The Selective Activation of the Glutamate Receptor GluR5 by ATPA Is Controlled by Serine 741 MAI MARIE NIELSEN, TOMMY LILJEFORS, POVL KROGSGAARD-LARSEN, and JAN EGEBJERG

Only a few agonists exhibit selectivity between the AMPA and the kainate subtypes of the glutamate receptor. The most commonly used kainate receptor preferring agonist, (S)-2-amino-3(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid [(S)-ATPA], is an (R,S)-2-amino-3-(5-methyl-3-hydroxy-4-isoxazolyl)propionic acid (AMPA) derivative in which the methyl group at the 5-position of the isoxazole ring has been replaced by a tertbutyl group. When characterized by the two-electrode voltage clamp method in Xenopus laevis oocytes, ATPA exhibits at least 50-fold higher potency on the kainate receptor subtype, GluR5, compared with the AMPA receptors. Through mutagenesis studies of GluR5 and the AMPA receptor subtype, GluR1, we demonstrate that this pronounced selectivity for ATPA can be ascribed to Ser741 in GluR5 and Met722 in GluR1. Examination of other aliphatic substitutions at the 5-position of the isoxazole ring revealed that (R,S)-2-amino-3-(5-isopropyl-3-hydroxy-4-isoxazolyl)propionic acid (isopropyl-AMPA) displayed a 6-fold higher potency for GluR5 than for GluR1, whereas the analogs, propyl-AMPA and isobutyl-AMPA, did not exhibit significantly different potencies. Our study suggests that the GluR5 selectivity was a result not only of steric interference between the bulky tert-butyl group in ATPA and the methionine (Met722) in GluR1 but also a serine-dependent stabilization of the active conformation of GluR5 induced by ATPA. The stabilization was agonist-dependent and observed only for ATPA and isopropyl-AMPA, not for other AMPA analogs with bulky substitutions at the 5-position of the isoxazole ring. Glutamate receptors are the most abundant excitatory receptors in the central nervous system. Activation and modulation of the glutamatergic system play a crucial role in our understanding of the neuronal activity in the healthy brain as well as for the mechanisms underlying various neurological and psychiatric disorders. The contributions of the different glutamate receptor subtypes to neuronal activity are to a large extent identified using subtype-selective compounds or, more recently, by studies of genetically modified animals (Bräuner-Osborne et al., 2000; Doherty and Collingridge, 2001). The traditional pharmacological division of the glutamate receptors into AMPA, kainate, and N-methyl-D-aspartic acid receptors based on the potencies of the respective agonists is reflected at the level of sequence identity between of the receptor subtypes forming the receptor complex. Thus, the subunits GluR1 to GluR4 form the AMPA receptors and the subunits GluR5 to GluR7 and KA1 and KA2 form the kainate receptors (Hollmann and Heinemann, 1994). The increasing understanding of the molecular diversity underlying the ionotropic glutamate receptor system has challenged the development of subtype-selective ligands (Dingledine et al., 1999; Bräuner-Osborne et al., 2000). AMPA activates the AMPA receptors expressed in oocytes with an EC50 in the range of 1.3 to 3.5 M (Vogensen et al., 2000), whereas kainate receptors formed from the GluR5 to GluR7 subunits are either activated with EC50 1 mM or not at all (Egebjerg et al., 1991; Sommer et al., 1992; Schiffer et al., 1997). Surprisingly, (S)-ATPA, an AMPA analog in which the methyl group at the 5-position in the isoxazole ring is replaced by a tert-butyl group (Lauridsen et al., 1985), exhibits a strong preference for GluR5 compared with the