studies performed on the GluR2- binding domain (44) show that upon agonist binding, residues in D1 are relatively fixed compared with considerable structural changes in D2. In concordance, a recent study of GluR2(L650T) leads to a model suggesting that AMPA binding is initiated by interaction with preorganized residues in D1 followed by a Glu-705 mediated reorganization of the residues in D2 (45). The latter conformational change induced the domain closure and was proposed to couple to the ion channel gating (46). The conformational flexibility of Leu-650 appears critical for the transitions because the L650T Fig. 3. Rapid application on GluR2i and GluR2o. Representative traces from partially trap the domain closure in an intermediate nonconducting outside-out patches pulled from Xenopus oocytes expressing GluR2i (Upper) state resulting in a submaximal current (45). Consequently, the and GluR2o (Lower) showing responses to 100-ms application of 2.5 mM S (S)-thio-ATPA (A), 10 mM (S)-glutamate (B), or 5 mM (RS)-Br-HIBO (C). All different conformation of Leu-650 observed in the ( )-thio-ATPA patches were clamped at Ϫ120 mV. Vertical and horizontal scale bars repre- and (S)-Br-HIBO complexes might change the gating kinetics of sent 500 pA and 20 ms, respectively, for all traces, except for 5 mM (RS)-Br- these agonists. HIBO on GluR2o where the vertical bar is 200 pA. Met-708 also shows a considerable conformational difference

Holm et al. PNAS ͉ August 23, 2005 ͉ vol. 102 ͉ no. 34 ͉ 12057 Downloaded by guest on September 30, 2021 between the (S)-thio-ATPA and (S)-Br-HIBO crystal structures D1 and D2 in the (S)-Br-HIBO complex as compared with the (Fig. 4). Based on high-resolution structures of the 5-substituted (S)-thio-ATPA complex (Fig. 4A). The twist also occurs to different willardiine derivatives, it has been proposed that conformational degree in other complexes, e.g., in GluR2–S1S2J:(S)-2-Me-Tet- changes in Met-708 might be transmitted to the channel region via AMPA and GluR2-S1S2J:(S)-glutamate, where the three different helix I (47). Analysis of complexes with the full agonists (S)- molecules show changing twists ranging from 1–7° and 0.5–3°, glutamate, (S)-AMPA, or (S)-ATPA shows that Met-708 adopts an respectively, even for the same ligand–receptor combination. Fur- induced fit involving rather large conformational changes without ther structural studies will be needed to elucidate the effect of the affecting the efficacy significantly (12, 34, 36) making conforma- twist on the kinetic properties. Interestingly, in accordance with the tional changes in Met-708 a less likely factor underlying the similar maximal current on GluR2o(L483Y) observed for the two observed kinetic differences. agonists, (S)-Br-HIBO and (S)-thio-ATPA, the twist does not alter NMR and UV spectroscopic methods indicate that agonist the distances between residues of the two protomers linked to the binding to the binding domain may induce changes in the ␤-sheet pore region in the receptor (Ile-633–Ile-633 distance of 34.8 and content or orientation in D2, which has been proposed as a 34.5Åinthe(S)-thio-ATPA and (S)-Br-HIBO complexes, respec- mechanism for coupling of the changes in the binding site to the tively; see Fig. 4A). In conclusion, we have used structurally channel activation (48). However, we are not able to detect signif- different agonists to identify residues and mechanisms apart from icant differences in the ␤-sheet contend in D2 for the agonists. domain closure that might influence the kinetic properties of GluR desensitization. Twist of D2 Relative to D1. In the crystals of GluR2–S1S2J complexed with (S)-thio-ATPA, a twofold dimer is formed of two We thank Eric Gouaux for generously providing the GluR2–S1S2J symmetry-related molecules with interface accessible surface area construct and protocols for refolding and purification; Tine B. Stensbøl of 866 Å2. This size is similar to those reported of other agonist (Danish University of Pharmaceutical Sciences, Copenhagen), Ulf Mad- complex structures (6, 12). A comprehensive mutagenesis study sen, and Povl Krogsgaard-Larsen for kindly providing the compounds; demonstrated that the stability of the interdomain interactions Jeremy R. Greenwood for guidance using computational techniques; and between helix D and helix J correlated strongly with the desensi- Dan A. Klærke (Copenhagen University, Copenhagen) and Marianne tization properties of glutamate at GluR2i (43). Similar interdo- Faber (Lundbeck, Valby, Denmark) for kindly providing some of the main interactions are found in the (S)-thio-ATPA, (S)-Br-HIBO, X. laevis oocytes. The work was supported by the University of Aarhus (M.M.H.) and by grants from NeuroScience PharmaBiotec (to M.M.H., and (S)-glutamate complexes, suggesting that the intersubunit M.-L.L., J.E., and J.S.K.), the Danish Centre for Synchrotron Based interface mainly formed between D1 in the two subunits (both Research (DANSYNC) (to M.-L.L. and J.S.K.), Apotekerfonden of 1991 helices D and J are located in D1) are of equal stability. However, (to M.-L.L. and J.S.K.), the National Institutes of Health (to S.F.T.), The the location of the two protomers comprising the (S)-thio-ATPA Danish Medical Research Council (to J.E., M.L.L., and J.S.K.), and The and (S)-Br-HIBO dimers is slightly different (see Fig. 4A). European Community: Access to Research Infrastructure action of the Alignment of the structures reveals a relative twist of 6° between Improving Human Potential Program.

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