Constitutive Activation of the N-Methyl-d-aspartate Receptor via Cleft-spanning Disulfide Bonds
2008; Elsevier BV; Volume: 283; Issue: 31 Linguagem: Inglês
10.1074/jbc.m709190200
ISSN1083-351X
AutoresMarie L. Blanke, Antonius M.J. VanDongen,
Tópico(s)Receptor Mechanisms and Signaling
ResumoAlthough the N-methyl-d-aspartate (NMDA) receptor plays a critical role in the central nervous system, many questions remain regarding the relationship between its structure and functional properties. In particular, the involvement of ligand-binding domain closure in determining agonist efficacy, which has been reported in other glutamate receptor subtypes, remains unresolved. To address this question, we designed dual cysteine point mutations spanning the NR1 and NR2 ligand-binding clefts, aiming to stabilize these domains in closed cleft conformations. Two mutants, E522C/I691C in NR1 (EI) and K487C/N687C in NR2 (KN) were found to exhibit significant glycine- and glutamate-independent activation, respectively, and co-expression of the two subunits produced a constitutively active channel. However, both individual mutants could be activated above constitutive levels in a concentration-dependent manner, indicating that cleft closure does not completely prevent agonist association. Interestingly, whereas the NR2 KN disulfide was found to potentiate channel gating and M3 accessibility, NR1 EI exhibited the opposite phenotype, suggesting that the EI disulfide may trap the NR1 ligand-binding domain in a lower efficacy conformation. Furthermore, both mutants affected agonist sensitivity at the opposing subunit, suggesting that closed cleft stabilization may contribute to coupling between the subunits. These results support a correlation between cleft stability and receptor activation, providing compelling evidence for the Venus flytrap mechanism of glutamate receptor domain closure. Although the N-methyl-d-aspartate (NMDA) receptor plays a critical role in the central nervous system, many questions remain regarding the relationship between its structure and functional properties. In particular, the involvement of ligand-binding domain closure in determining agonist efficacy, which has been reported in other glutamate receptor subtypes, remains unresolved. To address this question, we designed dual cysteine point mutations spanning the NR1 and NR2 ligand-binding clefts, aiming to stabilize these domains in closed cleft conformations. Two mutants, E522C/I691C in NR1 (EI) and K487C/N687C in NR2 (KN) were found to exhibit significant glycine- and glutamate-independent activation, respectively, and co-expression of the two subunits produced a constitutively active channel. However, both individual mutants could be activated above constitutive levels in a concentration-dependent manner, indicating that cleft closure does not completely prevent agonist association. Interestingly, whereas the NR2 KN disulfide was found to potentiate channel gating and M3 accessibility, NR1 EI exhibited the opposite phenotype, suggesting that the EI disulfide may trap the NR1 ligand-binding domain in a lower efficacy conformation. Furthermore, both mutants affected agonist sensitivity at the opposing subunit, suggesting that closed cleft stabilization may contribute to coupling between the subunits. These results support a correlation between cleft stability and receptor activation, providing compelling evidence for the Venus flytrap mechanism of glutamate receptor domain closure. Ionotropic glutamate receptors (iGluRs) 2The abbreviations used are: iGluR, ionotropic glutamate receptor; DCK, 5,7-dichlorokynurenic acid; NMDA, N-methyl-d-aspartate; DCS, d-cycloserine; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; LBD, ligand binding domain; D1 and D2, domain 1 and 2, respectively; MTSEA, methanethiosulfonate; BME, 2-mercaptoethanol; APV, 2-amino-5-phosphonovalerate; EI, E522C/I691C; KN, K487C/N687C; WT, wild type. 2The abbreviations used are: iGluR, ionotropic glutamate receptor; DCK, 5,7-dichlorokynurenic acid; NMDA, N-methyl-d-aspartate; DCS, d-cycloserine; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; LBD, ligand binding domain; D1 and D2, domain 1 and 2, respectively; MTSEA, methanethiosulfonate; BME, 2-mercaptoethanol; APV, 2-amino-5-phosphonovalerate; EI, E522C/I691C; KN, K487C/N687C; WT, wild type. are key mediators of excitatory neurotransmission, contributing to both neuronal development and adult neuroplasticity (1Bashir Z.I. 