The Journal of Neuroscience, July 15, 2002, 22(14):5955–5965 Mapping the Binding Site of the Neuroprotectant Ifenprodil on NMDA Receptors Florent Perin-Dureau, Julie Rachline, Jacques Neyton, and Pierre Paoletti Laboratoire de Neurobiologie, Centre National de la Recherche Scientifique, Unite´ Mixte de Recherche 8544, Ecole Normale Supe´ rieure, 75005 Paris, France Ifenprodil is a noncompetitive antagonist of NMDA receptors control ifenprodil inhibition. Their location in a modeled three- highly selective for the NMDA receptor 2B (NR2B) subunit. It is dimensional structure suggests that ifenprodil binds in the cleft widely used as a pharmacological tool to discriminate sub- of the LIVBP-like domain of NR2B by a mechanism (Venus- populations of NMDA receptors, and derivatives are currently flytrap) resembling that of the binding of Zn on the LIVBP-like being developed as candidate neuroprotectants. Despite nu- domain of NR2A. These results reinforce the proposal that the merous studies on the mechanism of action of ifenprodil on LIVBP-like domains of NMDA receptors, and possibly of other NMDA receptors, the structural determinants responsible for ionotropic glutamate receptors, bind modulatory ligands. More- the subunit selectivity have not been identified. By combining over, they identify the LIVBP-like domain of the NR2B subunit functional studies on recombinant NMDA receptors and bio- as a promising therapeutic target and provide a framework for chemical studies on isolated domains, we now show that ifen- designing structurally novel NR2B-selective antagonists. prodil binds to the N-terminal leucine/isoleucine/valine-binding protein (LIVBP)-like domain of NR2B. In this domain, several Key words: glutamate receptors; NMDA; ifenprodil; phe- residues, both hydrophilic and hydrophobic, were found to nylethanolamine; LIVBP; neuroprotection Ionotropic glutamate receptors (iGluRs) are made of subunits domain forms a high-affinity Zn binding site (Paoletti et al., 2000) sharing a common membrane topology: a large N-terminal extra- (also see Choi and Lipton, 1999; Fayyazuddin et al., 2000; Low et cellular region, three transmembrane segments (TM1, TM3, and al., 2000). TM4), a P loop region (initially called TM2) that forms the pore Ifenprodil is representative of a class of NMDA receptor selectivity filter, and a cytoplasmic C-terminal region (Dingle- antagonists (phenylethanolamines) with high selectivity for dine et al., 1999). The agonist binding domain, made of ϳ150 NR2B-containing receptors (Williams, 1993; Chenard and Men- amino acids preceding TM1 together with the extracellular loop niti, 1999). Several phenylethanolamines are neuroprotective between TM3 and TM4, is distantly related to the bacterial both in vitro and in in vivo models of a variety of neurological periplasmic-binding protein (PBP) glutamine binding protein disorders and lack many of the side effects associated with non- (GlnBP) (Stern-Bach et al., 1994). It has been crystallized in the subunit-selective NMDA receptor antagonists (references in Kew case of the AMPA subunit GluR2 and the prokaryotic glutamate and Kemp, 1998); they also produce antinociceptive effects receptor subunit GluR0, showing a bilobed structure with the (Chizh et al., 2001). Ifenprodil acts as a noncompetitive, partial, agonist bound in a central cleft (Armstrong et al., 1998; Mayer et and voltage-independent antagonist (Carter et al., 1988; Leg- al., 2001). endre and Westbrook, 1991; Williams, 1993). Its potency strongly Eukaryotic iGluR subunits possess an additional extracellular depends on the extracellular pH and is only weakly affected by ϳ N-terminal domain made of the first 380 amino acids that is the insertion of the NR1 exon 5 (Pahk and Williams, 1997; Mott weakly related to leucine/isoleucine/valine-binding protein et al., 1998). Finally, ifenprodil displays use dependence such that (LIVBP), another PBP (O’Hara et al., 1993). In AMPA and binding of glutamate increases binding of ifenprodil and vice kainate receptors, this domain participates in subunit oligomer- versa (Kew et al., 1996; Zheng et al., 2001). On the basis of ization (Kuusinen et al., 1999; Leuschner and Hoch, 1999; Ayalon binding experiments on chimeric NR2 subunits, Gallagher et al. and Stern-Bach, 2001). In NMDA receptors (NRs; heteromers (1996) have proposed that determinants of ifenprodil inhibition made of NR1 and NR2A–NR2D subunits), we have proposed locate to the N terminus of NR2B. However, using a mutagenesis recently that the LIVBP-like domains of the NR2 subunits also approach, Masuko et al. (1999) concluded in favor of a binding have a bilobed structure, and we have shown that in NR2A, this site located in the N terminus of NR1. Thus, despite the detailed functional characterization of the mechanism of ifenprodil inhi- Received Feb. 27, 2002; revised May 6, 2002; accepted May 7, 2002. This work was supported by Assistance Publique des Hoˆpitaux de Paris (F.P.D.), bition, the precise location of the ifenprodil binding site has Ministe`re