AMPA receptors (Clarke et al., 1997; Stensbøl et al., 1999). Analogs of AMPA with different 5-substitutions of the isoxazole ring have been studied extensively (Krogsgaard-Larsen et al., 1996). These derivatives seem to contribute to the selectivity and potency both within the AMPA receptor family and beThis study was supported by the Danish Medical Research Council, the Carlsberg Foundation, the Lundbeck Foundation, and the University of Aarhus (to M.M.N.). ABBREVIATIONS: AMPA, (R,S)-2-amino-3-(5-methyl-3-hydroxy-4-isoxazolyl)propionic acid; ATPA, (R,S)-2-amino-3-(5-tert-butyl-3-hydroxy-4isoxazolyl)propionic acid; TM, transmembrane; GluR, glutamate receptor; isopropyl-AMPA, (R,S)-2-amino-3-(5-isopropyl-3-hydroxy-4-isoxazolyl)propionic acid; propyl-AMPA, (R,S)-2-amino-3-(5-propyl-3-hydroxy-4-isoxazolyl)propionic acid; isobutyl-AMPA, (R,S)-2-amino-3-(5-isobutyl-3hydroxy-4-isoxazolyl)propionic acid; (S)-2-Me-Tet-AMPA, (S)-2-amino-3-(5-(2-methyltetrazolyl)-3-hydroxy-4-isoxazolyl)propionic acid; LCR, low Ca Ringer; conA, concanavalin A. 0026-895X/03/6301-19–25$7.00 MOLECULAR PHARMACOLOGY Vol. 63, No. 1 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 1811/1033805 Mol Pharmacol 63:19–25, 2003 Printed in U.S.A. 19 at A PE T Jornals on Sptem er 6, 2017 m oharm .aspeurnals.org D ow nladed from tween the AMPA and kainate receptors. In particular, the AMPA analog with a 2-methyltetrazolyl substituent at the 5-position increases the potency and activates GluR4 and GluR1 with EC50 values of 9 and 160 nM, respectively, but remains AMPA receptor-selective by activating the kainate receptor GluR5 with an EC50 of 9 M (Vogensen et al., 2000). In contrast, the tert-butyl substitution in ATPA resulted in 100-fold higher potency at GluR5 (0.66 M) compared with GluR1 (62 M) when expressed in Xenopus laevis oocytes (Stensbøl et al., 1999). The selectivity is even more pronounced (1000-fold) compared with the peak current in the AMPA receptors observed after fast application (Clarke et al., 1997). Studies performed on cortical wedges, which mainly reflect activation of the AMPA receptors, showed that the 5-ethyl analog of AMPA was more potent than AMPA, whereas the propyl and butyl analogs showed decreased potencies (Sløk et al., 1997). These observations and studies of willardiine analogs (Wong et al., 1994; Jane et al., 1997; Swanson et al., 1998) have resulted in a hypothesis proposing that the AMPA and kainate receptors might contain a hydrophobic cavity that can accommodate hydrophobic substituents to a certain size at the 5-position of the isoxazole ring of the AMPA molecule (Krogsgaard-Larsen et al., 1996). The current structural model of the ionotropic glutamate receptors suggests a tetrameric complex formed by two dimers (Armstrong and Gouaux, 2000; Robert et al., 2001). The membrane topology of each subunit is three transmembrane(TM) spanning segments, where the pore is formed by the two N-terminal TM-spanning segments and a re-entrant loop located between these TM segments. The ligand-binding domain is composed of two lobes formed from the part preceding the first TM and the extracellular region between the second and third TM (for review see Dingledine et al., 1999; Bräuner-Osborne et al., 2000). A soluble form of the ligandbinding domain of the GluR2 subunit has been expressed and crystallized in the apo form and also cocrystallized with a number of ligands, including the agonists kainate, glutamate, AMPA, and the antagonist 6,7-dinitro-2,3-quinoxalinedione (Armstrong and Gouaux, 2000; Armstrong et al., 1998). The crystal structure data suggest that the