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The iGluR family is composed of three subclasses, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, and NMDA receptors, all of which share the same modular domain structure. Two discontinuous segments, S1 and S2, form the ligand binding domain (LBD), whereas the ion channel portion contains three putative transmembrane segments, M1, M3, and M4, and a re-entrant loop, M2. NMDA receptors generally form as tetramers of two NR1 and two NR2 subunits, which bind glycine and glutamate, respectively (9Schorge S. Colquhoun D. J. Neurosci. 2003; 23: 1151-1158Crossref PubMed Google Scholar). At present, LBD crystal structures have been obtained for each iGluR subclass, revealing bilobed clamshell-shaped domains arranged as back-to-back dimers (10Furukawa H. Singh S.K. Mancusso R. Gouaux E. Nature. 2005; 438: 185-192Crossref PubMed Scopus (582) Google Scholar, 11Mayer M.L. Neuron. 2005; 45: 539-552Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 12Armstrong N. Sun Y. Chen G.-Q. Gouaux E. Nature. 1998; 395: 913-917Crossref PubMed Scopus (601) Google Scholar). Within each LBD, domain 1 (D1) is stabilized by cross-subunit interactions, whereas domain 2 (D2) rotates and closes around bound ligand. Extensive studies with the GluR2 AMPA receptor, in complex with a range of full and partial agonists, established a correlation between agonist efficacy and degree of LBD closure, and a similar relationship was observed in kainate receptors (11Mayer M.L. Neuron. 2005; 45: 539-552Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 13Jin R. Banke T.G. Mayer M.L. Traynelis S.F. Gouaux E. Nat. Neurosci. 2003; 6: 803-810Crossref PubMed Scopus (335) Google Scholar). These results led to a structural model of receptor activation, in which agonist binding promotes LBD closure by rotating D2 toward D1, separating the linker regions and promoting channel opening. According to this hypothesis, partial agonists produce less domain closure, leading to decreased linker separation and slowing a subunit-specific pregating conformational change (14Erreger K. Geballe M.T. Dravid S.M. Snyder J.P. Wyllie D.J.A. Traynelis S.F. J. Neurosci. 2005; 25: 7858-7866Crossref PubMed Scopus (58) Google Scholar). However, crystal structures obtained for the NR1 subunit in complex with full and partial agonists adopt a similar degree of domain closure; thus, it appears that a different structural mechanism is required to account for partial agonist action at the NMDA receptor (15Inanobe A. Furukawa H. Gouaux E. Neuron. 2005; 47: 71-84Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Structural and molecular dynamics data have implicated the conformation of the hinge region, particularly the second interdomain β strand, in sensing agonist efficacy (15Inanobe A. Furukawa H. Gouaux E. Neuron. 2005; 47: 71-84Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 16Kaye S.L. Sansom M.S.P. Biggin P.C. J. Biol. Chem. 2006; 281: 12736-12742Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Increased interpocket motion has also been associated with NR1 site partial agonism, and both glycine and DCS can reportedly move within the pocket without affecting domain closure (16Kaye S.L. Sansom M.S.P. Biggin P.C. J. Biol. Chem. 2006; 281: 12736-12742Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). However, it remains unclear whether this destabilization is transmitted to the ion channel via increased cleft opening, permitting faster agonist dissociation, or a more subtle relay mechanism. In the NR2 subunit, a recent study has demonstrated that strengthening the D1-D2 interaction can increase open probability, kinetically linking domain closure to channel gating (17Maier W. Schemm R. Grewer C. Laube B. J. Biol. Chem. 2007; 282: 1863-1872Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Furthermore, destabilizing the closed cleft state by engineering steric clashes has been shown to reduce agonist efficacy and apparent affinity in NR2, GluR2, and GluR6 (18Weston M.C. Gertler C. Mayer M.L. Rosenmund C. J. Neurosci. 