de la Recherche (J.R.), and Institut National de la Sante´etdela remained for the most part elusive. Recherche Me´dicale (P.P.). We are most grateful to Roderick MacKinnon and All the functional properties of the ifenprodil inhibition of members of his laboratory who helped much in starting the biochemical approach. We thank Philippe Ascher and Roderick MacKinnon for comments on this manu- NR2B-containing receptors listed above also apply to the high- script. We also thank Sanofi-Synthe´labo for the gift of ifenprodil. affinity Zn inhibition of NR2A-containing receptors (Westbrook Correspondence should be addressed to Dr. Pierre Paoletti, Laboratoire de and Mayer, 1987; Christine and Choi, 1990; Paoletti et al., 1997; Neurobiologie, Ecole Normale Supe´rieure, 46 rue d’Ulm, 75005 Paris, France. E-mail: [email protected]. Traynelis et al., 1998; Choi and Lipton, 1999; Low et al., 2000; Copyright © 2002 Society for Neuroscience 0270-6474/02/225955-11$15.00/0 Zheng et al., 2001). This striking similarity between both antag- 5956 J. Neurosci., July 15, 2002, 22(14):5955–5965 Perin-Dureau et al. • The Ifenprodil Binding Site on NMDA Receptors onisms suggests that Zn and ifenprodil share a common mecha- NR2B (LIVBP NR2A)-containing receptors at high (10 and 30 ␮M) Ϫ ϩ nism of modulation at the structural level. We now show that, ifenprodil concentrations, 2 sec 70/ 50 mV voltage ramps were used (capacitive and leakage currents were recorded before agonist applica- similarly to the Zn binding site on the NR2A LIVBP-like do- tion and subtracted from the agonist-induced currents). main, the LIVBP-like domain of NR2B forms in its central cleft Error bars represent the SD of the mean relative currents. a high-affinity binding site for ifenprodil. Production of isolated LIVBP-like domains in Escherichia coli and proteolysis experiments. LIVBP-like domains of the NR2A and of the MATERIALS AND METHODS NR2B subunits were produced as thrombin-cleavable glutathione S-transferase (GST) fusion proteins in E. coli. LIVBP-like domains of Molecular biology. The expression plasmids, mutagenesis strategy, RNA the rat NR2A (Glu 28-Val 375) and of the mouse NR2B (⑀2; Ser 28-Val 376) synthesis, and NR2A/NR2B chimera constructions have been described were subcloned in the pGEX-2T vector (Amersham Biosciences, Buck- previously by Paoletti et al. (1997, 2000). Each mutation was verified by ϳ inghamshire, UK). After transformation, BL21(DE3) cells were grown in sequencing across the mutated region ( 400–600 bp; Genome Express, 1 liter of Luria–Bertani medium supplemented with ampicillin (100 Montreuil, France). For each mutation strongly affecting ifenprodil in- ␮ g/ml) at 37°C until OD600 reached 0.7–0.8. Protein production was hibition, two independent clones were isolated, sequenced, and function- induced by 1 mM isopropyl-␤-D-thiogalactopyranoside (Roche Biochemi- ally characterized (except for NR2B-K234A, for which only one clone cals, Meylan, France) for 2.5 hr at 37°C. The following steps, adapted has been isolated). Point mutations in isolated LIVBP-like domains were from those of Chen and Gouaux (1997), were performed either on ice or made by using mismatch PCR (QuikChange; Stratagene Europe, Am- at 4°C. Cells were harvested and resuspended in buffer 1 (in mM: 200 sterdam, The Netherlands) and verified by sequencing the entire domain. NaCl, 20 Tris, and 1 EDTA, pH 7.5). The cells were sonicated for 2 min. Electrophysiology and data analysis. Xenopus laevis oocytes were pre- Inclusion bodies were collected by centrifugation (15,000 rpm, 20 min), pared, kept, injected with cRNAs, voltage-clamped, and superfused as resuspended in buffer 1 (20 ml), and purified by a first incubation (30 described by Paoletti et al. (1995, 1997). Oocytes were injected with min) in the presence of DNase1 (1 mg), deoxycholic acid (120 mg), and 30–40 nl of a mixture of NR1 and NR2 cRNAs (ratio, 1:2) at a final ␮ lysozyme (100 mg), followed by a second incubation (30 min) with 0.5% concentration of 100 ng/ l and recorded in the following 1–4d.The Triton X-100. Purified inclusion bodies were then solubilized overnight control solution superfusing the oocytes contained (in mM): 100 NaCl, 5 in buffer 2 (6 M GuHCl, 50 mM Tris, and 10 mM DTT, pH 8.0). Proteins HEPES, 0.3 BaCl2, and 10 Tricine (used to chelate traces amount of (ϳ1 mg/ml) were refolded by 16 hr of dialysis against a 20-fold higher contaminating Zn; Paoletti et al., 1997). The pH was adjusted to 7.3 with volume of buffer 3 (in mM: 500 NaCl, 50 Tris, and 1 DTT, pH 8.0), using KOH. Both L-glutamate and glycine were prepared as 250 ␮l aliquots (in Ϫ a membrane tube with a molecular mass cutoff of 15,000 Da (Fisher, bidistilled water) at 100 mM and stored at 20°C. NMDA currents were Illkirch, France). Buffer 3 was changed after 8 hr of incubation. Refolded induced by application of the agonist solution containing a saturating ␮ proteins were separated from the precipitate by centrifugation (1 hr, 40 concentration of both L-glutamate and glycine (100 M each).