agonistinduced closure of the binding domain gives rise to the opening of the channel and, furthermore, the degree of closure correlates with the agonist specific properties of the channel, including the degree of desensitization. In the current study, we attempt, based on mutagenesis, molecular modeling, and the use of AMPA analogs, to identify the amino acid(s) determining the difference in potency for ATPA on the AMPA receptor GluR1 and the kainate receptor GluR5. Materials and Methods Glutamate Receptor Ligands and Reagents. The AMPA analogs (S)-ATPA (Lauridsen et al., 1985; Stensbøl et al., 1999), isopropyl-AMPA, (R,S)-2-amino-3-(5-propyl-3-hydroxy-4-isoxazolyl)propionic acid (propyl-AMPA), (R,S)-2-amino-3-(5-isobutyl-3-hydroxy-4isoxazolyl)propionic acid (isobutyl-AMPA) (Sløk et al., 1997), and (S)-2-amino-3-(5-(2-methyltetrazolyl)-3-hydroxy-4-isoxazolyl)propionic acid [(S)-2-Me-Tet-AMPA] (Vogensen et al., 2000) were synthesized as described previously. All other pharmacological tools and reagents were purchased from regular commercial sources. Mutagenesis. The mutations were introduced by the standard overlap polymerase chain reaction method, using Pfu polymerase. The mutated polymerase chain reaction fragments were inserted between the BspEI and MluNI in GluR1flop and BlnI and EcoRI, BlnI, and XbaI or SalI and XbaI in GluR51a. The inserted fragments were sequenced. All constructs were inserted in the pGEMHE (Liman et al., 1992) oocyte expression vector. In Vitro cRNA Transcription. DNA (3 g) was linearized using the appropriate enzymes. Run-off transcription was performed for 2 h at 37°C in 100 l using the following concentrations; 7 mM MgCl2, 10 mM NaCl, 2 mM spermidine, 40 mM Tris-HCl, pH 8.0, 37.5 mM dithiothreitol, 0.5 mM ATP, 0.5 mM UTP, 0.5 mM CTP, 0.1 mM GTP, 0.5 mM CAP (GpppGTP), 400 U/ml RNase block, and 300 to 500 U/ml T7 RNA polymerase. Trace amounts of [ -P]UTP were included to allow quantification of the transcribed cRNA. Electrophysiology. A female Xenopus laevis frog was anesthetized and three to five ovarian lobes were surgically removed. The follicle layer was removed by washing twice in Barth’s solution (88.0 mM NaCl, 1.0 mM KCl, 2.4 mM NaHCO3, 15.0 mM HEPES pH 7.6, 0.30 mM CaNO3, 0.41 mM CaCl2, 0.82 mM MgSO4, 10 g/ml penicillin, and 10 g/ml streptomycin), once in OR-2 (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2 and 5 mM HEPES, pH 7.4) followed by treatment with collagenase A (1 mg/ml in OR-2) for 3 h at RT. Oocytes at stages 4 to 5 were isolated and injected the following day with 50 nl (5–50 ng) of cRNA. The oocytes were kept at 18°C in Barth’s medium before recordings were performed 3 to 12 days after injection, using a two-electrode voltage clamp (Warner OC-725C; Warner Instruments, Inc., Hamden, CT). The recording solution was low Ca Ringer (LCR; 10 mM HEPES-NaOH, pH 7.4, 115 mM NaCl, 0.1 mM CaCl2, 2.5 mM KCl, and 1.8 mM MgCl2). The LCR buffer was chosen to prevent activation of the endogenous Ca -activated Cl channel. The oocytes were clamped at 70 to 20 mV. Electrodes (borosilicate glass capillaries, outer diameter, 1.5 mm; inner diameter, 1.17 mm; with inner filament; Harvard apparatus LTD, Kent, UK) were filled by 3 M KCl and exhibited resistances around 0.7 to 2 M . Oocytes expressing GluR5 were treated with 1 mg/ml concanavalin A (type IV; Sigma Chemical, St. Louis, MO), in normal frog Ringer solution (10 mM HEPES-NaOH, pH 7.4, 115 mM NaCl, 1.8 mM CaCl2, 2.5 mM KCl, and 0.1 mM MgCl2) for 5 min, before recording. Stock solutions of the drugs were made in LCR at a concentration of 2.5 mM, pH adjusted