2006; 26: 7650-7658Crossref PubMed Scopus (70) Google Scholar, 19Hansen K.B. Clausen R.P. Bjerrum E.J. Bechmann C. Greenwood J.R. Christensen C. Kristensen J.L. Egebjerg J. Brauner-Osborne H. Mol. Pharmacol. 2005; 68: 1510-1523Crossref PubMed Scopus (22) Google Scholar, 20Robert A. Armstrong N. Gouaux J.E. Howe J.R. J. Neurosci. 2005; 25: 3752-3762Crossref PubMed Scopus (98) Google Scholar). However, partial agonist crystal structures have not yet been reported for NR2, so it remains to be determined whether the NR2 LBD behaves like NR1 or follows the AMPA/kainate receptor paradigm of partial agonist action. In the present study, we explored the relationship between cleft closure and activation in the NMDA receptor by stabilizing the NR1 and NR2 subunits in closed-cleft conformations via introduction of cross-cleft disulfide bonds. Both disulfide bonds induced significant constitutive activation, but although the NR2 LBD mutant appeared to be stabilized in a nearly full-agonist conformation, the disulfide-stabilized NR1 LBD displayed characteristics more indicative of a partial agonist. Our results suggest that although cleft closure is an important mediator of agonist efficacy, additional mechanisms are required to achieve full receptor activation, including a potential role for helix F. Additionally, both disulfide mutants increased agonist sensitivity at the opposing subunit, suggesting a possible intersubunit interaction between the NR1 and NR2 binding clefts. Molecular Modeling—Residue selection, mutation, and rotamer optimization were performed in the Swiss PDB Viewer (available on the World Wide Web), which was also used to detect hydrogen bonds and measure interatomic distances. The resulting molecular graphics were rendered in POV-Ray version 3.6. Site-directed Mutagenesis—Mutations in NR1 were generated using the megaprimer PCR method, as described previously (22Wood M.W. VanDongen H.M.A. VanDongen A.M.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4882-4886Crossref PubMed Scopus (136) Google Scholar), and subsequently confirmed by DNA sequencing of the cloned region. cRNA was prepared from linearized plasmids by in vitro transcription (22Wood M.W. VanDongen H.M.A. VanDongen A.M.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4882-4886Crossref PubMed Scopus (136) Google Scholar). Amino acids in both NR1 and NR2 are numbered from the initiator methionine. The NR2 subunit used in all experiments is an NR2A construct with a shortened 5′-untranslated region, previously shown to increase expression (23Wood M.W. VanDongen H.M.A. VanDongen A.M.J. J. Biol. Chem. 1996; 271: 8115-8120Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Oocyte Preparation and Injection—Stage V-VI oocytes were surgically removed from Xenopus laevis frogs under tricaine anesthesia, followed by manual defolliculation and treatment with collagenase type I (Invitrogen) (24Jones K.S. VanDongen H.M.A. VanDongen A.M.J. J. Neurosci. 2002; 22: 2044-2053Crossref PubMed Google Scholar). All frog work was carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Oocytes were injected with a 1:1 ratio of NR1 and NR2 cRNA (75 nl of a 10–100 ng/μl solution), transferred to SOS buffer, and incubated at 19 °C. SOS buffer consisted of 100 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 5 mm HEPES, pH-adjusted to 7.6 with NaOH, and supplemented with gentamicin (100 μg/ml). Two-electrode Voltage Clamp Electrophysiology—Functional expression was assessed 2–6 days after injection using a two-electrode voltage clamp amplifier (OC-725; Warner Instrument, Hamden, CT). The extracellular recording solution consisted of low barium Ringer's solution (Lobar; 100 mm NaCl, 5 mm KCl, 0.5 mm BaCl2, 10 mm HEPES, 10 μm EDTA), pH-adjusted to 7.35 with Tris base and maintained at room temperature. Barium was used as the divalent cation to minimize secondary activation of calcium-activated Cl- currents (25Leonard J.P. Kelso S.R. Neuron. 1990; 4: 53-60Abstract Full Text PDF PubMed Scopus (125) Google Scholar). EDTA was included to chelate trace amounts of the soft metal divalent cations Cd2+ and Zn2+, which have been reported to contaminate buffer solutions and inhibit the NMDA receptor by binding to a high affinity site (26Low C.-M. Zheng F. Lyuboslavsky P. Traynelis S.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11062-11067Crossref PubMed Scopus (151) Google Scholar, 27Paoletti P. Perin-Dureau F. Fayyazuddin A. Goff A.Le Callebaut I. Neyton J. Neuron. 2000; 28: 911-925Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). EDTA also removes a zinc-dependent component of desensitization (28Zheng F. Erreger K. Low C.M. Banke T. Lee C.J. Conn P.J. Traynelis S.F. Nat. Neurosci. 2001; 4: 894-901Crossref PubMed Scopus (104) Google Scholar). Maximal current response was elicited by application of 100 μm glycine and 100 μm l-glutamate, and agonists were co-applied unless stated otherwise. Voltage clamp recordings were performed in a perfusion chamber (Warner Instrument Corp., Hamden CT) optimized for laminar flow, and solution changes were accomplished using a gravity-fed, computer-controlled perfusion system, at a flow rate of ∼15 ml/min (24Jones K.S. VanDongen H.M.A. VanDongen A.M.J. J. Neurosci. 2002; 22: 2044-2053Crossref PubMed Google Scholar). Oocytes were impaled with low resistance glass microelectrodes (0.5–2.0 megaohms) filled with 3 m KCl and maintained at a holding potential of -60 mV. Data acquisition and voltage control were performed with PClamp hardware and software (Axon Instruments, Burlingame, CA). Measurement of Contaminating Glycine Concentration—The concentration of glycine in our Lobar solution, both in the presence and absence of glutamate, was evaluated via liquid chromatography-mass spectrometry using an ABI QStar electrospray mass spectrometer. The samples were run in positive mode, using a reverse phase column and 10–95% acetonitrile, 0.1% trifluoroacetic acid gradient. Glycine peaks (m/z = 76) were not observed in the total ion, total wavelength, or extracted ion chromatograms at a detection threshold of 1 pm. Curve Fitting and Statistics—Concentration-response curves were obtained by fitting data with a modified Hill equation: R/Rmax = Y0 + (1 - Y0)/(1 + (EC50/A)n), where R is the response for the given agonist concentration (A), Rmax is the maximum response, n is the Hill coefficient, EC50 is the concentration midpoint, and Y0 is the y intercept. Parameters were optimized by minimizing the residual sum of squares using the Solver function in Microsoft Excel. Each concentration point represents 4–8 oocytes, and error bars indicate S.E. All other data are presented as mean ± S.E. from 5–12 oocytes and analyzed statistically using one-way analysis of variance. For multiple comparisons, the data were initially subjected to a global analysis of variance incorporating all factors and measurements, and if this test showed a strong interaction between mutant and agonist response (p < 0.001), data were subdivided by agonist for lower order tests. Fischer's protected least significant difference test was then applied to compare the effects of the mutation on each response. Statistical significance is indicated with an asterisk for p < 0.05 (significant) or a double asterisk for p < 0.01 (highly significant). Time courses of methanethiosulfonate (MTSEA) modification and MK-801 block were fitted with first order exponential functions using the Clampfit module in pCLAMP 9.0. Reagents—All chemicals were purchased from Sigma with the exception of MTSEA (Toronto Research Chemicals), glycine (EMD Biochemicals), and l-glutamate (Invitrogen). DCK stock solutions were initially made in DMSO and diluted to their final concentrations in Lobar. For those experiments, DMSO was added to all recording solutions to maintain a standard concentration. Previous reports from our laboratory and others have suggested that the M3 domain functions as a transduction element, coupling ligand binding to channel opening, and agonist efficacy has been shown to correlate with M3 accessibility (24Jones K.S. VanDongen H.M.A. VanDongen A.M.J. J. Neurosci. 2002; 22: 2044-2053Crossref PubMed Google Scholar). To further explore the activation mechanism of the NMDA receptor, we set out to investigate the role of cleft closure in sensing partial agonist efficacy, specifically focusing on how cleft closure affects the accessibility of M3 and agonist sensitivity of both subunits. Closed cleft stabilization was achieved by engineering disulfide bonds, a technique commonly used to lock proteins in a defined conformation, spanning the NR1 and NR2 ligand-binding clefts. Designing the Disulfide Mutants—Complementary residues in the NR1 subunit were chosen by comparing the glycine- and antagonist-bound S1S2 crystal structures and identifying a cleft-spanning pair that could potentially interact only in the closed cleft conformation. Following cysteine substitution, the S-S distance within the Glu522/Ile691 pair (Fig. 1, A and B) was measured at 10.2 Å in the open cleft state and 3.5 Å after glycine binding, close enough to form a hydrogen bond and potentially a disulfide with a slight increase in cleft closure. Since an open cleft crystal structure for the NR2 S1S2 domain has not yet been published, pairs of NR2 residues were selected from the glutamate-bound structure and compared with full-agonist and antagonist-bound GluR2 crystal structures (29Armstrong N. Gouaux E. Neuron. 2000; 28: 165-181Abstract Full Text Full Text PDF PubMed Scopus (784) Google Scholar). One set of NR2 residues, K487C and N687C, was predicted to form a disulfide bond in the glutamate-bound NR2 crystal structure (Fig. 1C). The homologous GluR2 substitutions (A452C and S652C) were predicted to form a hydrogen bond in the AMPA-bound structure but moved to 5.66 Å apart in the antagonist-bound structure, suggesting the possibility of a cleft-closing disulfide interaction. The selected residues were then introduced into their respective subunits, both individually and in pairs, and expressed in Xenopus oocytes for functional characterization. Glycine-independent Activation of the NR1 Subunit—To test for the presence of a cleft-stabilizing interaction, glycine concentration-response curves were performed on the NR1 double mutant and the individual point mutants (Fig. 2A and Table 1). Both E522C and I691C exhibited an increased sensitivity to glycine compared with wild type, as evidenced by the left-shifted curves, but very little glycine-independent current. In contrast, the E522C/I691C double mutant (henceforth referred to as EI) displayed significant glycine-independent current, ∼87% activation in the presence of glutamate alone. A left-shifted concentration-response curve has been previously reported for the I691C mutant, attributed to the stabilizing effects of an interlobe hydrogen bond with Glu522 (30Kalbaugh T.L. VanDongen H.M.A. VanDongen A.M.J. Mol. Pharmacol. 2004; 66: 209-219Crossref PubMed Scopus (22) Google Scholar), and replacing Glu522 with the smaller cysteine residue appears to eliminate potential clashes with the cross-cleft isoleucine. However, although each single mutation favorably affects cleft stability, the phenotype of the double mutant is clearly synergistic. Trace amounts of glycine have been reported to contaminate buffer solutions (31Zhang L. Peoples R.W. Oz M. Harvey-White J. Weight F.F. Brauneis U. J. Neurophysiol. 1997; 78: 582-590Crossref PubMed Scopus (52) Google Scholar, 32Lerma J. Zukin R.S. Bennett M.V.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2354-2358Crossref PubMed Scopus (98) Google Scholar), resulting in transient glutamate-only currents, but since NR1 EI also exhibits decreased glycine sensitivity (Fig. 2A), glycine contamination cannot account for the substantial glutamate-only current observed in this mutant. Additionally, our buffer and glutamate-only solutions were assayed via liquid chromatography-mass spectrometry, which determined background glycine levels to be below a detection level of 1 pm.TABLE 1Concentration-response data for NR1 EI and NR2 KN Shown are values obtained from fitting concentration-response curves for glycine, glutamate, DCK, and APV with the Hill equation. Y0 represents the glycine- or glutamate-independent current, nH is the Hill coefficient, and EC50/IC50 is the concentration midpoint in μm. Note that the EI glycine and KN glutamate midpoints are not technically EC50 values, since the receptors are over 50% activated in the absence of agonist.GlyGluDCKAPVY0EC50nHY0EC50nHIC50nHIC50nHμmμmμmμmWT0 ± 0%1.161.247 ± 1%4.001.8441.21.9418.01.06NR1 EI87 ± 4%3.752.357 ± 2%0.581.642500.381161.60NR1 E522C4 ± 1%0.280.992 ± 1%1.111.45NR1 I691C8 ± 1%0.341.352 ± 1%4.641.94NR2 KN0 ± 0%0.761.9090 ± 1%0.020.9041.52.078.810.54NR2 K487C0 ± 0%0.972.005 ± 1%2.200.94NR2 N687C0 ± 0%1.202.455 ± 2%4.921.09 Open table in a new tab To examine whether the altered phenotype of the NR1 LBD had any intersubunit effects, glutamate concentration-response curves were also determined for all three mutants (Fig. 2B and Table 1). Both EI and E522C were significantly left-shifted relative to wild type, suggesting a potential positive coupling between two subunits reported to exhibit negative cooperativity (21Regalado M.P. Villarroel A. Lerma J. Neuron. 2001; 32: 1085-1096Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The source of the E522C phenotype is unclear but may be due to the removal of cross-cleft steric clashes observed in silico with the glutamate residue. The presence of a functional disulfide bond was tested initially with 1 and 10 mm dithiothreitol (data not shown), which had little effect on the agonist-independent current. A smaller reducing agent, 2-mercaptoethanol (BME), was successful at reducing the glycine-only current by 70% (Fig. 2C), thus confirming the presence of a cleft-stabilizing disulfide bond. The possibility that some cysteine residues do not form disulfide bonds has been reported for LBD-stabilizing disulfides in the GABA-B receptor (33Kniazeff J. Saintot P.-P. Goudet C. Liu J. Charnet A. Guillon G. Pin J.-P. J. Neurosci. 2004; 24: 370-377Crossref PubMed Scopus (73) Google Scholar); however, treatment with 0.1% H2O2, an oxidizing agent, and full agonist test pulses had no significant effect on the glycine-independent response of NR1 EI receptors (data not shown), indicating that unreacted cysteine residues are unlikely to contribute to the phenotype of this mutant. Glutamate-independent Activation of the NR2 Subunit—The same set of experiments was repeated for the NR2 cysteine mutants, K487C, N687C, and K487C/N687C (KN), to assess the degree of glutamate-independent activation. One of the individual cysteine mutants, K487C, exhibited a small leftward shift of the glutamate concentration-response curve, most likely due to relief of potential clashes observed between lysine and the cross-cleft asparagine (Fig. 2D). The NR2 KN mutant, however, was substantially more sensitive to glutamate, with a concentration midpoint of 21 nm. Furthermore, KN receptors were 90% activated by glycine alone, indicating a significant glutamate-independent response. Glycine concentration-response curves were also performed, in order to gauge the effects of the NR2 phenotype on the NR1 subunit (Fig. 2E). K487C and KN were both slightly left-shifted, with 1.2–1.5-fold decreases in the concentration midpoint, indicating that eliminating the steric clashes between Lys487 and Asn687, and thus facilitating NR2 LBD closure, is the most likely cause of increased glycine sensitivity. Reducing agents were used to probe the existence of a cross-cleft disulfide, which was also resistant to 1 and 10 mm DTT treatment). BME application, however, resulted in a 58% reduction in glycine-only current, strongly suggesting the presence of a cleft-stabilizing disulfide interaction (Fig. 2F). Full agonist test pulses had no effect on the constitutive activation of KN receptors; however, a small degree of potentiation (15%) was observed following H2O2 treatment (data not shown). Thus, we cannot rule out the possibility that unreacted cysteine residues contribute to the phenotype of NR2 KN. Decreased Sensitivity to Competitive Antagonists—The NR1 EI and NR2 KN mutants were further characterized using concentration-inhibition curves for 5,7-dichlorokynurenic acid (DCK), a competitive glycine-site antagonist, and 2-amino-5-phosphonovalerate (APV), a competitive glutamate site antagonist. NR2 KN displayed similar DCK sensitivity to wild type, whereas NR1 EI was completely immune to inhibition (Fig. 3A). NR2 KN was not only insensitive to APV inhibition; the concentration-response curve revealed a 33% potentiation with the competitive antagonist (Fig. 3B). Both results are consistent with stabilization of the LBDs in a closed cleft state, which could preclude antagonist access to the ligand-binding cleft and/or prevent antagonist-induced cleft opening. Interestingly, NR1 EI also exhibited a 6.5-fold increase in APV IC50, indicating a decreased sensitivity to glutamate site antagonism consistent with the observed increased sensitivity to glutamate. NR1 EI and NR2 KN Have Divergent Effects on Channel Activation—Further experiments were undertaken to determine the effect of our LBD disulfide mutants on the channel gate and the M3 segment, proposed to couple ligand binding to channel gating (24Jones K.S. VanDongen H.M.A. VanDongen A.M.J. J. Neurosci. 2002; 22: 2044-2053Crossref PubMed Google Scholar). The rate of inhibition by MK-801, an irreversible NMDA receptor open channel blocker, has been shown to depend on channel open probability (Po) and MK-801 binding rate (34Rosenmund C. Feltz A. Westbrook G.L. J. Neurosci. 1995; 15: 2788-2795Crossref PubMed Google Scholar). Given that MK-801 interacts with the channel pore, its binding rate should be unaffected by mutations within the ligand-binding clefts, and we therefore assumed that an altered block rate would reflect a change in Po. Both mutants were treated with 200 nm MK-801 in the presence of saturating glycine and/or glutamate (Fig. 4A); the MK-801 concentration was optimized to yield an inhibition time course of ∼10 s in wild type, facilitating comparison of blocking kinetics. Both disulfide mutants were fully inhibited by 200 nm MK-801 but displayed significant differences in block rate (Table 2). The NR1 EI mutant was found to block ∼2-fold slower than wild type, indicating an unexpected decrease in open probability. In the absence of glycine, the EI block rate decreased even further, but the difference between both agonists and glutamate alone was not statistically significant. Thus, closing the glycine-binding domain via an EI disulfide bond appears to inhibit channel gating, even in the presence of full agonist. In contrast, the MK-801 block rate in the NR2 KN mutant was increased by ∼2-fold relative to wild type, in both the presence and absence of glutamate. These results suggest that the KN disulfide bond has a potentiating effect on channel gating, such that the response elicited from KN with glycine alone has a higher Po than the wild type full agonist response.TABLE 2MTSEA modification and MK-801 block rates Shown are the rate and degree of potentation by 0.5 mm MTSEA and inhibition by 200 nm MK-801. MTSEA potentiation values for NR1 EI and NR2 KN indicate modification of the opposing subunit (NR2-A7C and NR1-A7C, respectively). Relative Po values were estimated from the ratio of MTSEA -fold potentiation or MK-801 block, based o
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